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Surface modification of an acrylate-based shape memory polymer to promote adsorption to biomarkers of eosinophilic esophagitis

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Title:
Surface modification of an acrylate-based shape memory polymer to promote adsorption to biomarkers of eosinophilic esophagitis
Creator:
Shah, Roopali R. ( author )
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
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English
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Subjects / Keywords:
Shape memory effect ( lcsh )
Esophagus -- Diseases ( lcsh )
Eosinophilia ( lcsh )
Adsorption (Biology) ( lcsh )
Adsorption (Biology) ( fast )
Eosinophilia ( fast )
Esophagus -- Diseases ( fast )
Shape memory effect ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Review:
The surface of an acrylate-based shape memory polymer ("SMP") was modified to capture cationic biomarkers of eosinophilic esophagitis ("EoE") to establish a better diagnostic and monitoring system of this disease. Incorporation of a negative charge into the SMP allows for specific adsorption of the cationic biomarkers with additional leverage from the shape memory effect to position the polymer near the lumen wall of the esophagus. SMP modification was performed by copolymerization with acrylate acids at increasing weight percentages to incorporate a negative, electrostatic charge onto the polymer surface, but still preserve and instill hydrophobic interactions. Because literature suggests that electrostatic, hydrophobic and hydrogen bonds are the major driving interactions for adsorption, the polymer was the target of modification to the system in the initial phases of the experimental design. Reproducible binding did not occur with in-vivo mimicking systems. Polystyrene microspheres, a commercially available positive control known to adsorb proteins, was introduced to the experimental process after controlling for other influential adsorption parameters (pH, protein, protein concentration, surface-to-volume ratios, pH, riddance of impurities or competing proteins, etc.) that also established no binding to the surface of the SMPs. The positive control facilitated a checkpoint for the development of SMP adsorption. The microspheres were used to check against all experimental parameters that were performed prior, to measure encouraging or discouraging protein adsorption factors. The polystyrene microspheres unveiled the importance of ionic strength, pure protein solutions and surfactants to adsorption. This illustrates that adsorption depends on multiple factors and that electrostatic charge and hydrophobicity on the polymer surface is just one such parameter. The system was revised and binding was performed with SMPs incorporated with 0, 0.5, 1 and 2.5 wt% of SEM. Results showed significant amounts of protein binding onto the surface of the SMP's. Additionally, adsorption of the SEM polymers was tested against the adsorption of polystyrene and nylon for comparative analysis of mainstream biomedical materials. SEM SMPs bind to protein preferentially over these materials suggesting our SMPs are better adsorbents, paving the way for many biomedical applications.
General Note:
Thesis (M.S.)--University of Colorado Denver. Bioengineering
Bibliography:
Includes bibliographic references.
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System requirements: Adobe Reader.
Numbering Peculiarities:
Department of Bioengineering
Statement of Responsibility:
by Roopali R. Shah.

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|University of Colorado Denver
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880150586 ( OCLC )
ocn880150586

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Full Text
SURFACE MODIFICATION OF AN ACRYLATE-BASED SHAPE MEMORY POLYMER TO
PROMOTE ADSORPTION TO BIOMARKERS OF EOSINOPHILIC ESOPHAGITIS
by
ROOPALIR. SHAH
B.A., University of Colorado Boulder, 2008
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Masters of Science
Bioengineering
2013


This thesis for the Masters of Science degree by
Roopali R. Shah
has been approved for the
Bioengineering Program
by
Robin Shandas, Chair
Glenn Furuta, Advisor
Dae Won Park, Advisor


Shah, Roopali, R (M.S., Bioengineering)
Surface Modification of an Acrylate-Based Shape Memory Polymer to Promote Adsorption to Biomarkers
of Eosinophilic Esophagitis
Thesis directed by Professor Robin Shandas
ABSTRACT
The surface of an acrylate-based shape memory polymer (SMP) was modified to capture cationic
biomarkers of eosinophilic esophagitis (EoE) to establish a better diagnostic and monitoring system of
this disease. Incorporation of a negative charge into the SMP allows for specific adsorption of the cationic
biomarkers with additional leverage from the shape memory effect to position the polymer near the lumen
wall of the esophagus. SMP modification was performed by copolymerization with acrylate acids at
increasing weight percentages to incorporate a negative, electrostatic charge onto the polymer surface, but
still preserve and instill hydrophobic interactions. Because literature suggests that electrostatic,
hydrophobic and hydrogen bonds are the major driving interactions for adsorption, the polymer was the
target of modification to the system in the initial phases of the experimental design. Reproducible binding
did not occur with in-vivo mimicking systems. Polystyrene microspheres, a commercially available
positive control known to adsorb proteins, was introduced to the experimental process after controlling for
other influential adsorption parameters (pH, protein, protein concentration, surface -to-volume ratios, pH,
riddance of impurities or competing proteins, etc.) that also established no binding to the surface of the
SMPs. The positive control facilitated a checkpoint for the development of SMP adsorption. The
microspheres were used to check against all experimental parameters that were performed prior, to measure
encouraging or discouraging protein adsorption factors. The polystyrene microspheres unveiled the
importance of ionic strength, pure protein solutions and surfactants to adsorption. This illustrates that
adsorption depends on multiple factors and that electrostatic charge and hydrophobicity on the polymer
surface is just one such parameter. The system was revised and binding was performed with SMPs
incorporated with 0, 0.5, 1 and 2.5 wt% of SEM. Results showed significant amounts of protein binding
onto the surface of the SMPs. Additionally, adsorption of the SEM polymers was tested against the
adsorption of polystyrene and nylon for comparative analysis of mainstream biomedical materials. SEM
m


SMPs bind to protein preferentially over these materials suggesting our SMPs are better adsorbents, paving
the way for many biomedical applications.
The form and content of this abstract are approved. I recommend its publication.
Approved: Robin Shandas
IV


ACKNOWLEDGMENTS
I would like to thank Kiran Dyamenahalli, Dr. Steve Ackerman and my committee members for
their time and helpful guidance through this process. A special thanks to my parental unit, Raj and Geeta
Shah for their encouragement and endless support.
v


TABLE OF CONTENTS
Chapter
1. Introduction.................................................................................1
1.1 Shape Memory Polymer: A Diagnostic Approach for Eosinophilic Esophagitis....................1
2. Background...................................................................................3
2.1 Eosinophilic Esophagitis and the Esophageal String Test.....................................3
2.2 Adsorption Principles.......................................................................4
2.3 SMPs and Surface Modification...............................................................6
3. Rational for the Experimental Design and Important Considerations for Adsorption Studies.....8
3.1 Prelude to Rationale........................................................................8
3.2 Polymer.....................................................................................8
3.2.1 Major Interactions Forces Involved in Adsorption..........................................8
3.2.2 Shape Memory Polymer.....................................................................10
3.2.3 Surface Area.............................................................................10
3.2.4 Impurities on the Polymeric Surface......................................................10
3.2.5 Postprocessing...........................................................................11
3.3 Protein....................................................................................13
3.3.1 General Considerations for the Study Protein.............................................13
3.3.2 Poly-L-Arginine..........................................................................14
3.3.3 Eosinophil Derived Granule Proteins......................................................14
3.3.4 Bovine Serum Albumin.....................................................................15
3.4 Solution.................................................................................15
3.4.1 Solutions Importance....................................................................15
3.4.2 Temperature..............................................................................15
3.4.3 pH.......................................................................................16
3.4.4 Ionic Strength...........................................................................16
vi


3.4.5 Purity..................................................................................16
4. Materials and Methods......................................................................17
4.1 Materials.................................................................................17
4.2 Methods...................................................................................17
4.2.1 Characterization........................................................................17
4.2.1.1 Contact Angle and Surface Free Energy..................................................17
4.2.1.2 pH Testing.............................................................................18
4.2.1.3 Water Equivalency Test.................................................................18
4.2.1.4 Dynamic Mechanical Analysis............................................................18
4.2.1.5 Fourier Transform Infrared Analysis....................................................19
4.2.2 Adsorption Testing......................................................................19
4.2.2.1 SMP Synthesis..........................................................................19
4.2.2.2 Binding Experiments....................................................................19
4.2.2.3 Experiment 1. MA.......................................................................20
4.2.2.4 Experiment 2-3. CEA....................................................................20
4.2.2.5 Experiment 4. Surface Area 132mm2/ml...................................................21
4.2.2.6 Experiment 5. Surface Area with SMP Particles..........................................21
4.2.2.7 Experiment 6. Surface Area with SMP Particles..........................................23
4.2.2.8 Experiment 7. Surface Area 648mm2/ml...................................................24
4.2.2.9 Experiment 8. Eosinophil Lysate........................................................24
4.2.2.10 Experiment 9-10. Eosinophil Lysate....................................................25
4.2.2.11 Experiment 11. Eosinophil Lysate......................................................25
4.2.2.12 Experiment 12. CEA....................................................................26
4.2.3 Positive Control- Optimization of Polystyrene Microspheres..............................26
4.2.3.1 General procedure......................................................................26
4.2.3.2 Surface Area and Surfactant Optimization...............................................27
4.2.3.3 Total Exclusion of Surfactants.........................................................27
vii


4.2.3.4 BSA Concentration Optimization...........................................................27
4.2.3.5 Buffer Solution Optimization.............................................................27
4.2.3.6 Reproducibility Testing..................................................................27
4.2.3.7 BSA Alexa Fluor 488......................................................................28
4.2.4 Positive Control Test Polystyrene Microspheres against Past Experiments................28
4.2.4.1 General Procedure........................................................................28
4.2.4.2 Experiments 1-7 Poly-L-Arginine..........................................................28
4.2.4.3 Experiments 8-11 Eosinophil Lysate.......................................................28
4.2.4.4 Experiment 12 BSA and Ionic strength.....................................................28
4.2.5 SEM Adsorption............................................................................29
5. Results and Discussion.......................................................................30
5.1 CEA Characterization........................................................................30
5.1.1 Contact Angle and Surface Energy..........................................................30
5.1.2 pH Testing................................................................................31
5.1.3 Dynamic Mechanical Analysis...............................................................34
5.1.4 Fourier Transform Infrared Spectroscopy...................................................34
5.2 SEM SMP Characterization....................................................................35
5.2.1 Contact Angle and Surface Energy..........................................................35
5.2.2 Reproducibility...........................................................................38
5.2.3 Water Equivalency Test....................................................................38
5.2.4 Reproducibility of Water Equivalency Test.................................................39
5.2.5 Dynamic Mechanical Analysis...............................................................39
5.2.6 Fourier Transform Infrared Spectroscopy...................................................43
5.3 Adsorption Testing..........................................................................45
5.3.1 Experiment 1. MA..........................................................................45
5.3.2 Experiment 2-3. CEA.......................................................................46
5.3.3 Experiment 4. Surface Area 132mm2/ml......................................................47
viii


5.3.4 Experiment 5. Surface Area with SMP Particles...........................................49
5.3.5 Experiment 6. Surface Area with SMP Particles- Complete Submersion......................52
5.3.6 Experiment 7. Surface Area 648mm2/ml....................................................53
5.3.7 Experiment 8. Eosinophil Lysate.........................................................55
5.3.8 Experiment 9-10. Eosinophil Lysate......................................................58
5.3.9 Experiment 11. Eosinophil Lysate........................................................61
5.3.10 Experiment 12. CEA.....................................................................63
5.4 Positive Control Optimization of Polystyrene Microspheres...............................64
5.4.1 Surface Area and Surfactant Optimization.................................................64
5.4.2 Total Exclusion of Surfactants..........................................................66
5.4.3 BSA Concentration Optimization..........................................................67
5.4.4 Buffer Solution Optimization............................................................68
5.4.5 Reproducibility Testing.................................................................69
5.4.6 BSA Alexa Fluor 488.....................................................................69
5.5 Positive Control Test Polystyrene Microspheres against Past Experiments.................70
5.5.1 Experiments 1-7 Poly-L-Arginine..........................................................70
5.5.2 Experiments 8-11 Eosinophil Lysate......................................................71
5.5.3 Experiment 12 BSA and Ionic strength....................................................72
5.6 SEM Adsorption............................................................................73
6. Conclusion.................................................................................79
References.....................................................................................82
IX


Appendix
A: Detection..................................................................................84
A.l Overview of Detection Methods..............................................................84
A.2 Micro BCA Assay............................................................................88
A.3 Bio-Rad Protein Assay......................................................................91
A.4 Spectroscopy...............................................................................92
A. 5 Flamingo Fluorescent Gel Stain...........................................................93
B: Protocols..................................................................................96
B. l Acetate Buffer...........................................................................96
B.2 Polystyrene Microspheres...................................................................97
B. 3 SEM B inding Experiment...................................................................98
B.4 Eosinophil Lysate.........................................................................100
x


LIST OF TABLES
Table
1 Protein Characterization.........................................................................14
2 Contact Angle and Surface Energy Measurements: Average of Ten Trials...........................31
3 Contact Angle and Surface Energy Measurements: Average of Ten Trials...........................37
4 Water Equivalency Test: Average of 2 Samples....................................................39
5 Thermal Mechanical Properties of SEM SMPs: Average of Two Trials.................................42
6 Protein Characterization.........................................................................56
7 % Protein Bound Relative to Total ECP............................................................57
8 % Protein Bound Relative to Total EDN............................................................57
9 % Protein Bound Relative to Total MBP1...........................................................57
10 % Protein Bound Relative to Total ECP..........................................................59
11 % Protein Bound Relative to Total EDN..........................................................59
12 % Protein Bound Relative to Total MBP1.........................................................60
13 % Protein Bound Relative to Total ECP..........................................................60
14 % Protein Bound Relative to Total EDN..........................................................61
15 BSA Characterization............................................................................63
16 % Protein Bound Relative to Total ECP..........................................................63
17 % Protein Bound Relative to Total BSA..........................................................66
18 % Protein Bound Relative to Total BSA..........................................................66
19 % Protein Bound Relative to Total BSA..........................................................67
20 % Protein Bound Relative to Total BSA..........................................................68
21 % Protein Bound Relative to Total BSA..........................................................69
22 % Protein Bound Relative to Total BSA-488......................................................70
23 % Protein Bound Relative to Total Poly-L-Arginine..............................................71
24 % Protein Bound Relative to Total BSA..........................................................73
25 % Protein Bound Relative to Total BSA..........................................................76
xi


26: Antibody-Based Assays.................................................................85
27: Absorbance-Based Assays...............................................................86
28: Fluorescence-Based Assays.............................................................87
29: BCA Assay with Hanks Balanced Salt Solution (HBSS)....................................89
30: BCA Assay withRPMI Diluted with Water (W) or PBS (P)..................................89
31: BSA Standard Curve Diluted in PBS (IX)................................................90
xii


LIST OF FIGURES
Figure
1 Important Protein Adsorption Determinants12..................................................5
2 Schematic of Maj or Adsorption Interaction6,7................................................9
3 Schematic of Surfactants Inhibiting Protein Binding..........................................11
4 Methanol Treatments to CEA SMPs..............................................................12
5 Sterilization Treatments to SEM SMPs and Polystyrene.........................................13
6 Processing of SMP Particles..................................................................23
7 Adsorption Optimization Techniques............................................................24
8 Contact Angle and Surface Energy Measurements of CEA SMPs....................................31
9 pH Testing of CEA SMPs.......................................................................33
10 DMA Results on CEA SMPs.....................................................................34
11 FT IR on CEA SMPs...........................................................................35
12 Contact Angle and Surface Energy with SEM SMPs..............................................36
13 Water Equivalency Test with SEM SMPs........................................................38
14 DMA Results on SEM SMPs.....................................................................41
15 Thermomechanical Reproducibility Analysis...................................................42
16 FTIR on SEM SMPs............................................................................44
17 Experiment 1 MA.............................................................................46
18 Experiment 2-3. CEA.........................................................................47
19 Surface Areas Implemented to Maximize Loading Capacity......................................48
20 Experiment 4. Surface Area 132mm2/ml........................................................48
21 SMP Particles...............................................................................50
22 Nylon Particles.............................................................................51
23 Experiment 5 Surface Area with SMP Particles................................................52
24 Experiment 6 Surface Area with SMP Particles-Complete Submersion............................53
25 Experiment 7 Surface Area 648mm2/ml.........................................................54
xiii


26 Experiment 8 Eosinophil Lysate. ECP Adsorption............................................56
27 Experiment 8 Eosinophil Lysate. EDN Adsorption............................................57
28 Experiment 8 Eosinophil Lysate. MBP1 Adsorption...........................................57
29 Experiment 9 Eosinophil Lysate. ECP Adsorption............................................59
30 Experiment 9 Eosinophil Lysate. EDN Adsorption............................................59
31 Experiment 9 Eosinophil Lysate. MBP1 Adsorption...........................................60
32 Experiment 10 Eosinophil Lysate. MBP1 Adsorption..........................................60
33 Experiment 10 Eosinophil Lysate. EDN Adsorption...........................................61
34 Experiment 11 Eosinophil Lysate. ECP Adsorption...........................................63
35 Experiment 12 CEA.........................................................................64
36 Surface Area and Surfactant Optimization..................................................65
37 Total Exclusion of Surfactants............................................................66
38 BSA Concentration Optimization............................................................67
39 Buffer Solution Optimization..............................................................68
40 Reproducibility Testing...................................................................69
41 BSA Alexa Fluor 488.......................................................................70
42 Experiments 1-7. Poly-L-Arginine..........................................................71
43 Experiments 8-11. Eosinophil Lysate.......................................................72
44 Experiments 12. BSA and Ionic Strength....................................................73
45 SEM Adsorption of BSA.....................................................................75
46 SEM Polymer after 3 Hour Incubation.......................................................77
47 Images of the SEM Polymer after 3 Horn Incubation.........................................78
48 Micro BCA Assay Performed on Experiment 12 CEA............................................91
49 Bio-Rad Assay Performed on Experiment 12 CEA..............................................91
50 Spectroscopy Performed on Experiment 12 CEA...............................................92
51 Spectroscopy Performed on Experiment 12 CEA................................................93
52 Flamingo Fluorescent Gel Stain on BSA Standards, Exposure with a Fluorometer at 532nm......94
53 Flamingo Fluorescent Gel Stain on BSA Standards, Exposure with UV Transilluminescence......94
xiv


54 BSA Standard Curve from Fluorescent Gel Stain.
95
xv


1. Introduction
1.1 Shape Memory Polymer: A Diagnostic Approach for Eosinophilic Esophagitis
In developed countries, 15% of all medical cases are misdiagnosed1. The Seattle Times reports
nearly one- third of the 2.7 trillion spent each year on healthcare in the U.S. are considered to be wasted
dollars. The current diagnostics set in place for eosinophilic esophagitis (EoE), a chronic inflammatory
allergic disease of the esophagus, contributes to this heavy expense. In this disease, eosinophils invade the
esophagus and wear away the esophageal lining. Biopsies are the gold standard for detecting EoE and
depend on measuring these eosinophils2. Since the eosinophils are not evenly distributed within the
esophagus, and the procedure is limited to acquiring less than a 0.7% sample size relative to the entire
esophageal surface area, the diagnosis can be missed3. One study reported a correlation between the
quantity of these eosinophils and highly cationic eosinophil derived granule proteins (EDGPs) they
secrete in the luminal mucosa4. Therefore, measuring mucosal inflammation can be one route to effectively
diagnose EoE patients and is under investigation through a novel esophageal string test-l. In this approach,
a swallowable nylon string is used to capture these cationic proteins in the lumen of the esophagus. The test
drives down the diagnostic costs and is minimally invasive. However, small sample sizes of the EDGPs
are an underlining issue due to small sample volume collected onto the string and the lack of specificity for
the EDGPs. With these limitations of current detection methods, patients of EOE are needlessly suffering
from symptoms that affect their quality of life.
The initial aim of this study was to chemically tailor a polymeric surface for the specific capture of
EDGPs. This contribution can aid in the development of a minimally invasive, inexpensive and reliable
diagnostic for EoE. Incorporation of anionically charged groups to the surface of a shape memory polymer
(SMP) can selectively adsorb these unique cationic proteins with additional leverage from the SMP to
position itself near the lumen wall of the esophagus in close proximity to the EDGPs. Additionally, the
shape memory effect of the polymer allows for substantial increase in recoverable surface area during
deployment, correlating to capturing greater sample sizes. Adsorption mechanisms have been extensively
studied and reported as a complex system, where the exact occurrences between the protein and surface
interfaces are unknown5-7. However, literature states the dominating factors that affect adsorption are
electrostatic, hydrogen and hydrophobic interaction forces5-9. Recent studies have found trends of
1


adsorption through electrostatic and hydrophobic interactions. These groups found that adsorption through
negative electrostatic interactions is higher relative to adsorption through hydrophobic interactions5,7. We
therefore hypothesized that the incorporation of negatively charged functional groups onto the surface of
the SMP would specifically aid in adsorption of cationic EDGPs.
The purpose of this study was to design a shape memory polymeric surface to promote protein
adsorption for the eventual binding of specific bio markers of EoE. The results of the study show significant
amounts of protein adsorption, specifically bovine serum albumin (BSA), onto the surface of the SMPs
after the incorporation of negatively charged sulfonic acid groups coupled with extensive adsorption system
adjustments.
2


2. Background
2.1 Eosinophilic Esophagitis and the Esophageal String Test
Eosinophilic esophagitis is an emerging chronic inflammatory disease of the esophagus effecting
thousands of Americans each year10. The disease is characterized by the infiltration of eosinophils to the
esophagus. These cells are a gateway to esophageal inflammation as they have the capacity to initiate an
inflammatory cascade causing a diverse set of symptoms. Serious complications include food impaction
and stricture formation that could require urgent removal of the food or endoscopic balloon dilatations10.
The current requirement for diagnosing this disease is through esophagogastroduodenoscopy (EGD) and
histological examination of esophageal mucosal biopsies10. These methods can be both inaccurate and
unreliable. Biopsies represent 0.7% of the esophageal surface area, a small enough sample size to miss the
disease3. Endoscopies depend on abnormal physical features of the lumen that may not be present in all
diseased patients2.
The Enterotest, performed originally to detect parasites in the intestine, is under experimentation
for capturing highly cationic EDGPs, biomarkers of EoE in the mucosa of the esophagus4. This esophageal
string test is a nylon string that is packed into a capsule. During the procedure, one end of the string is taped
to the cheek and the remaining string is carried through the GI tract, unraveling from the weighted capsule
upon swallowing. The capsule is dislodged from the string and mucosal remnants are captured onto the
material. Further in-vitro detection of EDGPs is performed via western blots, ELISAs and other antibody
driven tests. Although this is a great advancement in a noninvasive diagnostic for EoE, there is room for
improvement. In our application, we are altering the surface of the SMP to capture these cationic
biomarkers of EoE in a similar fashion to the Enterotest. The shape memory effect of the polymer will aid
in positioning the functionalized surface near the site of the biomarkers while the modified surface is
designed to specifically adsorb the EDGPs of EoE. This can improve diagnosis considerably by obtaining
greater sampling sizes and capturing specific proteins of the disease.
3


2.2 Adsorption Principles
Before the surface was modified, the principles of adsorption were defined and understood.
Adsorption is the process of particulate or molecular binding onto a surface. Surfaces are not fully bound
and consumed by their surrounding atoms as their bulk material; they have the capacity for binding atoms,
an energy favorable mechanism that lowers their energy state. Thus, surfaces usually possess higher surface
energy than their bulk material which we might identify with as a good adsorbent8.
Since the surfaces of polymers usually have low surface energy and are not as reactive as other
surfaces such as metal because of their low chemical potential, functional groups were incorporated to
facilitate pro-adsorption characteristics for EDGPs11. Protein adsorption is a complex mechanism primarily
because of the myriad of influential factors it encompasses, surface energy being one of them. It's a
function of the polymers surface, the protein being adsorbed, and the solution they are in. All of these
entities have their own list of influencing characteristics displayed in figure 1. The ones that overlap with
each other were heavily implemented in the final experimental design.
4


Adsorption determinants
Surface Free Energy
(hydro phillicity/hydrsph obi city)
Charge
Topography (micro and
nanoscale)
Surface impurities
Oxide
Ion Binding
Redox/corrosion potential
Hydrogen bonding capacity
Surface water
structure/binding strength
Acid/basa properties (PI)
Interactions with small
molecules
NetHydrophobicity
Charge and dipole
size and location
Prosthetic groups
ion binding
Hydrogen bonding
residues
Water binding and
localization
PI
Specific interacting
residues
Structure
Number of internal
disulfide bridges
Size (molecular weight)
Temperature
pH
I m panties
Ionic Strength
Figure 1 Important Protein Adsorption Determinants12
The stages that comprise protein adsorption onto a polymeric surface are: 1- the transport of
protein near the surface, 2- the attachment of protein onto the polymeric surface, 3- the rearrangement and
reorientation of the protein onto the surface, and 4- the desorption or permanent attachment of protein onto
the polymeric surface. The last stage is dependent on the extent of relaxation of the protein upon binding.
As the protein contacts the surface, interaction forces increase, and if large enough to compete with the
proteins intermolecular forces, the protein will start to denature, encouraging irreversible binding. If the
residency time is maximal, the protein will have sufficient time to relax before desorption permits712.
Minimal residency time is due to few and weak interaction forces upon initial binding or the interference
with other competing proteins.
In the first step of adsorption, the transport of protein near the surface of the polymer is due mostly
1
to coulombs interactions (electrostatic interactions) with an interaction force of where r is the distance of
5


the protein from the polymers surface. As the protein is drawn closer, Vander Waals forces, with an
1 12
interaction force of , persists as the driving force for the initial stages of adsorption .
The second stage, protein attachment, occurs if the polymer elicits active sites that compliment the
amino acids on the surface of the protein. Particularly functional groups such as COOH, OH, NH2, SH,
S03 and hydrophobic residues on both the surfaces of polymers and proteins, aid in the initial attachment
onto a polymeric biomaterial. Though this usually consists of multiple weak interactions, if more time is
allowed, they become stronger and irreversible which affect the third stage, reorientation and relaxation
onto the polymeric surface81213.
The influential factors in figure 1 can determine if the protein will remain bound irreversibly or
bind momentarily and desorb back into solution, the 4th stage in protein adsorption. If the adsorbent
competes with the intermolecular forces that keep the protein structurally sound, the protein is most likely
to bind irreversibly due to permanent conformational changes. For irreversible binding to occur, the protein
must unveil its hydrophobic core to the adsorbent. The more heterogeneous the intermolecular forces are on
the adsorbent, the more likely it will be able to unfold the protein and permit maximal residency time for
irreversible binding. It is for this reason, the SMP was modified to have a negative charge and accrue the
EDGPs to its surface, encouraging interaction with both electrostatic and hydrophobic chemical
constituents.
2.3 SMPs and Surface Modification
Biological responses can be reprogrammed from the surface of biomaterials rendering polymeric
surfaces important considerations to biomedical applications14. Surface texture, surface potential and
surface energy are mainstream surface features that can influence aggregation and/or binding to the surface
and trigger such host response15. Particularly, surface chemistry and topography dominate biological
responses to bio materials16. By altering the surface profile, biological responses can be manipulated
specifically to adsorb protein onto the surface, an important consideration in the field of biomedical
applications8. Concomitantly, bulk polymeric properties attribute to the core mechanical strength and
agility of the device, playing an integral role in establishing durability, flexibility and comfort. SMPs
extend this role much further as they are a class of polymeric materials with the ability to hold a fixed
6


temporary state and recover a memorized permanent state upon the introduction of an external stimulus
1 l 9. In our application, the SMP was utilized for the base component for this device because of this
intrinsic ability to memorize a predefined shape. An acrylate based SMP was used, with the ability to be
thermally stimulated to initiate shape transitions. Because the SMP can be stored in a temporary fixed state
for elongated periods of time, it can be packed and stored in a small capsule. Upon the introduction of the
polymer to the esophagus, the SMP is thermally stimulated and has the ability to undergo large recoverable
deformations. This characteristic can be easily tailored such that the polymer returns to a site near the
biomarkers and increases its surface area substantially. Together, these properties encourage adsorption by
increasing the loading capacity of the polymer and placing them in close proximity to the biomarkers to
shorten their transport path. The union of SMPs and refined surface properties can pave the way for a
myriad of biomedical devices. For this study, the focus is on designing an optimal SMP surface to promote
binding of unique cationic, EoE biomarkers for the development and improvement of a diagnostic for this
disease.
The base formulation of the SMP is mostly hydrophobic, an encouraging factor of protein
adsorption in general. Incorporation of negatively charged groups onto the surface can facilitate specific
and preferential binding to eosinophil derived granule proteins: major basic protein 1 (MBP1), eosinophil
cationic protein (ECP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPX).
7


3. Rational for the Experimental Design and Important Considerations for Adsorption Studies
3.1 Prelude to Rationale
In this study, the initial aim was to modify the surface of an acrylate-based SMP to adsorb specific
cationic proteins of EoE. Because protein adsorption is a complex mechanism, the initial experiments for
this goal yielded no binding. System adjustments were iterated throughout a major portion of this study
with a continued failure to adsorb proteins onto the SMP surface. The goals of the study broadened to
modifying the surface of the SMP to eventually adsorb cationic proteins. Until the core principles of protein
adsorption were understood, the specific adsorption of cationic proteins would be difficult to meet. A
commercially available adsorbent that served as a positive control was issued in the experimental process to
check against previous experiments and their environments. This analysis allowed us to see what particular
factors are encouraging or discouraging for protein adsorption. From this, the degree of complexity of
adsorption was revealed as multiple factors can significantly inhibit or attenuate binding. After final system
revisions were made from the positive control findings, significant amounts of BSA were adsorbed onto the
modified polymer surface. Because of the numerous system revisions that were implemented in this study,
detailed below is the rationale for considering these important factors that contribute to the multifaceted
mechanism of adsorption.
3.2 Polymer
3.2.1 Major Interactions Forces Involved in Adsorption
Electrostatic, hydrophobic and hydrogen bonds are cited as the major interaction forces in protein
adsorption5,7,812. However, the dominating factor is still under controversy, as the literature remains
inconsistent due to the complexity of adsorption. As such, all three interaction forces were studied. The
base formulation of our SMP is mostly hydrophobic, providing one of the major interaction forces. Tert-
Butyl Acrylate (tB A), the hydrophobic monomer in the SMP formula is displaced by the weight percent
of the new functional monomer being incorporated into the mixture, minimally decreasing the
hydrophobicity but increasing the other interaction forces.
For the initial goal of adsorbing cationic protein onto the surface of the polymer, negative
electrostatic interactions were implemented. Methacrylic acid (MA), 2-carboxyethyl acrylate (CEA)
and 2-sulfoethyl methacrylate (SEM) were chosen as monomers that would exhibit an electrostatic
8


charge under physiological pH and were incorporated into the SMP at various weight percents. Below is a
schematic of how the polymer surface would exhibit key interaction forces of adsorption. At pH 7, the
hydrogen on the carboxylic acid of MA and CEA would disassociate and leave the oxygen species
negatively charged. The negatively charge surface of the polymer can provide interaction sites for the
cationic proteins, encouraging adsorption through both electrostatic and hydrophobic (Van der Waals and
London type) interactions.
h COO-
CH
CM
h COOH
CH
CH
SO3 -
CM
CH
\ coo-
\ COOH
SO3-
I
CM
CM
CH
CH
CH
CH
Hcoo-
COOH
--5Q3 _
-------- Hydrophobic interaction ...........Electrostatic Interaction ------------Hydrogen Bonding
Figure 2 Schematic of Major Adsorption Interaction6,7
Methacrylic acid, 2-carboxyethyl acrylate and 2-sulfoethyl methacrylate was incorporated into the SMP at
increasing weight percentages to facilitate major interaction forces of adsorption.
Because the incorporation of MA and CEA at pH 7 did not yield protein binding, the system was adjusted
such that the pH matched the isoelectric point of the study protein. At the new pH (4.5), the hydrogen atom
on the carboxylic acid group should not disassociate, thus leaving the hydrogen intact to facilitate hydrogen
bonding.
After these polymeric alterations, binding still did not persist. The last modification was made
with SEM. We proposed this new monomer would work because it was a stronger acid than carboxylic
acids used previously and may possess a larger interaction force for protein capture. Literature also verified
using S03 functional groups for protein adsorption5.
9


3.2.2 Shape Memory Polymer
An acrylate based SMP was employed as the base polymer mainly because of its ability to self
deploy in the esophagus near the site of the EoE biomarkers and its high specific surface area after shape
recovery.
Additional benefits are its ability to be packed into a small capsule in its temporary state for an
elongated period of time. The SMP is also easily processed, cost efficient, non invasive and has great
mechanical properties.
3.2.3 Surface Area
Surface area directly correlates to the loading capacity of the polymer for protein. If the surface
area is minimal, small samples sizes are retrieved, and make detection difficult. As such, a course of action
was taken to increase surface area substantially and remained a focus for many experiments.
3.2.4 Impurities on the Polymeric Surface
Careful processing of the SMP is crucial for binding experiments as the surface is highly reactive
and contamination is fairly easy. The polymer samples were handled with gloves and under clean
conditions. The samples were always stored in a Ziploc bag, away from air particulates, before the binding
experiments.
During the initial experiments, the shape memory coupons were washed with Sparkleen. Because
this is an amphiphilic molecule, one concern was the hydrophobic portion of the molecule binding
irreversibly to the polymer surface. Even if the bond was reversible, impurities such as this become part of
the adsorption system and could interfere with binding by taking up sites on the protein or polymer that
shield the interaction forces we were counting on and altering the adsorption kinetics. To prevent
occupancy of the binding sites with such impurities, the polymers were switched to being washed with
clean nano pure or distilled (DI) water. The water source is important as well because organic and ionic
species can bind to the polymer surface, altering the charge or taking vacancy on limited binding sites. All
surfaces and equipment the polymeric samples came into contact with were wiped down with methanol or
ethanol and DI water.
10


Protein
M
.-? j
Surfactants
I
Figure 3 Schematic of Surfactants Inhibiting Protein Binding
3.2.5 Post Processing
Initially, all polymers were post cured for lhour at 90C Since binding did not occur, one
explanation was the possibility of free monomer that was not evaporated during the post cure. As
mentioned above, these impurities can take up valuable binding sites on the protein and polymer while
shielding important interaction forces. Thus, methanol treatments were carried out to swell the polymer and
eliminate leachable content. However, methanol treatments were swelling the polymer to approximately
1/3 of its own size, which ultimately could irreversibly expand the pore sizes of the polymer networks and
influence adsorption. Since our goal was to adsorb protein through electrostatic charge, the altered pore size
added an additional avenue of protein adsorption. Additionally, when methanol treatments were carried out
with CEA at 5 and 15wt%, the samples cracked after vacuum drying them for 48hours. In effect, methanol
treatments were stopped.
11


Figure 4 Methanol Treatments to CEA SMPs
48hour vacuum drying and methanol treatments on 5 and 15 wt% CEA causes polymeric cracks.
Sterilization was examined to ensure riddance of impurities. Polystyrene and SEM samples (0, 0.5,
1 and 2.5 wt %) were placed in an autoclave after DI washing. The 2.5 and 1% SEM samples did not
survive this process as they encountered numerous cracks. Additionally, the polystyrene samples became
distorted so we reverted back to the original post erne of lhour at 90C.
12


Figure 5 Sterilization Treatments to SEM SMPs and Polystyrene
Polymeric breaks and distortion were created from autoclaving samples at 250F for 15minutes. The first
box shows 0, 2.5 and 0.5% SEM SMPs while the second encompasses polystyrene and 1% SEM SMP
samples.
3.3 Protein
3.3.1 General Considerations for the Study Protein
The structural mobility, compactness, size, intramolecular forces and charge were all considered in
picking a study protein.
High structural mobility encourages irreversible binding onto the polymer surface by readily
making conformational changes upon adsorption. This mobility allows for increased contact numbers onto
the polymeric surface and thus heightens the affinity of the protein for the polymer. Internal mobility also
increases the rate of binding.
The intramolecular forces maintain structurally stability of the protein; hence, less internal motion
occurs. Therefore, these intramolecular forces are what the adsorbent is competing against for final
conformational changes to take place that lead to irreversible binding. If the intramolecular forces are
strong and plentiful, more energy is required from the adsorbent to denature the protein. Therefore, less
intramolecular forces are better for this application. The number of disulfide bonds and apolar groups are
13


key contributors to the proteins stability. Apolar groups also contribute to the compactness of the protein,
an anti-adsorption variable.
The size of the protein is directly correlated to the number of interaction forces it can make. Since
a larger protein can make more contacts onto the polymer surface, the strength of the total adsorption bond
is stronger and will trend toward irreversible binding. A charged protein is structurally loose because of
intramolecular repulsion between the charged residues. This provides extra molecular mobility. Since the
target proteins are EDGPs, a positively charged analog was used. Below are the proteins picked for this
study and why.
Table 1 illustrates major protein characteristics that influence protein adsorption.
Table 1 Protein Characterization
Protein MW (Id) a) PI % Hydrophobicity % Charged Sites # disulfide bonds
Poly L Arginine 5-15 10.76 100 0
ECP 18.39 11.4 43.16 20.63 9
EDN 18.35 8.9 41.62 15.53 9
EPX 81.04 10.8 43.24 24.2 17
MBP 25.21 10.9 40.54 26.12 12
BSA 69.29 4.7 42.83 33.27 35
3.3.2 Poly-L-Arginine
Poly-L-arginine was chosen because it is a cationic protein with a similar isoelectric point (10.76)
to the EDGPs.
3.3.3 Eosinophil Derived Granule Proteins
Poly-L-arginine did not bind to the negatively charged polymeric surface. Since these synthetic
analogs did not have hydrophobic domains to make irreversible interactions, EDGPs were used in place.
These are the actual target proteins for the diagnosis and will provide the correct intramolecular forces that
we will be competing against. They were also chosen for the study protein after poly-L-arginine because of
the high density of interaction forces these native proteins intrinsically have on their surface and core to
promote irreversible binding.
14


3.3.4 Bovine Serum Albumin
BSA was primarily used because it is widely characterized and was the main study protein in other
adsorption studies.
Careful consideration of the experiments was taken because of the proteins tendency to
oligomerize over time or during elevated temperatures. Because oligomerization creates a bigger molecule,
it may adsorb more readily (quickly and firmly) than single BSA molecules. BSA also possesses pro-
adsorption characteristics because of its 1) high structural mobility, 2) hydrophobic cleft on its surface and
3) moderate size for more interactions (larger than most EDGPs).
3.4 Solution
3.4.1 Solutions Importance
The solution is especially important in our experiments as it controls the ionization states of both
the protein and polymeric surfaces. In essence, the negative, electrostatic interactions incorporated on the
polymeric surface are dependent on the pH and ionic strength of the solution. Because the negative charges
on the SMP specifically serve to bind the target, cationic proteins, it illustrates how critical the solution is
to EDGP adsorption. Similarly, if the pH and competing ions in solution displace the cationic charge on the
proteins surface, the specificity of the SMP device is eliminated.
Initially, solutions mimicking physiological parameters were implemented but because of the high
ionic strength, amino acid content and other interfering components, other buffers were considered. After
the process of elimination, ionic strength and the pH of the solution deemed the most important factors to
consider and were adjusted appropriately.
3.4.2 Temperature
All the adsorption studies were executed at physiological temperature (37C). Increased
temperatures accelerate the transport of the protein near the interfacial region of the polymer surface and
contribute to internal mobility of the protein.
15


3.4.3 pH
The pH influences charged groups onto the surface of the polymer and protein. If the pH is under
the isoelectric point of the species, it acquires a net positive charge. If the pH is above, the species has a net
negative charge and if the pH is at the PI of the species, it is neutral. This is a central component to protein
adsorption as it can impose repulsion forces between proteins and can change the acid/base nature of the
polymer surface.
3.4.4 Ionic Strength
Ionic strength is crucial to protein adsorption as it defines the degree of interfering counter ions in
solution. These ions can shield the charge that was designed for EDGP attachment or consume binding sites
on the protein itself. In this way, ions have the strength to weaken or persuade protein adsorption one way
or the other. The final experiments were performed in low ionic strength conditions (0.01M).
3.4.5 Purity
The cleanest reagents, including water, must be used. Ions and organic species compete with the
target proteins to adsorb onto the polymer surface. Not only do they consume valuable binding sites in lieu
of the targeted protein but also they can alter the charges of both the protein and polymer in which the SMP
device is reliant on for specific EDGP adsorption.
DI water was passed through a 0.2um filter and used to make up all solutions. All the beakers and
materials that came into contact with the adsorbents were autoclaved or sterilized by methanol/ethanol prior
to use.
16


4. Materials and Methods
4.1 Materials
Tert-butyl acrylate monomer, polyethylene glycol) dimethacrylate (PEGDMA) (Mn=550)
crosslinker, CEA monomer (552348-50ML), MA monomer and the photo initiator 2, 2-dimethoxy-2-
phenylacetophenone (DMPA)were ordered from Sigma Aldrich. SEM monomer was ordered from
polysciences (cat 02597-50). Aliquots of poly-L-glutamic acid (Sigma Aldrich MW 3,000-15,000, Stock
p4636-25mg) and poly-L-arginine (Sigma Aldrich MW 5,000-1500, Stock P4663) were prepared at
2,500ug/ml in PBS (Invitrogen) diluent. Nylon (ASTM D4066 PA-0114, White Amazon Supply) and
polystyrene sheets (McMaster-Carr 8734K39 1/16" Thick) were purchased to examine their adsorbent
potential compared to SMPs. Nylon is the composite material of the esophageal string test and polystyrene
is a known adsorbent that was utilized as a positive control. ECP and ECP ELISAs were purchased from
MBL International Corporation. The Micro BCA Protein Assay Kit (Thermo Scientific / Pierce 23235) was
the primary protein detection method.
4.2 Methods
4.2.1 Characterization
4.2.1.1 Contact Angle and Surface Free Energy
Polymer surfaces were examined through a goniometer. A flat polymer was placed on a stage and
a volume of DI water or Diiodomethane (Sigma-Aldrich 158429-25G) was slowly dropped onto the
surface. The droplet was photo documented and the angle between the interfacial solid and liquid was
calculated through the automated software. The surface energies were calculated through Fowkes equation
depicted below. The surface energies of the associated liquids are listed as well. Note: Because surfaces are
active species, precaution was taken in handling and processing them to avoid contamination or destructs
within its smooth surface.
17


Fowkes Equation12,20,21:
Ys = Yd + Yp
Yi = Yd + Yp
Ys = Ys = 0.25y;(l + cosG)2
p [0.5yi(l+cos9p)-{yiyf) |
Ys ~ v
n
H20 = y? = 21.8m//mA2
H20 = yp = 51.0 mj/m2
Diiodomethane = Yi = Yi = 50.8 mj/m2
4.2.1.2 pH Testing
12x59xlmm CEA SMP samples were placed in lOmL of Acetate buffer at a pH of 4.45. Stir bars
were added to each sample and were placed on a stir plate. The pH was recorded every hour for 4 hours.
The experiment was performed in duplicates.
4.2.1.3 Water Equivalency Test
SEM polymers, nylon and polystyrene were dehydrated in a vacuum oven for 48 horns at 60 C
and weighed for a baseline measurement. The polymers were incubated in excess DI water at 37C. The
polymers were weighed and recorded every 12 hours until they reached equilibrium, at 24hours. The
experiments were performed in duplicate.
4.2.1.4 Dynamic Mechanical Analysis
All CEA and SEM SMP samples were sized to 5x30xlmm and their edges were sanded with 600-
grit sand paper. The samples were cycled at 0.1HZ, with a heating rate of 3C/min with the testing
temperature ranging from O-IOOC0. The glass transition temperature (Tg) was determined by the peak of
the tan delta curves.
18


4.2.1.5 Fourier Transform Infrared Analysis
All CEA and SEM samples were polymerized between two glass slides with a thin spacer <
0.5mm. Free radical polymerization was initiated and propagated with a UV source (black ray) at an
intensity of ~1 lmW/cm2 for 30 minutes. The samples were carefully removed and heat treated for 1 horn at
90C. Fourier transform infrared spectroscopy (FTIR) spectra were taken and the disappearance of the
alkenes peak at 1610-1680cm_1 was used to determined convergence.
4.2.2 Adsorption Testing
4.2.2.1 SMP Synthesis
SMPs were synthesized using tBA monomer, PEGDMA crosslinker and DMPA photinitiator.
MA, CEA or SEM monomers were added to the base formulation listed initially at different mass fractions
displacing tBA fractions. Solutions were made by mixing desirable weight percentages of monomers, tBA,
PEGDMA and 0. lwt% of DMPA in a glass vial. The solutions were injected into a pre-casted mold made
from two glass slides separated with 1mm spacers. A UV lamp (Black-Ray) was used to polymerize the
solutions at an intensity of ~1 lmW/cm2 for 30 minutes. After polymerization, the polymer coupons were
removed from its cast and were heat treated at 90C for 1 hour to evaporate unreacted monomers. The
samples were sized and their edges sanded with 600 grit sand paper to even out texture introduced from
sizing. Afterwards, the samples were methodically washed with DI water.
4.2.2.2 Binding Experiments
SMP samples were incubated in protein solution for 1 hour at 37C. A protein only condition was
incubated alone for quantification of initial protein concentrations. Media only (no SMP added) and SMP
incubated in the media (no protein) are additional conditions that were used as background controls. All
experimental conditions were performed in triplicate. Supernatant was collected in separate eppendorfs and
frozen in a -20C freezer until further use. Either the solution depletion method was used to quantitate
protein bound indirectly to the samples or an extra elution step was performed to quantitate protein bound
directly. With the elution step, the polymer sample was rinsed with PBS and dabbed with a Kim wipe
before incubation with EST buffer. The samples were incubated for 30 minutes with a 30 second vortex
step after ten minute intervals. The free solution was collected and frozen until further use. An alternative is
19


the solution depletion method where the concentration of free protein after incubation with the SMP sample
was subtracted from initial protein concentration.
*Any deviances from these methods are noted in the experiment itself below.
4.2.2.3 Experiment 1. MA
SMP coupons were prepared with 0, 5 and 15 wt% of MA (0:80:20, 5:75:20, 15:65:20 [MA: tBA:
PEGDMA]). A UV source was used for polymerization with the intensity ~20mW/cm2. Circular discs were
formed with a 6mm dye (McMaster-Carr 3418A6). The discs were cleaned with methanol and DI water
several times and dried at 90 C for lhour. Samples were parafdmed in glass beakers overnight and used
the next day for the binding experiment.
SMP discs were incubated in 44ug/ml of poly-L-arginine and poly-L-glutamic acid for 5 minutes,
30minutes and 2hours at 37C in 24 well plates. PBS was used because of its compatibility with the BCA
assay and the surface to volume ratio was 37.68mm2. The BCA assay was ran on all samples after the free
supernatant was collected and the solution depletion method was used for quantification of protein bound.
4.2.2.4 Experiment 2-3. CEA
SMP coupons were prepared with 0, 5 and 15 wt% of CEA (0:80:20, 5:75:20, 15:65:20 [CEA:
tBA: PEGDMA]). A UV source was used for polymerization with the intensity ~20mW/cm2. Circular discs
were formed with a 10mm dye (McMaster-Carr 3418A1). The discs were cleaned with Sparkleen and DI
water several times and left out to dry overnight. Nylon was sized, sanded, washed and dried in the same
manner as the SMPs for experiment 3.
SMP discs were incubated in 44ug/ml of poly-L-arginine and poly-L-glutamic acid for 1 hour at
37C in 24 well plates. PBS was used because of its compatibility with the BCA assay and the surface to
volume ratio was 94.2mm2. Six experimental repeats were performed per condition to help decrease the
error bars. The BCA assay was run on all samples after the free supernatant was collected and the solution
depletion method was used for quantification of protein bound.
20


4.2.2.5 Experiment 4. Surface Area 132mm2/ml
SMP coupons were prepared from the base formulation 80:20 (tBA: PEGDMA). A UV source
was used for polymerization with the intensity ~20mW/cm2. Square samples were formed with the
dimensions of 20x20mm. The samples were cleaned with Sparkleen and DI water several times and left out
to dry overnight. Nylon was sized, sanded, washed and dried in the same manner as the SMPs for
experiment 3.
SMP discs were incubated in 44ug/ml of poly-L-arginine for 1 horn at 37C in glass beakers. PBS
was used because of its compatibility with the BCA assay. Three square samples were added per condition.
Because of the increased surface area, more protein solution had to be added (20mL) to cover the surfaces.
As such, the surface to volume ratio was 132mm2/ml. There were no experimental replicates because of the
large polymer samples. The BCA assay was run on all samples after the free supernatant was collected and
the solution depletion method was used for quantification of protein bound.
4.2.2.6 Experiment 5. Surface Area with SMP Particles
SMP coupons were prepared from the base formulation (80:20 tBA: PEGDMA) in. A UV source
was used for polymerization with the intensity ~20mW/cm2. The coupons were cleaned with Sparkleen and
DI water several times and left out to dry overnight. Polymer and nylon particles were created from a
dremel and collected into a vacuum device depicted below. The samples were imaged using an optical
microscope at 6X magnification to see the range of sizes created. Small particles were disposed of through
centrifugation steps (particles were passed through a 230um sieve to rid small particles but this did not
work well) so as not to interfere with the BSA assay and elicit a false positive result. One gram of SMP
particles were measured in a polypropylene 50mL conical. Ultra pure H20 was added to the conical and
spun at 1325 RCF at RT for lOminutes. The smaller particles were found on top and were decanted. Three
of these washes were performed as displayed below. The particles were vacuum dried for 2.5 horns. SMP
particles were weighed at 0.08, 0.04, 0.02, 0.01, 0.005 and 0.0025 grams and added to an ultra low
attachment (ULA) 6well plate. To determine the maximal surface to volume ratio, lmL volumes of PBS
were added to each sample until 8mL was reached per well. With the findings, 0.06 and 0.04g were used
for the binding experiment.
21


A. Vacuum System to Collect Polymer Particles
Polymer Particles are fed into the
50mL eppendorf tube
B. SMP Particle wash step
22


D. PBS added to Polymer Samples c. SMP particles weighed out and
Figure 6 Processing of SMP Particles
The particles were weighed at 0.04 and 0.06 grams and added to a ULA 6 well plate (Costar
Product 3471). 5mL of 44ug/ml of poly-L-arginine was added and the samples were incubated for 1 hour at
37C in a shaker at 115RMP. PBS was used because of its compatibility with the BCA assay. After the
incubations, lmL of supernatant was aspirated from each condition into separate eppendorfs and spun at
13,000 g for 10 minutes at RT. The BCA assay was ran on all samples after the free supernatant was
collected and the solution depletion method was used for quantification of protein bound.
4.2.2.7 Experiment 6. Surface Area with SMP Particles Complete Submersion
Refer to Experiment 5. 1.22 grams of SMP particles were weighed and placed in glass cryovials.
1.7mL of 44ug/ml of poly-L-arginine was added and the samples were incubated for 1 hour at 37 C in a
shaker at 200RMP. PBS was used because of its compatibility with the BCA assay. After the incubations,
lmL of supernatant was aspirated from each condition into separate eppendorfs and spun at 13,000 g for 10
minutes at RT. The BCA assay was ran on all samples after the free supernatant was collected and the
solution depletion method was used for quantification of protein bound.
23


4.2.2.8 Experiment 7. Surface Area 648mm2/ml
SMP coupons were prepared from the base formulation 80:20 (tBA: PEGDMA). A UV source
was used for polymerization with the intensity ~20mW/cm2. Square samples were sized to 20x20mm and
cut further into ~5x6mm samples. The samples were cleaned with Sparkleen and DI water several times
and left out to dry overnight. Nylon was sized, sanded, washed and dried in the same manner as the SMPs.
Next, the samples were incubated in methanol for 48hours to alleviate competing leachables that may still
be present after post heat treatments. Afterwards the samples were washed with DI water and dried in a
vacuum oven at 60C for 48 horns.
SMP pieces were incubated in 44ug/ml of poly-L-arginine for 1 hour at 37C in glass vials. PBS
was used because of its compatibility with the BCA assay. Three 20x20mm square samples each cut into
5X6mm samples were added per condition. 5mL of protein solution was added per condition for a final
surface to volume ratio of ~648mm2/ml. The samples were incubated at 37C and shaking at 200RPMs.
The samples were specifically oriented upright to limit them from floating to the top. The BCA assay was
ran on all samples after the free supernatant was collected and the solution depletion method was used for
quantification of protein bound.
Figure 7 Adsorption Optimization Techniques
Orient the vials so they are standing up to limit polymers from floating to the top
4.2.2.9 Experiment 8. Eosinophil Lysate
SMP coupons were prepared from the base formulation 80:20 (tBA: PEGDMA). A UV source
was used for polymerization with the intensity ~20mW/cm2. Coupons were washed with Sparkleen and DI
water and dried at room temperature. The coupons were cut into ~6X30mm wide strips and submerged in
24


methanol for 48hours. The strips were vacuum dried for 48hours at 60C. Afterwards samples were sized to
5X12mm and their edges were sanded with 600-grit sand paper. Next, the samples were washed with DI
water and dried once more for lhour at 60C in a vacuum oven. Samples were stored in a Ziploc bag until
further use. Nylon was treated in the same throughout all experiments. It was cleaned, cut and sized in the
same manner as the SMP samples. No post treatments were necessary except for lhour incubation at 90C
after it was washed.
Eosinophils (EOs) were isolated from peripheral blood. The cells were suspended in 0.025M of
sodium acetate buffer (pH of 4.3) with 10% protease inhibitor (Roche) at a concentration of 5X106 cells/ml.
The cells were sonicated to create EO lysate and spun down at 300g for 10 minutes. The supernatant was
collected and frozen at -80C until use.
SMP samples were incubated in 500k/ml of blood eosinophil lysate per RPMI + 8% FBS buffer
for lhour at 37C, final pH 7. The surface to volume ratio was 154mm2/ml. An elution step was carried out
after the binding experiment. The supernatant was collected and ECP, EDN and MBP1 ELIS As were
performed.
4.2.2.10 Experiment 9-10. Eosinophil Lysate
The experimental parameters remained the same as experiment 8. The only exception is the
polymer samples did not undergo methanol treatments. The polymer samples were cleaned with Sparkleen
and DI water. After this washing step, the polymer was vacuum dried at 90C for 3.5 hours. The coupon
was cut, sized to 5x12mm samples and sanded. Next, they were washed with DI water and heat treated
once more for lhour at 60C in a vacuum oven to ensure the riddance of water. They samples were placed
in a Ziploc bag for storage.
4.2.2.11 Experiment 11. Eosinophil Lysate
To compare heat treated to methanol treated polymers, both post processing techniques were
administered. See experiment 8 and 9-10 for polymer preparation.
All parameters stayed the same with the EO prep except the low 300g spin was switched to a high
speed spin at 13,000g to ensure not sonicated cellular debris was pulled down and excluded from the lysate
for binding.
25


4.2.2.12 Experiment 12. CEA
SMP coupons were prepared with 0, 5 and 15 wt% of CEA (0:80:20, 5:75:20, 15:65:20 [CEA:
tBA: PEGDMA]). After the standard polymerization and heat treatment, the samples were washed with
Sparkleen and DI water. SMPs were sized to 5X12mm dimensions and their edges were sanded with 600-
grit sand paper. Finally, samples were washed with DI water several times and dried at 90C for one horn.
SMP samples were incubated in lmg/ml of BSA (Sigma) for 3 hours at 37C in 1.5mL eppendorf
tubes. Acetate buffer (40% of 0.1M acetic acid + 60% 0.1M sodium acetate) was used at a pH of 4.5. The
surface to volume ratio was 15mm2/ml. Three experimental repeats were performed per condition. The
BCA assay was ran on all samples after the free supernatant was collected and the solution depletion
method was used for quantification of protein bound.
4.2.3 Positive Control- Optimization of Polystyrene Microspheres
4.2.3.1 General procedure
Polystyrene microspheres were purchased from Bangs Laboratories, Inc (Catalog code DS03V,
0.5 lum). The polystyrene microspheres were initially optimized for the appropriate quantity-to-surfactant
ratio with respect to adsorption. Afterwards adsorption was tested against protein concentration, buffer
solution, reproducibility and surfactant-less solution. All the experimental groups remained the same from
the previous binding experiments: protein only, media only (no microspheres added), microspheres
incubated in media (no protein) and microspheres incubated with protein. The groups were performed in
triplicate. The samples were incubated in 120ug/ml for 3 horns at 37C unless indicated otherwise. After
the incubation, the microspheres were spun at 9300 G for 15minutes at 10C. The supernatant was carefully
collected in a separate eppendorf tube and spun again at 9300G for 15min at 10C. The supernatant was
collected and the BCA assay was ran. The solution depletion method was used for quantification.
26


4.2.3.2 Surface Area and Surfactant Optimization
Microspheres were diluted 1:10, 1:100 and 1:1000 in acetate buffer (pH 4.45, Ionic strength
0.01M, reagent grade materials used with fdtered DIH20) and incubated in BSA with a final concentration
of 120ug/ml for 3 hours at 37C. *
*First 1:10 dilution was made by adding 300ul of microspheres into 2,700ul of acetate buffer
4.2.3.3 Total Exclusion of Surfactants
40ul of microspheres were placed in a centrifuge tube and diluted with acetate buffer. The tube
was vortexed to wash the microspheres and spun at 14,000 g for 5 minutes at RT. The supernatant was
discarded and one more wash was performed on the spheres. Next, the microspheres were diluted 1:100 in
acetate buffer (pH 4.45, Ionic strength 0.01M, reagent grade materials used with fdtered DI H20) and
incubated in BSA with a final concentration of 120ug/ml for 3hours at 37C.
4.2.3.4 BSA Concentration Optimization
The microspheres were diluted 1:100 in acetate buffer (pH 4.45, Ionic strength 0.01M, reagent
grade materials used with nano-pure H20) and incubated in BSA with a final concentration of 120ug/ml or
40ug/ml for 3hours at 37C.
4.2.3.5 Buffer Solution Optimization
The microspheres were diluted 1:100 in either acetate buffer made from nano pure water or DI
water passed through a 0.2um filter. The diluted spheres were incubated in BSA with a final volume of
120ug/ml for 3 hours at 37C.
4.2.3.6 Reproducibility Testing
The reproducibility of adsorption with polystyrene microspheres was tested with pipetting small
versus large quantities of the microspheres when the microsphere dilutions were prepared. The
microspheres were diluted 1:100 by two 1:10 serial dilutions. The first 1:10 dilution determined the error as
the second dilution stayed the same for both conditions. The small quantity was prepared by adding 40ul
of microspheres into 360ul of acetate buffer (pH 4.45, Ionic strength 0.01M, reagent grade materials used
with filtered DI H20) for the first 1:10 dilution, the large quantity was prepared by adding 300ul into
27


2700ul of acetate buffer for its first 1:10 dilution. The second dilution remained the same for both
conditions; a 1:10 was performed by adding 300ul of the already prepared 1:10 dilution into 2700ul of
acetate buffer. The final diluted microspheres (1:100) were incubated in BSA with a final concentration of
120ug/ml for 3 hours at 37C.
4.2.3.7 BSA Alexa Fluor 488
Polystyrene microspheres were diluted 1:100 in acetate buffer (pH 4.45, Ionic strength 0.01M,
reagent grade materials used with filtered DIH20) and incubated in BSA alexa fluor 488 (Invitrogen
A13100) at a final concentration of 120ug/ml for 3 hours at 37C.
4.2.4 Positive Control Test Polystyrene Microspheres against Past Experiments
4.2.4.1 General Procedure
The polystyrene microspheres were treated the same as before. The microspheres were diluted
1:100 in the appropriate buffer specified and incubated with the study protein. The microspheres were spun
at 9300 G for 15minutes at 10C. The supernatant was carefully collected in a separate eppendorf tube and
spun again at 9300G for 15min at 10C. The supernatant was collected and the BCA assay was ran. The
solution depletion method was used for quantification.
4.2.4.2 Experiments 1-7 Poly-L-Arginine
Polystyrene microspheres were diluted 1:100 in PBS and incubated in 44ug/ml of poly-L-arginine
for 30 minutes, 1 hour, 2 hours and 3 hours at 37C.
4.2.4.3 Experiments 8-11 Eosinophil Lysate
Polystyrene microspheres were diluted 1:100 inRPMI with 8%FBS and incubated in 500k of EO
lysate (lysate was suspended in 0.25M sodium acetate buffer + 10% PI) for 1 hour at 37C.
4.2.4.4 Experiment 12 BSA and Ionic strength
Polystyrene microspheres were diluted 1:100 in either high ionic strength acetate buffer (0.1M) or
low ionic strength acetate buffer (0.01M). The spheres were incubated in BSA at a final concentration of
28


lmg/ml for the high ionic strength condition and 120ug/ml for the low ionic strength condition. The
samples were incubated for 3 hours at 37C.
4.2.5 SEM Adsorption
SEM polymers were synthesized by free radical polymerization using 0.4 wt% of DMPA photo
initiator. Mixtures of SEM monomer, the PEGDMA crosslinker and tBA monomer were injected between
the glass mold mentioned previously with the exception of a 1.5mm spacer. Polymerization was performed
under a UV lamp source with an intensity of ~1 lmW/cm2 for 30 minutes. After polymerization, the
polymer coupons were removed from their cast and were heat treated at 90 C for 1 hour to evaporate
unreacted monomers. The samples were sized to 5x19mm2 and their edges sanded with 600 grit sand paper
to even out texture introduced from sizing. Polystyrene sheets (McMaster-Carr) and nylon samples were
sized and sanded in the same manner for a comparative analysis against our SMPs; Afterwards, all
adsorbents were methodically washed with DI water.
SMP samples were incubated in 120ug/ml of BSA for 3 hours. Acetate buffer (pH 4.45, Ionic
strength 0.01M and reagent grade materials used with filtered DI H20) was used as the media with
0.0048%SDS and 0.0002% NAN3. The samples had a surface to volume ratio of 262mm2/ml. The BCA
assay was ran on all samples after the free supernatant was collected and the solution depletion method was
used for quantification of protein bound.
29


5. Results and Discussion
5.1 CEA Characterization
5.1.1 Contact Angle and Surface Energy
Water contact angle measurements in air were performed to test the incorporation of CEA, a
hydrophilic monomer, onto the polymeric surface of the SMP. Results show decreasing hydrophobicity
with increasing CEA wt%. In contrast, Diiodomethane, a nonpolar reagent, was used to measure
hydrophobicity at the SMP surfaces. Figure 8 shows increasing hydrophobicity with increasing CEA
percentages, an opposite trend of what was shown with water. Because surfaces are highly reactive, they
tend to be labile, which may begin to explain the adverse trend. The surfaces may have adapted to their
environment to stay at their most thermodynamically stable state.
Surface free energy was calculated from Fowkes equation found in the methodology section. By
partitioning the components of interaction forces between the solid and measuring liquid, both dispersive
and polar interactions were derived although they are not completely divorced entities20. The surface free
energy results disclose the increase in both dispersive and polar forces with increasing CEA wt%. These
increases are advantageous to protein adsorption as electrostatic and hydrophobic domains are the
dominating interactions.
30


Carboxyethyl Acrylate Influence on Contact Angle
H20
________________________________________________ Dioodomethane
0% CEA 5% CEA 10% CEA 15%CEA
s 60
1 50

40
30
W
d M 20
10
GO 0
Surface Free Energy
0% CEA 5% CEA 10% CEA
Dispersive
Polar
15% CEA
Figure 8 Contact Angle and Surface Energy Measurements of CEA SMPs
Water contact angles show decreasing hydrophobicity at the polymeric surfaces with increasing
incorporation of the hydrophilic monomer, CEA.
Table 2 Contact Angle and Surface Energy Measurements: Average of Ten Trials
Sample Code On 0a yt* (mj/m2) yI (mj/m2) Ys (mj/m2)
0% CEA 89.2 5.3 59.11 4.6 29.1 2.7 31.8
5% CEA 81.57 4.2 49.52 6.9 34.5 4.0 38.5
10% CEA 74.79 7.1 43.8 6.8 37.6 5.9 43.5
15% CEA 61.84 3.9 15.67 4.0 48.9 8.6 57.5
0p = Water contact angle 0d = Diiodomethane contact angle yf = Dispersive surface free energy
p
ys = Polar surface free energy ys = Total surface free energy
5.1.2 pH Testing
CEA was incorporated into the SMP to possess a negative charge at the polymers surface under
physiological pH. CEA SMPs were also studied at pH 4.5 to investigate hydrogen bond influences to
protein adsorption because hydrogen bonds are another major interaction force driving adsorption7. To
ensure hydrogen atoms were not disassociating from carboxylic acid functional groups, and were available
on the surface of the CEA SMPs, the pH was monitored over the course of 4 horns. The decline of the
31


solutions pH during the time course, from its baseline pH would illustrate hydrogen disassociation. Results
show very little variance from the initial pH for all CEA SMP polymers. Acetate buffer was used as a
control to gauge general fluctuations that may occur without the polymer influence. The acetate buffer
incubated with the polymer maintains a stable pH relative to that of the acetate buffer alone. These results
validate that the hydrogen atom does not disassociate from the carboxylic acid group, rendering it free for
hydrogen bonding during adsorption testing.
32


Level of COO-H Dissociation
4.5
X
a.
4.45
#G%CEA
H 15% CEA
Acetate Buffer
120 ISO
Time (min)
=£. 4.45
5. 4.45 _ _
Time (min)
150
Time (min}
100 ISO 200
Time {min}
ISO
Time (min)
4.5
= 4.-15
*
4.4
Acetate Buffer



Tim. (mini
Figure 9 pH Testing of CEA SMPs
The pH tests were carried out to measure the disassociation of hydrogen atoms from carboxylic acid
functional groups on the surface of CEA SMPs. Results show the preservation of the hydrogen atom onto
the polymer, thus ability to form hydrogen bonds.
33


5.1.3 Dynamic Mechanical Analysis
CEA SMPs were tested with dynamic mechanical analysis (DMA) to show preservation of the
shape memory effect after CEA incorporation. All the polymers still retain their shape memory effect
evidenced by the enormous temperature dependence illustrated by the 2-3 orders of magnitude drop in
storage modulus at its glass transition temperature. This indicates the recovery ability of the SMP. The
results display a 2-3 degree increase in the glass transition temperature per 5% CEA incorporation. The
slope of the storage modulus and width of the tan delta curve represents similar shape memory recovery to
the base formulation 0% CEA or 80:20 tBA: PEGDMA. These small deviances from the base polymer
are insignificant and could still provide proper mechanical properties for packing the SMP into a small
capsule and recover large deformations.
Figure 10 DMA Results on CEA SMPs
Shape memory effect is preserved by CEA incorporation.
5.1.4 Fourier Transform Infrared Spectroscopy
FTIR spectra of the CEA SMPs show the disappearance of the alkene peak at 1680-1610cm_1. This
validates consumption of reactive alkene groups during free radical polymerization and concurrently,
convergence to alkanes through the 2850-2970cm"1 peak.
34


Carboxyettiyl Acrylate
Figure 11 FTIR on CEA SMPs
The reactive alkene groups were consumed during polymerization of CEA SMPs
5.2 SEM SMP Characterization
5.2.1 Contact Angle and Surface Energy
Water contact angle measurements in air display slight increases in hydrophobicity with 1% and
2.5% SEM incorporation. Due to the low density of polymers, the flip-flop of molecules on the surface is
not uncommon4. Atmospheric exposure and consequently adaptation, can lead to reversible changes on the
surface to reduce high energy states. As such, the O-H of SEM could be embedded in the bulk of the
polymer when exposed to air. 0.5% SEM has a similar hydrophobicity to 0% SEM due to the low number
density of functional groups introduced. Both water and diiodomethane were used as measuring fluids to
departmentalize the interfacial interaction forces into dispersion or polar components. From the contact
angles alone, diiodomethane reveals the materials higher interactions with dispersive forces translating to
their hydrophobic nature. Nylon has substantial polar and dispersive forces, which may be an artifact from
poor manufacturing and thus surface impurities and imperfections that easily sway the results. The
hydrophobicity of a material extensively influences protein adsorption as it allows water to organize
loosely on the surface, granting a more energetically favorable displacement of water upon protein
adsorption. A hydrophilic surface binds water tightly and would require more energy to displace water for
protein5. From this, the SEM polymers would encourage protein adsorption but because there are many
other influential factors, contact angle measurements alone do not directly correlate to binding efficacy.
35


Contact Angle Measurements
Bat chi
Contact Angle Measurements
Batch2
105 90
1 60
O 45
cd 30
o u 15
0
DH20
Diiodomethane
i . b-
1 IH
L L 1 ~ ifer
ii v ii B
PS Nylon 0% 0.5% 1% 2.5%
SMP SMP SMP SMP
DH20
Diiodomethane
PS Nylon 0% 0.5% 1% 2.5%
SMP SMP SMP SMP
Surface Energy. Batch 1
Surface Energy. Batch 2
Dispersive Forces
^ Polar Forces
^ 25 -|-----------------------------------
PS Nylon 0% 0.5% 1% 2.5%
SEM SEM SEM SEM
J5 20
15
S-H w 10
1) o ,cs 5
GO 0
Dispersive Forces
Polar Forces
PS Nylon 0% 0.5% 1% 2.5%
SEM SEM SEM SEM
Figure 12 Contact Angle and Surface Energy with SEM SMPs
Contact angle measurements of SEM SMPs do not display SEM functional groups at the surface. Small
percentages of SEM were incorporated and may fall outside of the sensitivity of contact angle
measurements. This method may not display detailed chemical constituents.
36


Table 3 Contact Angle and Surface Energy Measurements: Average of Ten Trials
Sample Code. Batch 1 Or, erf Ys (mj/m2) Ys (mj/m2) Ys (mj/m2)
Polystyrene 91.2 3.0 42.4 3.3 18.8 0.4 19.2
Nylon 57.3 4.4 46.2 2.7 19.6 11.8 31.4
0% SEM 74.5 3.5 53.4 7.0 17.2 5.4 22.6
0.5% SEM 73.6 4.6 58.3 4.2 16.2 6.3 22.5
1% SEM 77.8 2.5 55 3.6 16.9 4.3 21.2
2.5% SEM 85.5 2.8 52.3 7.3 17.4 1.8 19.2

Sample Code. Batch 2 ev 0a Vs (mf/m2) YPs (mj/m2) Ys (mf/m2)
Polystyrene 91.2 3.0 42.4 3.3 18.8 0.4 19.2
Nylon 57.3 4.4 46.2 2.7 19.6 11.8 31.4
0% SEM 82.4 2.1 61.7 5.1 15.6 3.4 18.9
0.5% SEM 79.2 3.3 60.2 5.3 15.8 4.3 20.2
1% SEM 83.1 1.9 58.8 2.6 16.1 2.9 19.0
2.5% SEM 83.9 3.8 60.8 2.6 15.7 2.8 18.6
dp = Water contact angle Qd = Diiodomethane contact angle y = Dispersive surface free energy
p
ys = Polar surface free energy ys = Total surface free energy
Surface layers have more surface free energy than their bulk because their valence electrons are
not shared by their neighboring atoms1. As a result, surfaces are reactive and exist in an energetically
unfavorable state. In this way, surface free energy can represent the affinity of the surfaces for adsorption.
Fowkes partitioned the surface free energy into two components, dispersive and polar forces. From table 3,
batch 1 shows increasing dispersive or nonpolar surface free energy in the polymers with SEM
incorporation. However, 2.5% SEM in Batch 2 shows a decrease in dispersive surface free energy relative
to the other SEM polymers. Of die total surface free energies in the SEM polymers, the trend from greatest
to lowest surface energy is 0.5% SEM< 1% SEM< 2.5% SEM. Since the dispersive forces are pro
adsorption, these may be the ones to consider for protein adsorption. Strangely, nylon has more dispersive
and polar forces dian all other materials. Because non-uniform surface heterogeneity and surface mobility
can alter contact angle measurements representing the material, surface energies of nylon and between
SEM batches are not representative of detailed chemical compositions1.
37


5.2.2 Reproducibility
Between batches, the SMPs vary from approximately 0.5- 8.5 degrees and can subsequently alter
the trends seen within the SEM samples. The variability can be attributed to topographical imperfections on
the surface, non-uniform chemical heterogeneity and/or surface mobility 1 7. Performing more
measurements on a greater surface area may contribute to lowering these differences.
5.2.3 Water Equivalency Test
As evidenced with water equivalency tests, shown in figure 13, SEM was incorporated into the
polymer, as the hydrophilicity increased with increasing wt % of SEM. Nylon did not equilibrate at
24hours suggesting it could be more hydrophilic than the SEM samples. Due to its high physical and/or
chemical crosslink density, diffusion of water into the polymer system is much slower8. Since we
performed a 3 hour time point for the binding studies, the trends before the 24hour time point are more
relevant.
0 5 10 15 20 25 30
Nylon
Polystyrene
-*-0% SMP
-*-0.5% SMP
-*-l%SMP
--2.5% SMP
Time (hrs)
0 5 10 15 20 25 30
Nylon
Polystyrene
*-0% SMP
-*-0.5% SMP
-*-l%SMP
--2.5% SMP
Time (hrs)
Figure 13 Water Equivalency Test with SEM SMPs
Bulk absorption validates that the SEM monomer was incorporated into die SMP.
38


Table 4 Water Equivalency Test: Average of 2 Samples
Sample Code. Batch 1 % Water Uptake (12 HR) % Water Uptake (24 HR)
Polystyrene 0.1 0.2 0.1 0.2
Nylon 3.3 0.0 5.1 0.0
0% SMP 1.4 0.1 1.4 0.0
0.5% SMP 4.8 0.0 5.3 0.0
1% SMP 7.8 0.2 8.2 0.3
2.5% SMP 10.6 0.5 10.1 0.6

Sample Code. Batch 2 % Water Uptake (12 HR) % Water Uptake (24 HR)
Polystyrene 0.1 0.2 0.1 0.2
Nylon 3.3 0.0 5.1 0.0
0% SMP 1.3 0.1 1.4 0.2
0.5% SMP 4.2 0.2 4.7 0.0
1% SMP 7.4 0.0 7.8 0.0
2.5% SMP 11.1 0.2 11.0 0.1
5.2.4 Reproducibility of Water Equivalency Test
Both batches follow the same trends and have percent differences less than 1. 2.5% SEM
consistently shows more variance between batch 1 and 2 in the water equivalency and contact angle tests.
The monomer mixture may have small differences of SEM content with an error in the hundredths or
thousandths place.
5.2.5 Dynamic Mechanical Analysis
5.2.5.1 Shape Memory Effect
Figure 14 shows the temperature dependence of the polymers by a 1-2 orders of magnitude
decrease in storage modulus at their glass transition temperatures. This transition or switching effect,
allows the recovery of the polymer once it has been stored in its temporary state. The elasticity or storage
modulus gives the polymer its memory and allows it to return to its original shape2 Above the Tg, about
60-100C0, the SEM polymers should experience immediate elasticity as all the polymers are still in their
rubbery phase. The plateau of the storage modulus shown above the Tg can indicate physical cross linking
or increased crystalline formations from secondary intermolecular interactions between chains1,23. Since
SEM introduces polarity to the SMP, more secondary intermolecular interactions between chains can take
place in addition to chain realignment for crystalline/physical cross linkage. Incorporation of SEM shows


kinetically different transitions or switching rates as their slopes are moderate compared to 0% SEM. Thus,
the ability of the former to recover to its memorized shape is slower. Additionally, the weak mechanical
properties of the SEM polymers may hinder specific deformations or programming to its temporary shape
such that manufacturing processes would need to be carefully planned out.
5.2.5.2 Storage Modulus
SEM incorporation leads to a reduced storage modulus below Tg. Because chemical constituents
have the largest effect on altering thermal mechanical properties, it is reason to believe the C-0 bonds from
SEM allow for increased chain/bond flexibility below the Tg1. Tert-Butyl Acrylate (tBA) is also displaced
by the weight percent of SEM during monomer mixture preparations, contributing to lower Tg/storage
modulus values. Less free volume from the bulky tert-Butyl groups of tBA restrict large molecular
movements and as such, more energy requirements are needed for thermo mechanical transitions.
0.5 and 2.5% SEM have similar glass transition temperature slopes, regressing more gradually
from their glassy to rubbery phase relative to 0% SEM while 1% SEM has the slowest transition phase. The
storage modulus of 1% SEM is the highest of the SEM incorporated polymers, theoretically possessing
better elastic properties for shape recovery. 0.5 and 2.5% exhibit similar storage moduli and switching
stages but in batch two, 2.5% SEM possesses better elastic recovery than 0.5%. This may be a
reproducibility error and should be repeated. The trends within the SEM samples are less evident, but may
be contributed to the polymerization process itself as molecular weight, crystallinity and cross link density
affect the storage modulus and Tg values as well as chemical composition 1 3.
5.2.5.3 Tan Delta Peak
SEM incorporation into the SMP causes decreased crosslink density, increased crystallinity as
seen by the lower amplitude, and a left shift of the tan delta peak relative to the 0% SEM polymer. Thus,
SEM polymers have less mechanical strength or damping abilities that may permit low impact breaks
within the polymer system. Additionally, the broad peaks of the SEM polymers dictate slower glass
transition phases, in tune with the storage moduli slopes. Two transitions are seen in 0.5% SEM that may
be a caustic response from the annealing of polymeric chains through increased molecular movement after
its first glass transition phase. The additional secondary intermolecular interactions yield crystallinity above
40


the first glass transition temperature but can slip as the temperature increases further illustrating the second
glass transition temperature1. 2.5% SEM has the greatest cross linking density and amorphous content
compared to the other SEM incorporated polymers and thus has better damping abilities at its glass
transition temperature. The tan delta peak of 1% SEM may indicate a more uniform increase in molecular
weight during its polymerization process as it transitions slowly from its glassy to rubbery state with a tan
delta peak spanning the largest temperature range.
Batch 1 Batch 2
Figure 14 DMA Results on SEM SMPs
DMA storage modulus and tan delta graphs of SMPs with 0, 0.5, 1 and 2.5 wt% incorporation of SEM. The
SEM samples have depreciated mechanical and shape memory properties.
41


Table 5 Thermal Mechanical Properties of SEM SMPs: Average of Two Trials
Sample Code. Batch 1 Glassy Modulus (Mpa) Rubbery Modulus (Mpa) Tan Delta Peak Tg (C)
0% SEM 1551.45 473.41 4.12 0.08 1.75 0.00 51.87 1.21
0.5% SEM 850.56 31.28 5.03 0.17 0.52 0.00 29.63 1.02
1% SEM 1214.05 264.81 5.27 0.59 0.52 0.02 40.6 0.59
2.5% SEM 650.33 9.61 4.47 0.08 0.78 0.16 32.39 5.42
Sample Code. Batch 2 Glassy Modulus (Mpa) Rubbery Modulus (Mpa) Tan Delta Peak Tg (C)
0% SEM 1817.20 195.73 4.35 0.07 1.72 0.03 52.33 2.35
0.5% SEM 718.41 41.21 4.81 0.08 0.52 0.00 30.28 0.26
1% SEM 1212.45 71.63 5.32 0.10 0.48 0.01 42.29 0.42
2.5% SEM 1015.69 254.29 5.15 0.80 0.74 0.01 34.01 1.11
2500
m 2 2000
1500
1 1000
m m cz 500
3 0
m 1)
*3 S
O
H
60
50
40
30
20
10
0
Glassy Modulus
Glass Transition Temperature
Batch 1
Batch 2
Rubbery Modulus
Batch 1
Batch 2
Tan Delta Peak
Batch 1
Batch 2
2 i-----------------------------------
Figure 15 Thennomechanical Reproducibility Analysis
The stiffness between batches is the most variable but the other properties (Tg and tan delta peak) have
good reproducibility.
42


5.2.5.4 Reproducibility of Thermomechanical Properties
Figure 15 illustrates reproducibility of thermo mechanical properties between batches. The glassy
modulus is most prone to fluctuations between batches. Literature suggests these variations may be due to
the delicate process of polymerization, including pre and post processing. Variables such as aging,
atmospheric exposure and post heat treatments after polymerization can contribute to molecular
rearrangements and alter mechanical properties The tan delta peak and Tg between both batches have
similar trends with relatively low standard deviations. However, the rubbery moduli of 2.5% SEM
polymers potentially elicit different trends of stiffness in account of the standard deviations. Since binding
occurred at this rubbery state and stiffness is an influential factor in protein adsorption, this small trend
change could directly affect binding abilities and could start to explain the variable binding trend between
batches for 2.5% SEM. If heat pockets were introduced in the polymer coupon during the polymerization
process by the UV source, non-uniform cross linkage, molecular rearrangements and stresses could have
occurred in the 2.5% samples leading to different thermo mechanical properties. Note that the coupons used
for DMA analysis were the same coupons used to prepare the samples for the binding experiment.
An important distinction in the batches is the modulus switch between the 2.5% SEM and 0.5%
SEM sample in batch 2. In batch one, the 2.5% and 0.5% SEM samples are similar but the glassy and
rubbery modulus of 2.5% is slightly lower than 0.5% SEM. However, in batch 2, the glassy moduli and
glass transition temperature phase of 2.5% SEM is higher than 0.5%. Since stiffness could play a role in
adsorption, this may be a reason in a drop in adsorption at 2.5% SEM in batch 1.
5.2.6 Fourier Transform Infrared Spectroscopy
Fourier Transform Infrared Spectroscopy was utilized to confirm complete polymerization. The
SEM polymers show the disappearance of a peak near 1610-1680cm_1, the alkene range. Additionally a
large peak is shown in the 2850-2970cm_1 range, depicting convergence of alkene bonds to alkane bonds.
43


Absorbance Absorbance
Batch 1
Batch 2
Figure 16 FTIR on SEM SMPs
The reactive alkene groups during SEM polymerization were consumed.


5.3 Adsorption Testing
5.3.1 Experiment 1. MA
Figure 17 shows the adsorption ability of MA at 5 and 15wt% compared to the non functionalized
base polymer (0%) to poly-L-amino acids. poly-L-arginine was used as an EDGP analog with a PI of 10.76
and poly-L-glutamic acid was introduced as a negative control with a PI of approximately 3. Significant
binding onto the polymeric surfaces did not occur. Although the 15% MA SMP condition shows adsorption
of poly-L-glutamic acid during the 2 hour time point, its binding is variable. One explanation of why poly-
L-glutamic acid may bind to the modified polymer is the presence of counter ions from PBS binding to the
surface of the polymer first, encouraging poly-L-glutamic acid to bind secondary onto the new monolayer.
The large error bars and negative values indicate the levels of binding are beneath the sensitivity threshold
of the assay. 5 minute and 30minute incubations were inaccurate time points as the post and pre incubations
of SMP in protein solution were of the duration of the time point itself or exceeded beyond this point. Since
MA is a small monomer with its functional site near its vinyl group steric hindrance may attenuate binding.
In essence, because of the pure randomness and noise from the assay, a better monomer may contribute to
higher levels of detectable binding coupled with an increase in surface area to provide more available
binding sites for binding.
45


JP
T3 ,
.p
5?
C
o
a
a
15
10
5
0
-5
10
Poly-L Amino Acid adsorbed. 120 min Incubation
Poly-L-Arginine
Poly-L-Glutamic Acid
0 5 15
% Metliacrylic Acid
g
'Sh
T3 ^
D V
X> S
t; o
o &
in CL
S S
< CO
Poly-L-Amino Acid adsorbed. 5 min Incubation
Poly-L-Arginine
_______________________________________________ Poly-L-Glutainic Acid
-10
-15
-20
-25
0 5 15
% Methacrylic Acid
Figure 17 Experiment 1 MA
Time points of poly-L amino Acids adsorbed onto MA SMPs (Surface to volume ratio: 37.68mm2/ml, poly-
L-arginine concentration: 44ug/ml, pH 7 and temperature: 37C).
5.3.2 Experiment 2-3. CEA
Figure 18 shows Poly-L-amino acid adsorption onto CEA SMP surfaces or nylon. Since the base
material of the esophageal string test is made from nylon, it was introduced in the binding experiment for a
comparative analysis against the modified SMP samples. Experimental repeats were increased from 3 to 6
and the surface- area- to- volume ratio was increased approximately 3 fold to decrease the error and to
supply more binding support for detectable binding. As such, the standard deviations and negative values
still exist. Two alternative approaches to have detectable binding are by: 1) picking a more sensitive
detection method or 2) increasing the surface area further. The data shows no appreciable amounts of
binding with CEA incorporation. Although the base formulation of the SMP (0%) mildly binds poly-L-
arginine, the results of this are variable. Nylon also does not show detectable amounts of binding
suggesting the esophageal string test may work in capturing proteins through high specific surface area.
46


Therefore, increasing the surface area further for all samples was employed in the next experiments to
ensure enough support for detectable binding.
Amino Acid adsorbed. 1 HR Incubation
% 2- Carboxyethyl Acrylate
Poly-L-Arginine
Poly-L-Glutamic
Acid
cb '5b 20
10
d £ * o o
o (*) a -io
T3
< 00 -20
o -30
S-H CLh -40
Amino Acid adsorbed. 1 HR Incubation
1 1
0% CEA 5% CEA 15% CEA Nylon
Poly-L-Arginine
Poly-L-Glutamic
Acid
Figure 18 Experiment 2-3. CEA
Time points of poly-L amino acids adsorbed onto CEA SMPs and/or nylon (surface- to- volume ratio:
94.2mm2/ml, poly-L-arginine concentration: 44ug/ml, pH 7 and temperature: 37C).
5.3.3 Experiment 4. Surface Area 132mm2/ml
High specific surface area is important as it relates to the total loading capacity of the polymer for
protein. As such, figure 19 summarizes the trials of surface areas examined (some of which are outlined
below). SMP particles were also created but exhibited no observable appreciation in poly-L-amino acid
adsorption.
47



6mm 10mm
SA: 75.36mm2 SA: 188.4mm2
*3
X=Y=20mm
SA: 2640mm2


> ^
*3
x
X=5mm Y=6.66rnm
SA: ~3240mm2
5x12mm
SA: 154mm2
Figure 19 Surface Areas Implemented to Maximize Loading Capacity
Figure 20 displays poly-L-arginine binding onto SMP (80:20- tBA: PEGDMA) and nylon
samples. The surface area was increased to 2640mm2 per condition from 188.4mm2 per condition to absorb
more protein onto the surface. The surface to volume ratio only increased from 94.2mm2/ml to 132mm2/ml
because more volume was needed to cover the larger samples. Nonetheless, after increasing the surface
area beyond previous experiments, there are still negative readings and large standard deviations. The assay
sensitivity threshold could be an avenue to consider but because the surface to volume ratio was not much
more than the previous experiment, additional efforts were made to minimize the volume the adsorbents
were incubated in and increase the surface area for the next experiments.
2
3*
T3
1)
£
O
isi
S3
t:
a
a
CO
1.5
1
0.5
0
-0.5
Poly-L-Amino Acid adsorbed. 1 HR Incubation
SMP Nylon
Poly-L-Arginine
Poly-L-Glutamic
Acid
Figure 20 Experiment 4. Surface Area 132mm2/ml
Poly-L amino acids adsorbed onto SMP and nylon (surface- to- volume ratio: 132mm2/ml, poly-L-arginine
concentration: 44ug/ml, pH 7 and temperature: 37C).
48


5.3.4 Experiment 5. Surface Area with SMP Particles
Polymer particles were created to increase the loading capacity for the protein far beyond the
surface areas introduced in the previous experiments. High surface areas are usually necessary for
implementing adsorption. Many studies use particulate or beaded adsorbents for large surface area
exposure, for instance, Sepharose 4B (agarose beads) used for adsorption in chromatographic methods
have a surface area of 8m2/ml13. The largest surface to volume ratio achieved thus far is 0.132m2/ml,
reaching nowhere near this level. To attain a quantifiable amount of protein binding onto our polymeric
surface, particulates may need to be created. Even nylon has not adsorbed appreciable amounts of protein,
and since this is the material the esophageal string test is made from, known to capture protein, the small
surface area may be the underlying issue. Therefore creating particles was the new concentration. A dremel
was used to create the adsorbent particles but in the process, various particle sizes were generated. Figure
21 and 22 display the large range of the sizes created (.01-1.2mm SMP and 0.1-13.857mm nylon). Because
nylon sample sizes were significantly larger than the SMPs, they could not be used for comparison during
the binding experiments. These SMP particles were washed several times and centrifuged to remove
extremely small particles that could be difficult to remove after the binding procedure. Centrifugation
should pool the larger particles to the bottom, and the residing supernatant should contain the smaller sized
particles. This supernatant was decanted to remove these small particles. After the particles were correctly
processed and dried, the adsorption experiment was carried out and the results are displayed in figure 23.
49


Figure 21 SMP Particles
80:20 (tBA: PEGDMA) SMP particles imaged through an optical microscope. Large ranges exist; some
particles are below the detection limit of the scope. The sizes ranged from 0.01-1.2mm. Particles smaller
than 0.01mm could not be measured using this method.
50


Figure 22 Nylon Particles
Nylon particles imaged through an optical microscope. Nylon particles are larger than SMP; therefore, the
two cannot be compared in further polymer particle adsorption studies. The sizes ranged from 0.1-
13.857mm.
The exact surface area per condition is unknown due to the large variation of sizes created during
the generation of the SMP samples. As such, the weight of the particles was used to control for a fixed
amount per condition. The most saturated samples with the polymer particles were at 0.06g and a condition
at 0.04g was also tested. Poly-L-amino acids were the study protein at a pH of 7 in PBS buffer. The results
show that even though the surface to volume ratio is maximal in this experiment relative to the others,
appreciable amounts of binding did not occur. The problems associated with creating SMP particles is the
high variability of particulate size, the uncertainty of false negative results, the aggregation of the
hydrophobic particles together in solution, and the high buoyancy of the particles. It can be speculated that
the large error bars resulted from the large range of sizes produced when creating the SMP particles.
Initially, the particles were passed through a 230um mesh sieve to try to eliminate some of this variation
but most of the sample sizes were so small that >80% of the particulate passed through. Thus,
centrifugation was implemented to remove the particles but further optimization to create tighter particle
sizes should be implemented. There is uncertainty that all the small particles were in fact removed by this
method. This can lead to false negative results if the particles have bound protein on its surface and the
51


solution depletion method is performed. In this method, we only want to measure free bulk protein that was
not bound to the polymer surface and total protein. The difference of the two values yield the protein bound
to the polymer surface. If the polymer particle with protein bound is not properly removed from the free
protein being measured, there is a large possibility of a false negative result. The shape of the polymers also
added variability to each condition. Another concern is full exposure of the particles to the protein solution.
The purpose of creating SMP particles was to increase surface area, but because of the hydrophobicity of
the polymers, they clumped together to reach a more thermodynamically favorable state during aqueous
incubation. The SMP particles also have a density similar to water such that they tend to float in PBS.
Together, these findings show that the total surface areas of all the particles are not available for the protein
to adsorb, defeating the overall purpose in creating these particles. One solution to this problem is forcing
the solution to pass through all the particles. This can be done by a rotary device or through vigorous
shaking in a closed vial bringing us to experiment 6 where this was carried out.
Amino Acid adsorbed. 1 HR Incubation
Poly-L-Arginine
Si Poly-L-Glutamic Acid
B 30 i---------------------------------------------------------------:----------------
T3
0.04 g SMP 0.06g SMP
Figure 23 Experiment 5 Surface Area with SMP Particles
Poly-L amino acids adsorbed onto 0.04g and 0.06g of SMP (poly-L-arginine concentration: 44ug/ml, pH 7
and temperature: 37C).
5.3.5 Experiment 6. Surface Area with SMP Particles- Complete Submersion
Figure 24 displays poly-L-amino acid adsorption onto SMP particles with an increase in surface-
to- volume ratio. The particles were incubated in a closed vial and were vigorously shaken throughout the
incubation period to ensure full exposure of the polymeric particles to the protein solution. However,
minimal adsorption took place with a high deviation for poly-L-arginine. Because poly-L-glutamic acid has
a negative value but a low standard deviation, this may be the limit of detection of the assay. It is still not
52


clear if the particles were removed before the BCA assay was performed which could interfere with the
results as a false negative reading. Therefore, we reverted back to creating square samples to increase the
surface area.
^ 30
20
^ g 10
0
B -10
£ -20
Figure 24 Experiment 6 Surface Area with SMP Particles-Complete Submersion
Poly-L amino acids adsorbed onto 0.122g of SMP (poly-L-arginine concentration: 44ug/ml, pH 7 and
temperature: 37C).
5.3.6 Experiment 7. Surface Area 648mm2/ml
Figme 25 displays protein adsorption after exposing 648mm2 SMP surface area per lrnL of
protein solution. Poly-L-glutamic acid, the negative control binds to the polymeric surface better than poly-
L-arginine. Our aim is to specifically bind highly cationic proteins of EoE onto the SMP through anionic
charged groups on the polymer surface. Because the polymer used for this experiment was the base
formulation with mostly hydrophobic bonds, it will bind protein more nonspecifically. In addition, glutamic
acid was attached to sodium as purchased from Sigma. The sodiums positive charge allows this to bind to
the negative groups on the polymer surface negating its repulsion and serving as a negative control. After
performing several trials to increase the protein capacity of the polymer with little improvement, surface
area considerations, although important, may not be the factor causing minimal binding.
Amino Acid adsorbed. 1 HR Incubation
Poly-L-Arginine
Poly-L-Glutamic Acid
0.112 g SMP
53


Poly-L-Amino Acid adsorbed. 1 HR Incubation
'3b
Poly-L-Arginine
Poly-L-Glutainic Acid
SMP
Nylon
Figure 25 Experiment 7 Surface Area 648mm2/ml
Poly-L amino acids adsorbed onto SMP and nylon (Surface area to volume ratio: 648mm2/ml, poly-L-
arginine concentration: 44ug/ml, pH 7 and temperature: 37C).
After more consideration for maximizing surface to volmne ratios, the best approach was
submerging 5xl2xlmm SMP sample in lmL volume, specifically in a 1.5mL eppendorf tube. This
combination allows the entire polymer surface to be submerged in solution and does not consume
expensive processing time with creating numerous coupons. The problem associated with incubating
numerous SMP samples in one condition is the uncertainty of a fully exposed surface. The polymers lie on
top of each other and can hide a large surface area. One solid SMP support eliminates this issue and still
exposes large surface area to the protein solution.
Many studies refer to hydrophobic forces as the dominate interactions for binding proteins more
firmly 22x'12. Dehydration of the polymer and protein surfaces is a prerequisite for protein adsorption.
Water that resides on the surface of both the hydrophobic protein domains and hydrophobic polymer
surfaces are highly ordered (loosely bound) and require less energy to displace than with hydrophilic
surfaces where the water is tightly bound12. In this way, hydrophobic domains increase polymer-protein
interactions and trend toward irreversible binding. The hydrophobic forces were considered for the polymer
surface but the protein needs complimentary hydrophobic domains for the interaction to take place. Poly-L-
arginine was chosen because of its net positive charge that resembles the unique cationic biomarkers of
EoE. However, the synthetic analog does not emulate physiological proteins; it is just composed of one
type of amino acid. Although the proteins charged outer surface allows specific binding onto the
anionically charged polymer surface, it lacks a hydrophobic core to create long lasting, densely packed
contacts. Therefore, the study protein was changed to native EDGPs for the next series of experiments. The
54


diversity of amino acids, particularly hydrophobic domains, and their increased interaction forces can
facilitate irreversibly binding.
5.3.7 Experiment 8. Eosinophil Lysate
Eosinophil cationic protein, eosinophil derived neurotoxin and major basic rotein 1 were tested for
adsorption onto SMPs without functional group incorporation. We decided to optimize protein adsorption
by using the base SMP formulation because of its ability to preference cationic proteins because of its slight
negative charge from the crosslinker, PEGDMA. The base SMP formulation also facilitates hydrophobic
interactions from tB A methyl groups (CH3). For interpretation of protein binding to our polymer surface,
amino acid analysis was performed for each protein. Protein properties listed below in table 6 can affect
surface interactions during adsorption. Since there are many protein adsorption factors it is hard to predict
which ones are most influential in protein adsorption, thus 3 of the 4 proteins were quantified to determine
binding. For instance, Larger molecules tend to bind better because they have more sites of contact for the
polymer surface8. ECP, EDN and MBP1 have small molecular weights, reducing the strength of the
interaction to the polymeric surface. ECP, EPX and MBP1 all share high cationic charges that creates a
looser structural construct from the repulsion between adjacent charged residues. Thus, the internal
mobility needed upon adsorption and relaxation is greater for these proteins, encouraging protein
adsorption. EPX and MBP1 have the highest quantity of disulfide bonds that ultimately hold the protein in
its tertiary structure and aid in its stability, a discouraging protein adsorption factor.
55


Table 6 Protein Characterization
% Charged
Protein MW (kDa) PI % Hydrophobicity Sites # disulfide bonds
ECP 18.39 11.4 43.16 20.63 9
EDN 18.35 8.9 41.62 15.53 9
EPX 81.04 10.8 43.24 24.2 17
MBP1 25.21 10.9 40.54 26.12 12
After performing adsorption testing, select EDGP binding onto the surface of the methanol treated
SMP (80:20 tBA: PEGDMA) was quantified. ECP binds both nylon and SMP polymers the best over EDN
and MBP1. Two reasons why ECP may bind preferentially is its high isoelectric point and low disulfide
bond count relative to the other proteins. These factors place it in a looser construct with increased internal
mobility. To fully study this binding phenomenon, one variable should be eliminated that could cause
increased binding through pore size and not through chemical constituents on the surface. The polymer was
methanol treated for 48hours, which causes substantial swelling of the polymer. This affect could stretch
pore sizes beyond recovery through the dehydration step. To eliminate pore size from interfering with
binding, SMP samples were heat-treated in lieu of methanol treatments to rid of unpolymerized monomers.
Figure 26 Experiment 8 Eosinophil Lysate. ECP Adsorption
Eosinophil cationic protein (ECP) adsorption onto SMP methanol treated samples (Surface area to volume
ratio: 154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 1).
56


Table 7 % Protein Bound Relative to Total ECP
Sample Code % Bound
SMP + Lysate 63.51 20.35
Nylon + Lysate 50.23 11.85
5 o
l a
m ?
t/3
8 "Sb
Ph fl
70
60
50
40
30
20
10
0
EDN Adsorption
SMP + Lysate
Nylon + Lysate
Figure 27 Experiment 8 Eosinophil Lysate. EDN Adsorption
Eosinophil derived neurotoxin (EDN) adsorption onto SMP methanol treated samples (Surface area to
volume ratio: 154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 1).
Table 8 % Protein Bound Relative to Total EDN
Sample Code % Bound
SMP + Lysate 2.89 0.04
Nylon + Lysate 2.89 0.02
*2 t? 375
£ o o Oh 360
CQ "o & CO 345 330
8 £ 315
Ph £ 300
285
MBP1 Adsorption
SMP + Lysate Nylon + Lysate
Figure 28 Experiment 8 Eosinophil Lysate. MBP1 Adsorption
Major Basic Protein 1 (MBP1) adsorption onto SMP methanol treated samples (Surface area to volume
ratio: 154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 1).
Table 9 % Protein Bound Relative to Total MBP1
Sample Code % Bound
SMP + Lysate 1.91 0.07
Nylon + Lysate 1.96 0.12
57


5.3.8 Experiment 9-10. Eosinophil Lysate
After changing the post processing method of the SMP from methanol treatments to purely heat
treatments, appreciable amounts of binding did not occur. This suggests that if methanol did alter the
polymers pore size, ECP may have been absorbed through the pores or adsorbed at the surface of
increased surface area. The adsorption mechanism is unknown but the results below imply it was not a
function of purely non-covalent binding of the equivalent surface area. The % protein bound was
normalized to each donors specific total protein content such that comparisons between donors could be
made. That said, after comparing nylon between experiments, it has variable binding which could be
because of the adsorbent itself or reproducibility error of the assay. Nylon exhibited approximately 30%
more ECP binding in experiment 9 than experiment 8 but dropped to nearly 58% comparing experiment 9
to 10. Nylon was purchased with surface scratches from the manufacturer, altering uniformity from sample
to sample. This alone could influence adsorption as the scratches could facilitate sites for physical
entrapment of the protein and increased surface area. EDN and MBP1 minimally bind to the polymer
surface and do not need to be repeated again. To test the reproducibility of the ECP assay and if methanol
treatments sincerely encourage binding, the experiment was performed once more with both heat-treated,
methanol treated and nylon samples.
58


s
3 a
o a
03 5
c 1/3
8 ~bi)
Ph S=
1500
1250
1000
750
500
250
0
ECP Adsorption
SMP + Lysate
Nylon + Lysate
Figure 29 Experiment 9 Eosinophil Lysate. ECP Adsorption
Eosinophil cationic protein (ECP) adsorption onto SMP heat-treated samples (Surface area to volume ratio:
154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 1).
Table 10 % Protein Bound Relative to Total ECP
Sample Code % Bound
SMP + Lysate 8.61 1.92
Nylon + Lysate 86.21 8.49
Figure 30 Experiment 9 Eosinophil Lysate. EDN Adsorption
Eosinophil derived neurotoxin (EDN) adsorption onto SMP heat-treated samples (surface area-to-volume
ratio: 154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 1).
Table 11 % Protein Bound Relative to Total EDN
Sample Code % Bound
SMP + Lysate 0.43 0.01
Nylon + Lysate 0.43 0.00
59


Figure 31 Experiment 9 Eosinophil Lysate. MBP1 Adsorption
Major Basic Protein 1 (MBP1) adsorption onto SMP heat treated samples (surface area-to-volume ratio:
154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 1)
Table 12 % Protein Bomid Relative to Total MBP1
Sample Code % Bound
SMP + Lysate 0.73 0.01
Nylon + Lysate 0.77 0.03
-S
S o
13 a
o a
m a
8 ~5fc
Ph c
600
400
200
0
ECP Adsorption
SMP + Lysate
Nylon + Lysate
Figure 32 Experiment 10 Eosinophil Lysate. MBP1 Adsorption
Eosinophil cationic protein (ECP) adsorption onto SMP heat-treated samples (surface area-to-volume ratio:
154mm2/ml, EO lysate cone.: 500k/ml. pH 7 and temperature: 37C, Donor 2).
Table 13 % Protein Bound Relative to Total ECP
Sample Code % Bound
SMP + Lysate 3.71 1.71
Nylon + Lysate 27.90 7.30
60


Figure 33 Experiment 10 Eosinophil Lysate. EDN Adsorption
Eosinophil derived neurotoxin (EDN) adsorption onto SMP heat- treated samples (surface area- to- volume
ratio: 154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 2).
Table 14 % Protein Bomid Relative to Total EDN
Sample Code % Bound
SMP + Lysate 0.42 0.02
Nylon + Lysate 0.43 0.03
5.3.9 Experiment 11. Eosinophil Lysate
Minimal ECP adsorption onto SMP was found for both heat-treated and methanol treated samples.
ECP bound the nylon samples at a higher degree but again, this could be because of slight imperfections
that exist on the nylon surface. The results for the methanol treated SMPs were not reproducible. This may
be explained by delicate changes between the methanol treatments for the two experiments, as the smallest
change in polymer processing could alter the surface severely.
Going back to the literature, adsorption is maximal if the pH of the solution is equal to or near die
isoelectric point of the protein5'7,812. This can be explained in a couple of different ways. The protein and
polymeric surface should not be thought of in isolation, rather protein -protein interactions occur frequently
in addition to polymer-protein interactions. In our study, with a pH of 7, the eosinophil derived granule
proteins are positively charged and can attribute lateral repulsion upon binding to adjacent binding sites; the
proteins may need to be uncharged to decrease protein-protein interactions and promote polymer-protein
interactions. In addition, charged proteins take up more occupancy onto the polymer surface, reducing the
amount of protein that can bind per meter squared of surface area.
Studies have also shown that adsorbents introduced to multi component protein solutions tend to
adsorb one protein or molecular species preferentially I3. This is dependent on the concentration and
61


affinity of the protein for the polymeric surface. If the concentration of one protein is higher than the rest, it
will have a higher probability of interacting with the surface and adsorbing over rare proteins in the mix.
Increased concentration also promotes stacking of proteins next to one another by decreasing the residency
time of the protein onto the polymer surface, discouraging over-relaxation states that could otherwise take
up ample space6. As such, the concentration of the study protein was increased to lmg/ml to decrease
residency time on the polymer surface. For the affinity of a protein, if one protein has the correct surface
amino acids that compliment the interaction forces produced by the polymer surface, that protein will bind
preferentially. In the last set of experiments with the EO lysate, a pool of EO proteins were competing for
the surface but the media itself was infused with 8% FBS that could have bound preferentially as well.
Thus, in the next experiment, only purified protein was introduced so we could study the mechanism
involved in protein adsorption and control for competition that may be taking place and altering adsorption
of the EDGPs.
The study protein was also changed to BSA. The main justification is that BSA is frequently used
as the study protein for adsorptions studies. BSA is well characterized, has the high internal mobility, is a
moderately sized protein that could bind irreversibly and is easily available in pure delipidated form. Below
are its specific characteristics that aid in its pro-adsorption characteristics. In correlation, the solution was
switched from PBS to acetate buffer with a pH of 4.5 to bring the net charge of BSA to zero.
62


Table 15 BSA Characterization
Protein MW (kDa) PI % Hydrophobicity % Charged Sites # disulfide bonds
BSA 69.29 4.7 42.83 33.27 35
a
a
CO
S "bfc
Ph C
250
200
150
100
50
0
ECP Adsorption
SMP+ Lysate SMP Metlianol + Lysate Nylon + Lysate
Figure 34 Experiment 11 Eosinophil Lysate. ECP Adsorption
Eosinophil cationic protein (ECP) adsorption onto SMP heat-treated samples (surface area- to- volume
ratio: 154mm2/ml, EO lysate cone.: 500k/ml, pH 7 and temperature: 37C, Donor 3).
Table 16 % Protein Bound Relative to Total ECP
Sample Code % Bound
SMP + Lysate 0.56 0.26
SMP Methanol + Lysate 2.04 1.41
Nylon + Lysate 27.00 2.29
5.3.10 Experiment 12. CEA
After incorporating COO" functional groups to the polymeric surface, switching the study protein
to BSA, increasing the concentration to lmg/ml and adjusting the pH of the solution buffer to the pi of the
study protein, no protein was found bormd to the CEA SMPs shown in figure 35. One key consideration of
why the no binding occurred onto the CEA polymers is the high ionic strength of the solution. The ionic
strength of the acetate buffer used was at 0.1M. This can have an enormous degree of control over protein
adsorption as the counter ions in solution weaken electrostatic interactions both on the protein and polymer
surfaces by competing with the same binding sites. Since these molecules are small, they are transported to
these sites on the protein and polymer faster and can consume critical sites ultimately influencing
adsorption and adsorption kenetics7. Therefore, for future experiments, the ionic strength was adjusted to
0.01M to correct for any competing counterions.
63


Since the mechanism of adsorption is complex, and there are a multihide of factors that could
ultimately influence this mechanism, a positive control was needed to test if the correct experimental
parameters were set in place for pro-adsorption. Otherwise, the iterations of system adjustments could go
beyond the period of this project.
Figure 35 Experiment 12 CEA
Bovine serum albumin (BSA) adsorption onto SMP samples (surface area- to-volume ratio: 154mm2/ml,
cone.: lmg/ml, pH 4.5 and temperature: 37C).
5.4 Positive Control Optimization of Polystyrene Microspheres
Polystyrene microspheres are an established adsorbent utilized in nmnerous studies to investigate
protein adsorption7. As such, 0.5 lmn microspheres were purchased to validate experimental parameters set
forth previously and serve as a positive control. These spheres have a high specific surface area and are
hydrophobic with a smooth surface to promote protein adsorption.
5.4.1 Surface Area and Surfactant Optimization
The surfactants in which the microspheres were suspended in, were diluted several folds to find
the best surface area- to- surfactant ratio for maximal adsorption. Results show 92.4% adsorption of BSA to
polystyrene microspheres with a 1:100 dilution, the best choice for further adsorption studies with these
spheres. Though the 1:1000 dilutions yielded more protein bound per surface area, more error was
associated with this condition. One reason for this may be because of the minimal amounts of SDS in
solution. SDS prevents the microspheres from clumping. The hydrophobic end of the surfactant resides on
the polymers surface while the flanking end is negatively charged creating a negatively charged polymeric
surface. The negative charges of the polymer particles cause repulsion from each other, thus controlling the
64


dispersion of the polymers in solution. The high standard deviation could be a result of non-uniform
clumping from sample to sample and thus more error in pipetting the spheres to the protein solution for the
actual binding experiment. Diluting the polymers ultimately leave more room on the polymer surface for
protein adsorption but can also cause polymer-polymer interactions as these spheres are free floating.
Absolute BSA adsorbed of the 1:10 and 1:1000 diluted microspheres amounted to only 0% and
3.06%. The final SDS concentration of the 1:10 dilution was 0.048%, high enough to interfere with
binding. Because SDS is an anionic surfactant, it could occupy the binding sites of the polymer or protein,
this illustrates that too much SDS is not good either for our purposes. The 1:1000 dilutions reduce the
surface area for protein to adsorb onto which may be the reason for such a low adsorption yield. Here, only
5.57X108 microspheres were added per condition compared to the 1:100 dilutions where 5.5760X109
microspheres were added. The 1:100 diluted microspheres seem to meet the perfect balance between
surface area and surfactant levels.
a
a
t/2
a
40
30
20
10
0
-10
Surfactant Influence on BSA Adsorption
Surfactant 1:10 Surfactant 1:100 Surfactant 1:1000
Figure 36 Surface Area and Surfactant Optimization
Optimal surface area-to-surfactant ratios for BSA adsorption onto polystyrene microspheres (surface area-
to-volume ratio 1:10: 185263mm2/ml, surface area- to-volume ratio l:100:1852mm2/ml, surface area- to-
volume ratio l:1000:18.52mm2/ml, BSA cone.: 120ug/ml, pH 4.5 and temperature: 37C).
65


Table 17 % Protein Bound Relative to Total BSA
Sample Code % Bound
% Bound- Surfactant 1:10 - 10.23 0.25
% Bound- Surfactant 1:100 92.40 1.47
% Bound- Surfactant 1:1000 3.06 1.76
5.4.2 Total Exclusion of Surfactants
To clarity if the same optimal surface area to volume ratio (1852mm2/ml) without any interference
of surfactants would bind even more, microspheres were washed once in acetate buffer to rid SDS from
solution. Interestingly, adsorption decreased by approximately 85%. This suggests a role surfactants may
have in adsorption if the correct balance exists. SDS, the anionic surfactant the microspheres were shipped
in, may have a role in attracting the BSA near the interfacial region of the polymer since the surfactants are
utilized to charge the microspheres and prevent clumping. Adversely, free SDS could weaken
intramolecular forces of protein providing structural mobility. In any event, the microspheres were not
washed but only diluted 1:100 in future experiments.
BSA Adsorbed to Washed Polystyrene Microspheres
Washed polystrene microspheres
Figure 37 Total Exclusion of Surfactants
Washed polystyrenes affect on BSA adsorption onto polystyrene microspheres (surface area- to-volume
ratio l:100:1852mm2/ml, BSA cone.: 120ug/ml, pH 4.5 and temperature: 37C).
Table 18 % Protein Bound Relative to Total BSA
Sample Code % Bound
Washed polystyrene beads 6.73 0.98
66


5.4.3 BSA Concentration Optimization
Next, the optimal concentration of BSA was tested. One source discussed the optimal protein
concentration range for adsorption studies to be 200- 40ug/mL12. Thus, the concentrations were chosen
within this range. As mentioned before, concentration can determine the residency time a protein spends on
the polymer surface and as such, the degree to relaxation of the protein. A more packed orientation could
aid in more binding. Results show higher binding per meter squared for high protein concentrations with
~52ug of protein bound where the low protein concentration had approximately 25ug bound. From these
results, the high protein concentration was used in the next experiments.
It should be noted that the adsorption dropped in the 120ug/ml BSA samples by nearly 35%
relative to the surface area to surfactant experiment with the same experimental parameters. The only
minute alteration was the preparation of the acetate buffer wherein it was prepared from DI water passed
through a 0.2um filter before, the acetate buffer was made from nano-pure water in this experiment. The
next parameter of optimization tests these influences.
s
s a
o a
m a
b
8 Ta
Ph S3
Protein Concentration Influence on Protein Adsorption
High BSA Concentration (120ug/mL)
Low BSA Concentration (40ug/mL)
Figure 38 BSA Concentration Optimization
Optimal bovine serum albumin (BSA) concentration for maximal adsorption onto polystyrene microspheres
(surface area- to-volume ratio 1:100:1852mm2/ml, BSA cone.: 120ug/ml, pH 4.5 and temperature: 37C).
Table 19 % Protein Bound Relative to Total BSA
Sample Code % Bound
High Concentration 56.52 5.21
Low Concentration 63.47 1.98
67


5.4.4 Buffer Solution Optimization
The buffer solution was tested to see if its water base composite effected adsorption. Acetate
buffer was made from nano-pure water or fdtered DI water. Since the microspheres are highly reactive and
are prone to adsorb nonspecifically to ions, organic species and microbes that could be contaminants of the
water, the degree to this effect was examined. The results show that the microspheres suspended in acetate
buffer made from fdtered water does adsorb BSA better dian die acetate buffer made from nano-pure water.
Therefore, acetate made from fdtered water was implemented in the future studies. However, the degree to
binding depreciated much further. The variability amongst the positive control illustrates how sensitive
adsorption is and the degree of detail that needs to be considered. Numerous factors could have prevented
maximal protein binding on either the solution, protein or polymer end. For this reason, the study was
continued by examining one potential cause, pipetting error.
Figure 39 Buffer Solution Optimization
Optimal purity for Bovine Serum Albumin (BSA) adsorption onto polystyrene microspheres (surface area-
to- volume ratio 1:100:1852mm2/ml, BSA cone.: 120ug/ml, pH 4.5 and temperature: 37C).
Table 20 % Protein Bound Relative to Total BSA
Sample Code_________________% Bound________
Nano-Acetate Buffer 8.67 2.31
Filtered Acetate Buffer 19.71 3.18
68


5.4.5 Reproducibility Testing
The microspheres have a high initial concentration of 1.394X1012 microspheres per ml. The total
amomit of microspheres after a 1:100 dilution, introduced to each experimental condition, amounts to only
4ul of the undiluted stock. Pipetting could contribute to enormous error if the spheres were clumped
together or not vortexed properly to disperse the spheres and get a uniform amomit each time. The degree
to pipetting error was measured to see if this was problematic once methodical vortexing was controlled
for. The microspheres had an 8% difference of BSA adsorption when larger volumes were aspirated over
smaller volumes. Thus, larger sample dilutions were prepared for future experiments. The exact reason why
BSA adsorption decreased substantially previously is unknown, but meticulous precautions was taken to
treat all the base parameters the same.
ts o 7.5
o CQ Qh & 7
3 GO C-4 6.5
*53 +-> 8 a W) 6
Ph 3 5.5
BSA Adsorption onto Polystyrene Microspheres: Pipette Error
Considerations
Small Volumes Larger Volumes
Figure 40 Reproducibility Testing
Reproducibility of Bovine Serum Albumin (BSA) adsorption onto polystyrene microspheres (surface area-
to-volume ratio l:100:1852mm2/ml, BSA cone.: 120ug/ml, pH 4.5 and temperature: 37C).
Table 21 % Protein Bound Relative to Total BSA
Sample Code % Bound
Small Volumes 80.47 0.51
Large Volumes 88.62 0.75
5.4.6 BSA Alexa Fluor 488
Polystyrene microspheres were optimized to bind abundant amounts of purified BSA in the
previous experiments. The spheres have an extremely high specific surface area and are hydrophobic to
facilitate irreversible binding. Since the surface area may still be an underlining issue for the SMP system, a
more sensitive detection method was tested against the microspheres. BSA conjugated to alexa fluor 488
69


was purchased as the study protein with the ability to be quantified through its fluorescent tag up to
nanogram levels. The results show that the tagged surface interfered with adsorption significantly, as no
protein was adsorbed onto the microspheres. The modified surface of the protein may hide specific
interacting amino acid residues that were before interacting with the polymeric surface. The method was
therefore not used in future SMP experiments.
BSA Alexa Flour 488 Adsorption
0.5 t-------------------------------------------------
-2 J--------------------------------------------------------
Polystyrene Micropheres
Figure 41 BSA Alexa Fluor 488
The affect of BSA 488 adsorption onto polystyrene microspheres (surface area- to- volume ratio
l:100:1852mm2/ml, BSA cone.: 120ug/ml, pH 4.5 and temperature: 37C).
Table 22 % Protein Bound Relative to Total BSA-488
Sample Code_________________% Bound________
Polystyrene Microspheres -10,46 15,01
5.5 Positive Control Test Polystyrene Microspheres against Past Experiments
5.5.1 Experiments 1-7 Poly-L-Arginine
After the proper adsorption parameters were established to get ~90% binding, the positive control
was used against previous experimental enviromnents to justify if they were in fact, pro or anti-adsorption
systems. The polystyrene beads were incubated in 44ug/ml of poly-L-arginine in PBS buffer for lhour at
37C. Poly-L-arginine was proposed to discourage binding because of the lack of complimentary
hydrophobic domains to facilitate irreversible binding. However, the positive control unveiled prevalent
amounts of binding to its surface. Because the polystyrene spheres are a different polymeric system,
adsorption mechanisms may not be completely translational. This data shows that our polymer could have
been the anti-adsorption factor in the series of experiments with the amino acids.
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Poly-L-Arginine Bound
Figure 42 Experiments 1-7. Poly-L-Arginine
Poly-L-arginine adsorption onto polystyrene microspheres (surface area-to-volume ratio
l:100:1852nun2/ml, poly-L-arginine cone.: 44ug/ml, pH 7 and Temperature: 37C).
Table 23 % Protein Bomid Relative to Total Poly-L-Arginine
Sample Code % Bound
0.5 HR 91.79 3.84
1 HR 79.77 4.17
2 HR 96.07 1.82
3 HR 93.63 2.52
5.5.2 Experiments 8-11 Eosinophil Lysate
The exact system from the previous EO lysate experiments were tested with the polystyrene
microspheres and the BCA assay was performed to quantify a total protein change. The BCA absorbance
values in figure 43, at 562nm show little differences between each conditions, remembering that the 0
and 0+B conditions do not contain EO lysate but are purely media and media + adsorbent conditions.
This is an indication that FBS saturates the solution and inhibits EDGP adsorption, by masking the binding
sites on the polymer surface before EDGPs can arrive at the polymer surface. An additional consideration is
that RPMI, the media, is flushed with amino acids, vitamins, glucose, inorganic salts and substances that
could consume binding sites as well, or create a large background in colorimetric assays such that the
protein signal is hidden. In this experiment, the Bio-Rad protein assay was purposely chosen to have more
leniencies with the buffer and to pick up more of a protein signal. As a result, it unveiled the media
complications.
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Sample No Dilution Absorbance (562nm) 1:10 Absorbance (562nm) 1:50 Absorbance (562nm) 1:100 Absorbance (562nm)
500k 2.115 1.854 1.04 0.796
2.12 1.776 0.984 0.703
2.152 1.661 0.989 0.752
500k+B 2.24 1.77 1.074 0.756
2.27 1.752 0.99 0.701
2.27 1.637 0.951 0.722
0 2.007 1.77 0.992 0.849
2.095 1.801 0.982 0.688
2.255 1.689 0.916 0.707
0+B 2.235 1.684 0.923 0.622
2.189 1.692 1.042 0.648
2.151 1.647 1.048 0.735
Figure 43 Experiments 8-11. Eosinophil Lysate
Bio-Rad protein assay results of EO Lysate adsorption onto polystyrene microspheres (surface area-to-
volume ratio l:100:1852mm2/ml, EO lysate cone.: 500k/ml, pH 7 and Temperature: 37C).
5.5.3 Experiment 12 BSA and Ionic strength
Finally, polystyrene microspheres were incubated in low and high ionic strength solutions with
BSA. The results illustrate how important ionic strength is to adsorption. In low ionic strength
environments, 90% of BSA was bound to the microspheres. Adversely, a 10-fold increase in ionic strength
leads to 0% adsorption. In the next experiments to follow, ionic strength will be highly considered and
adjusted to 0.01M.
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t? O O
o Oh
CQ 3
GO
cb
o "w)
S-H
Ph 3
Ionic Strength influences on BSA Adsorption onto Microspheres
5 -
0 -
-5 -
Low Ionic Strength
High Ionic Strength
Figure 44 Experiments 12. BSA and Ionic Strength
Ionic strength influence on BSA adsorption onto polystyrene microspheres (surface area-to-volume ratio
l:100:1852mm2/ml, BSA cone.: lmg/ml or 120ug/ml, pH 4.5 and Temperature: 37C).
Table 24 % Protein Bomid Relative to Total BSA
Sample Code % Bound
Low Ionic Strength 90.97 0.57
High Ionic Strength -3.74 0.14
5.6 SEM Adsorption
Since protein adsorption is a complex mechanism, the positive control provided guidance toward a
working system and insight into why the previous experiments did not work. As such, the findings were
implemented and adjusted on a new system to encourage protein binding. 2-sulfoethyl methacrylate (SEM)
was copolymerized with tB A, PEGDMA and DMPA at different weight percents to leave the SMP samples
with negative functional groups on their surfaces. Binding experiments were carried out with BSA at its
isoelectric charge.
Results show that SEM polymers bind more protein per surface area than the other adsorbent
samples. Polystyrene microspheres were used as a positive control but because of its highly concentrated
state, the surface- to-volume ratio for the spheres was 1852mm2/ml contrary to the other samples that were
262mm2/ml. Thus, polystyrene sheets were purchased and sized as an additional control to eliminate
surface area bias to binding and other variables introduced from free floating microspheres rather than a
solid stationary adsorbent support. Nonetheless, binding per surface area was normalized as seen below.
From the polymer characterization data, the 2.5% SEM samples between batches possess differences that
could ultimately influence binding. The surface of 2.5% from batch 1 has a larger dispersive (non polar)
component than that of batch 2 shown in table 3. Though contact angle in air can be misrepresentative of
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the same surface in aqueous solutions, the trend change in hydrophobicity exists with increasing wt % of
SEM. Thus, the hydrophobicity could potentially translate to the number density of SEM methyl groups at
the surface in lieu of their OH groups. If this is the case, the increase in polarity between adjacent binding
sites of the polymer under aqueous solutions could lead to lateral repulsion upon protein binding and thus
less binding. The differences in the rubbery modulus between batches in 2.5% SEM could also affect
protein binding as cells bind to stiffer materials over softer materials in cell culture. The higher polar
groups could alter the number of cross links formed during the polymerization processes by repulsion.
Phase changes could exist within each polymer coupon. Nonetheless, figure 45 shows that the SEM SMPs
dominate BSA adsorption over the other adsorbents, including polystyrene, which is known to adsorb
protein readily and is in fact used to develop binding assays used for biomedical applications. Nylon is the
base component of the Enterotest, which is used in preliminary clinical tests to capture biomarkers of EoE.
The results below suggest that our system may bind protein better than nylon, and with development, has
the propensity to bind substantial amounts of EDGPs. Small differences of negatively charged functional
groups on the SMP surface can greatly affect protein adsorption. From 0% SEM to 2.5% SEM, there is
>50% change in BSA adsorption. Figure 45 illustrates how hydrophobicity (polystyrene, gold SMP) or
polarity alone (nylon) does not yield maximal BSA adsorption. The spacing of the functional groups
relative to each other and the surface potential (electrostatic charge) along with hydrophobicity influence
binding.
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45
a 40
a CO 35
"a 30
25
T3 D 20
£ O uy 15
PQ 0
a a
& a
f s
_£* s-l
o .a
BSA Adsorption. Batch 1
BSA Adsorption. Batch 2
^ 45
o
Figure 45 SEM Adsorption of BSA
Adsorbents from two different batches were tested for adsorption of BSA. Comparisons were made against
polystyrene microspheres with *P< 0.01.
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Table 25 % Protein Bound Relative to Total BSA
Sample Code. Batch 1 % Protein Bound Sample Code. Batch 2 % Protein Bound
Microspheres 39.89 Microspheres 83.31
Polystyrene 11.82 Polystyrene 11.18
Nylon 2.25 Nylon 2.72
Gold 6.10 0% SEM 7.26
0% SEM 9.44 0.5% SEM 18.77
0.5% SEM 29.32 1% SEM 71.47
1% SEM 65.62 2.5% SEM 78.73
2.5% SEM 61.54
After the binding experiment, the SEM SMP samples were collected, washed and visualized for
further evidence of protein bound to their surfaces. Figure 46 shows BSA bound to the polymer surface
after its incubation step compared to a polymer incubated in media alone. Figure 47 shows a closer look of
the polymer surfaces under a 10X magnification captured on an optical bench microscope. Visually and
through the values obtained by the colorimetric assay, the saturation point of the polymers may be near the
1% SEM SMP sample as there is not much change between 1% and 2.5%. In addition, it should be noted
that little SEM monomer was incorporated into the SMP system but drastic changes occurred between
samples. The 0% SEM bound <10% of the total protein in solution where the mere 1% addition of the SEM
monomer yielded >65% adsorption of BSA. In the previous experiments, CEA was incorporated at 5%,
10% and 15%, which could have over populated the polymer surface. The distances of the functional
groups from each other are an important consideration in adsorption studies, as proteins interact not only
with the polymer surface, but also with each other. If two proteins are binding to adjacent binding sites,
repulsion can occur if they are too close to one another. This event is reduced by neutralizing the protein
through the pH of the media but this only adjusts the net charge, such that repulsion is minimized but can
still occur.
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Figure 46 SEM Polymer after 3 Hour Incubation
SEM polymer incubated in media alone (Left) compared to SEM polymer incubated in BSA (Right)
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oi SEM No Protein 0.5% SEM No Protein ilSEM No Protein 2.5I SEM No Protein
:
o:HSEM Protein c.r^SEM Protein :coSEiii P'otc-in :.y -SEM Protein
I IVII II
Figure 47 Images of the SEM Polymer after 3 Hour Incubation
BSA adsorption onto the SEM SMP imaged with an optical microscope at 10X magnification.
78


6. Conclusion
The results of the present study demonstrate that altering the surface chemistry of an acrylate-
based SMP can encourage protein binding onto its surface with additional system adjustments. SEM SMPs
had a higher yield of protein adsorption than known adsorbents used currently in biomedical applications
such as polystyrene. Additionally our 1% and 2.5% SEM polymers bound BSA at least 60% better than
nylon, the base material of the esophageal string test with a known capacity to capture proteins and cells in-
vivo. This suggests the SEM polymers have potential to improve current diagnostics of EoE by adsorbing
substantial quantities of protein in a minimally invasive way with development. Further tests and
optimization of the SEM SMP system for specific adsorption to biomarkers of EoE would increase the
sensitivity of the eventual device.
During the developmental process, difficulties in improving the current diagnostics by depending
purely on electrostatic and hydrophobic interactions for adsorption of EoE biomarkers onto our SMP
became evident. The complexity of this aim originates from the vast collection of dominating factors for
adsorption that compete with the surface chemistry of our biomaterial and the relatively weak non-covalent
interactions we are basing specific adsorption on. The combination of surface chemistry and high surface
area could strengthen these weak interactions and facilitate more adsorption.
Chemical modification of the SMP through copolymerization was performed rather than other
surface alteration techniques such as texturing, ligand attachment or thin film deposition. Because of the
eventual desire to adsorb proteins specifically, and the complexity that arises from processing, validation of
the modified surface and additional multi-procedural stages associated with these other techniques, which
would ultimately reside outside the time frame for this study, chemical modification was pursued and
optimized.
Since EoE is a chronic inflammatory disease of the esophagus with a high rapport for
misdiagnosis through the invasive procedure of endoscopy, the progression toward a better diagnostic for
this disease is well justified. While the esophageal string test (EST) is a vast improvement to a minimally
invasive diagnostic for this disease, the surface of the SEM polymer already binds substantially more
protein than nylon, the base material of the EST. The reason for EST capturing protein in-vivo may be due
to its large surface area. Together, with the surface chemistry of our functionalized SMP and the addition of
79


large surface area, our SMP system could be an even greater improvement to the diagnostics in place.
Through the developmental stages of adsorption testing, electrostatic interactions were highly influenced by
the media. Since these are the interactions we are depending on for specific interactions, optimization and
more development of the polymer surface under physiological mimicking systems should be implemented.
Since the SEM SMP was tested under a pH that matched the isoelectric point of BSA, the study protein, it
would be beneficial to test if the SEM polymers adsorb cationic proteins of EoE at a pH of 7. This would
unveil if the active functional groups on the surface of the polymer encourage specific binding with the
electrostatic interactions in play on the protein surface. It was also found that competing proteins, ions and
molecular species could bind preferentially to our polymer system and consume binding sites originally
meant for the target protein. Additional optimization and tests could be performed to correct for this event.
It may be that we do not need specific binding of the EDGPs as long as they are captured at a high degree
along with other esophageal proteins. If the surface area of our polymer system were increased
substantially or specific texture or nano pockets were incorporated for capture of the EDGPs in
combination of the tailored SEM SMP, the polymer could be corrected for competing proteins and
molecules. The SEM polymer would consequently be tested under pH 7 with a mixture of proteins
including EDGPs to find the optimal loading capacity of our polymer system.
The SEM SMP could also be valuable for in-vitro medical research. We have shown SEM SMP
binds more effectively than polystyrene, a common polymer used in biomedical applications for attaching
cells to the bottom of cell culture plates, affinity chromatography or immunoprecipitation to purify and
study biological samples and microspheres used to optimize multiplex assays. Our SEM SMPs can pave the
way for similar applications with development.
In summary, the results of the present study illustrate how the right balance of electrostatic and
hydrophobic functional groups on the SMP could influence protein adsorption for the eventual
development of a diagnostic for EoE. Through the experimental process, the dependencies of protein
adsorption were discovered. The mechanism is based on weak, non-covalent interactions. As such,
chemical modification of SMPs coupled with high surface area may provide better specificity strength and
greater binding of the target proteins. In future work, the SMP can be altered for the capture of EDGPs
through physical entrapment between micro-crevasses, which match the size of the target protein as
80


described briefly above. Another route could be incorporating active biological species into the polymer
such as antibodies or amino acid peptide sequences to facilitate a specific response in-vivo.
81


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Appendix A: Detection
A.1 Overview of Detection Methods
Many protein quantification methods were considered in the initial stages of the experimental
design, illustrated in tables 26-28. Based on these findings, ELISAs and the micro BCA assay were mainly
used because of their high sensitivity and reproducibility. ELISAs were performed when quantifying one
protein species from a pool of proteins in solution and the micro BCA assay was used when quantifying
purified protein *.
Although there are various other methods for protein detection onto polymer surfaces such as
Fourier transform-attenuated total reflection infrared spectroscopy (ATR- FTIR), ellipsometry, total
internal reflection fluorescence spectroscopy (TIRF) or using radioisotope-labeled proteins, they were not
implemented in the experimental design because of their availability and the limited time frame of this
project913. Instead, UV absorption, fluorescence, colorimetric assays and antibody-based assays were
utilized as our choices to measure protein concentrations. These choices are suitable for both elution or
solution depletion methods but the critical determinants for these assays are their level of sensitivity
because of our low polymeric surface area. At these low protein levels, the assay is required to be sensitive
enough to discern low protein concentrations and have reproducible results at these small scales.
*The only exception was in the positive control experiment: Experiments 8-11 Eosinophil Lysate, where
a Bio-Rad protein assay was used, analogous to the Bradford assay. Because of media interference with the
micro BCA assay and the high costs associated with the multiple ELISAs, the Bio-Rad protein assay was
substituted.
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Table 26: Antibody-Based Assays.
Antibody based assays Sensitivity Comments
ELISA22 Concentrations <1.0 pg/rnl $$$ Multi-step procedure (time expensive)
Western Blot- Mini Gels23 Concentrations < 100 pg $$$ High variability from gel to gel Multi-step procedure (time expensive)
Antibody technology grants high specificity and sensitivity but can be expensive.
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Full Text

PAGE 1

SURFACE MODIFICATION OF AN ACRYLATE BASED S HAPE M EMORY P OLYMER TO PROMOTE ADSORPTION TO BIOMARKERS OF EOSINOPHILIC ESOPHAGITIS by ROOPALI R. SHAH B.A., University of Colorado Boulder 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Masters of Science Bioengineering 2013

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ii This the sis for the Masters of Science degree by Roopali R. Shah has been approved for the Bioengineering P rogram by Robin Shandas Chair Glenn Furuta Advisor Dae Won Park Advisor June 19, 2013

PAGE 3

iii Shah, Roopali, R (M.S. Bioengineering ) Surf ace Modification of an Acrylate Based S hape M emory P olymer to Promote Adsorption to Biomarkers of Eosinophilic Esophagitis Thesis directed by Professor Robin Shandas ABSTRACT The surface of an acrylate based s hape m emory p olymer ( SMP ) was modified to capture cationic biomarkers of eosinophilic e sophagitis ( EoE ) to establish a better diagnostic and moni toring system of this disease. Incorporation of a negative charge into the SMP allows for specific adsorption of the cationic biomarkers with additional leverage from t he shape memory effect to position the polymer near the lumen wall of the esophagus. SMP modification was performed by copolymerization with acrylate acids at increasing weight percentages to incorporate a negative, electrostatic charge onto the polymer su rface, but still preserve and ins till hydrophobic interactions. Because literature suggests that electrostatic, hydrophobic and hydrogen bonds are the major driving interactions for adsorption, the polymer was the target of modification to the system in th e initial phase s of the experimental design. Reproducible binding did not occur with in vivo mimicking systems. Polystyrene microspheres, a commercially available positive control known to adsorb proteins was introduced to the experimental process after c ontrolling for other influential adsorption parameters (pH, protein, protein concentration, surface to volume ratios, pH, riddance of im purities or competing proteins, etc.) that also established no bindi ng to the surface of the SMPs. The positive control facilitated a checkpoint for the development of SMP adsorption. The microspheres were used to check against all experimental parameters that were performed prior, to measure encouraging or discouragi ng protein adsorption factors. The polystyrene microspher es unveiled the importance of ionic strength, pure protein solutions and surfactants to adsorption. This illustrates that adsorption depends on multiple factors and that electrostatic charge and hydrophobicity on the polymer surface is just one such parame ter. The system was revised and binding was performed with SMPs incorporated with 0, 0.5, 1 and 2.5 wt % of SEM Results showed significant amounts of protein binding Additionally, adsorption of the SEM polymers was tested aga inst the adsorption of polystyrene and nylon for comparative analysis of ma instream biomedical materials. SEM

PAGE 4

iv SMPs bind to protein preferentially over these materials suggesting our SMPs are better adsorbents, paving the way for many biomedical application s. The form and content of this abstract are approved I recommend its publication. Approved: Robin Shandas

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v ACKNOWLEDGMENTS I would like to thank Kiran Dyamenahalli, Dr. Steve Ackerman and my committee members for thei r time and helpful guidance through this process A special thanks to my parental unit, Raj and Geeta Shah for their encouragement and endless support.

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vi TABLE OF CONTENTS C hapter 1. Introduction ................................ ................................ ................................ ................................ ................. 1 1.1 Shape Memory Polymer: A Diagnostic Approach for Eosinophilic Esophagitis ................................ ...... 1 2. Background ................................ ................................ ................................ ................................ ................. 3 2.1 Eosinophilic Esophagitis and the Esophageal String Test ................................ ................................ ......... 3 2.2 Adsorption Principles ................................ ................................ ................................ ................................ 4 2.3 SMPs and Surface Modification ................................ ................................ ................................ ................ 6 3. Rational for the Experimental Design and Important Considerations for Adsorption Studies .................... 8 3.1 Prelude to Rationale ................................ ................................ ................................ ................................ .. 8 3.2 Polymer ................................ ................................ ................................ ......................... 8 3.2.1 Major Interactions Forces Involved in Adsorption ................................ ................................ ................. 8 3.2.2 Shape Memory Polymer ................................ ................................ ................................ ....................... 10 3.2.3 Surface Area ................................ ................................ ................................ ................................ ......... 10 3.2.4 Impurities on the Polymeric Surface ................................ ................................ ................................ .... 10 3.2.5 Post P rocessing ................................ ................................ ................................ ................................ ..... 11 3.3 Protein ................................ ................................ ................................ ................................ ....... 13 3.3.1 General Considerations for the Study Protein ................................ ................................ ...................... 13 3.3.2 Poly L Arginine ................................ ................................ ................................ ................................ ... 14 3.3.3 Eosinophil Derived Granule Proteins ................................ ................................ ................................ ... 14 3.3.4 Bovine Serum Albumin ................................ ................................ ................................ ........................ 15 3.4 Solution ................................ ................................ ................................ ................................ ..... 15 ................................ ................................ ................................ .......................... 15 3.4.2 Temperature ................................ ................................ ................................ ................................ ......... 15 3.4.3 pH ................................ ................................ ................................ ................................ ......................... 16 3.4.4 Ionic Strength ................................ ................................ ................................ ................................ ....... 16

PAGE 7

vii 3.4.5 Purity ................................ ................................ ................................ ................................ .................... 16 4. Materials and Methods ................................ ................................ ................................ .............................. 17 4.1 Materials ................................ ................................ ................................ ................................ .................. 17 4.2 Methods ................................ ................................ ................................ ................................ ................... 17 4.2.1 Characterization ................................ ................................ ................................ ................................ .... 17 4.2.1.1 Contact Angle and Surface Free Energy ................................ ................................ ........................... 17 4.2.1.2 pH Testing ................................ ................................ ................................ ................................ ......... 18 4.2.1.3 Water Equivalency Test ................................ ................................ ................................ .................... 18 4.2.1.4 Dynamic Mechanical Analysis ................................ ................................ ................................ .......... 18 4.2.1.5 Fourier Transform Infrared Analysis ................................ ................................ ................................ 19 4.2.2 Adsorption Testing ................................ ................................ ................................ ............................... 19 4.2.2.1 SMP Synthesis ................................ ................................ ................................ ................................ ... 19 4.2.2.2 Binding Experiments ................................ ................................ ................................ ......................... 19 4.2.2.3 Experiment 1. MA ................................ ................................ ................................ ............................. 20 4.2.2.4 Experiment 2 3. CEA ................................ ................................ ................................ ........................ 20 4.2. 2.5 Experiment 4. Surface Area 132mm 2 /ml ................................ ................................ ........................... 21 4.2.2.6 Experiment 5. Surface Area with SMP Particles ................................ ................................ ............... 21 4.2.2.7 Experiment 6. Surface Area with SMP Particles ................................ ................................ ............... 23 4.2.2.8 Experiment 7. Surface Area 648mm 2 /ml ................................ ................................ ........................... 24 4.2.2.9 Experiment 8. Eosinophil Lysate ................................ ................................ ................................ ....... 24 4.2.2.10 Experiment 9 10. Eosinophil Lysate ................................ ................................ ............................... 25 4.2.2.11 Experiment 11. Eosinophil Lysate ................................ ................................ ................................ ... 25 4.2.2.12 Experiment 12. CEA ................................ ................................ ................................ ....................... 26 4.2.3 Positive Control Optimization of Polystyrene Microspheres ................................ .............................. 26 4.2.3.1 General procedure ................................ ................................ ................................ ............................. 26 4.2.3.2 Surface Area and Surfactant Op timization ................................ ................................ ........................ 27 4.2.3.3 Total Exclusion of Surfactants ................................ ................................ ................................ .......... 27

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viii 4.2.3.4 BSA Concentration Optimization ................................ ................................ ................................ ...... 27 4.2.3.5 Buffer Solution Optimization ................................ ................................ ................................ ............ 27 4.2.3.6 Reproducibility Testing ................................ ................................ ................................ ..................... 27 4.2.3.7 BSA Alexa Fluor 488 ................................ ................................ ................................ ........................ 28 4.2.4 Positive Control Test Polystyrene Microspheres against Past Experiments ................................ ...... 28 4.2.4.1 General Procedure ................................ ................................ ................................ ............................. 28 4.2.4.2 Experiments 1 7 Poly L Arginine ................................ ................................ ................................ ..... 28 4.2.4.3 Experiments 8 11 Eosinophil Lysate ................................ ................................ ................................ 28 4.2.4.4 Experiment 12 BSA and Ionic strength ................................ ................................ ............................. 28 4.2.5 SEM Adsorption ................................ ................................ ................................ ................................ ... 29 5. Results and Discussion ................................ ................................ ................................ .............................. 30 5.1 CEA Characterization ................................ ................................ ................................ .............................. 30 5.1.1 Contact Angle and Surface Energy ................................ ................................ ................................ ...... 30 5.1.2 pH Testing ................................ ................................ ................................ ................................ ............ 31 5.1.3 Dynamic Mechanical Analysis ................................ ................................ ................................ ............. 34 5.1.4 Fourier Transform Infrared Spectroscopy ................................ ................................ ............................ 34 5.2 SEM SMP Characterization ................................ ................................ ................................ .................... 35 5.2.1 Contact Angle and Surface Energy ................................ ................................ ................................ ...... 35 5.2.2 Reproducibility ................................ ................................ ................................ ................................ ..... 38 5.2.3 Water Equivalency Test ................................ ................................ ................................ ....................... 38 5.2.4 Reproducibility of Water Equivalen cy Test ................................ ................................ ......................... 39 5.2.5 Dynamic Mechanical Analysis ................................ ................................ ................................ ............. 39 5.2.6 Fourier Transform Infrared Spectroscopy ................................ ................................ ............................ 43 5.3 Adsorption Testing ................................ ................................ ................................ ................................ .. 45 5.3.1 Experiment 1. MA ................................ ................................ ................................ ................................ 45 5.3.2 Experiment 2 3. CEA ................................ ................................ ................................ ........................... 46 5.3.3 Experiment 4. Surface Area 132mm 2 /ml ................................ ................................ .............................. 47

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ix 5.3.4 Experiment 5. Surface Area with SMP Particles ................................ ................................ .................. 49 5.3.5 Experiment 6. Surface Area with SMP Particles Complete Submersion ................................ ............ 52 5.3.6 Experiment 7. Surface Area 648mm 2 /ml ................................ ................................ .............................. 53 5.3.7 Experiment 8. Eosinophil Lysate ................................ ................................ ................................ .......... 55 5.3.8 Experiment 9 10. Eosinophil Lysate ................................ ................................ ................................ .... 58 5.3.9 Experiment 11. Eosinophil Lysate ................................ ................................ ................................ ........ 61 5.3.10 Experiment 12. CEA ................................ ................................ ................................ .......................... 63 5.4 Positive Control Optimization of Polystyrene Microspheres ................................ ............................... 64 5.4.1 Surface Area and Surfactant Optimization ................................ ................................ ........................... 64 5.4.2 Total Exclusion of Surfactants ................................ ................................ ................................ ............. 66 5.4.3 BSA Concentration Optimization ................................ ................................ ................................ ......... 67 5.4.4 Buffer Solution Optimization ................................ ................................ ................................ ............... 68 5.4.5 Reproducibility Testing ................................ ................................ ................................ ........................ 69 5.4.6 BSA Alexa Fluor 488 ................................ ................................ ................................ ........................... 69 5.5 Positive Control Test Polystyrene Microspheres against Past Experiments ................................ ......... 70 5.5.1 Experiments 1 7 Poly L Arginine ................................ ................................ ................................ ........ 70 5.5.2 Experiments 8 11 Eosinophil Lysate ................................ ................................ ................................ .... 71 5.5.3 Experiment 12 BSA and Ionic strength ................................ ................................ ................................ 72 5.6 SEM Adsorption ................................ ................................ ................................ ................................ ...... 73 6. Conclusion ................................ ................................ ................................ ................................ ................. 79 References 82

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x Appendix A: Detection ................................ ................................ ................................ ................................ .................. 84 A.1 Overview of Detection Methods ................................ ................................ ................................ ............. 84 A.2 Micro BCA Assay ................................ ................................ ................................ ................................ .. 88 A.3 Bio Rad Protein Assay ................................ ................................ ................................ ........................... 91 A.4 Spectroscopy ................................ ................................ ................................ ................................ .......... 92 A.5 Flamingo Fluorescent Gel Stain ................................ ................................ ................................ ............. 93 B: Protocols ................................ ................................ ................................ ................................ ................... 96 B.1 Acetate Buffer ................................ ................................ ................................ ................................ ......... 96 B.2 Polystyren e Microspheres ................................ ................................ ................................ ....................... 97 B.3 SEM Binding Experiment ................................ ................................ ................................ ....................... 98 B.4 Eosinophil Lysate ................................ ................................ ................................ ................................ 100

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xi LIST OF TABLES Table 1 Protein Characterization ................................ ................................ ................................ ............................ 14 2 Contact Angle and Surface Energy Measurements: Average of Ten Trials ................................ .............. 31 3 Contact Angle and Surface Energy Measurements: Average of Ten Trials ................................ .............. 37 4 Water Equivalency Test: Average of 2 Samples ................................ ................................ ....................... 39 5 Thermal Mechanical Properties of SEM SMPs: Average of Two Trials ................................ .................. 42 6 Protein C haracterization ................................ ................................ ................................ ............................ 56 7 % Protein Bound Relative to Total ECP ................................ ................................ ................................ ... 57 8 % Protein Bound Relative to Total EDN ................................ ................................ ................................ ... 57 9 % Protein Bound Relative to Total MBP1 ................................ ................................ ................................ 57 10 % Protein Bound Relative to Total ECP ................................ ................................ ................................ 59 11 % Protein Bound Relative to Total EDN ................................ ................................ ................................ 59 12 % Protein Bound Relative to Total MBP1 ................................ ................................ .............................. 60 13 % Protein Bound Relative to Total ECP ................................ ................................ ................................ 60 14 % Protein Bound Relative to Total EDN ................................ ................................ ................................ 61 15 BSA Characterization ................................ ................................ ................................ .............................. 63 16 % Protein Bound Relative to Total ECP ................................ ................................ ................................ 63 17 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 66 18 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 66 19 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 67 20 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 68 21 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 69 22 % Protein Bound Relative to Total BSA 488 ................................ ................................ .......................... 70 23 % Protein Bound Relative to Total Poly L Arginine ................................ ................................ .............. 71 24 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 73 25 % Protein Bound Relative to Total BSA ................................ ................................ ................................ 76

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xii 26: Antibody Based Assays. ................................ ................................ ................................ ......................... 85 27: Absorbance Based Assays ................................ ................................ ................................ ...................... 86 28: Fluorescence Based Assays. ................................ ................................ ................................ ................... 87 29 : BCA Assay with Hanks Balanced Salt Solution (HBSS) ................................ ................................ ........ 89 30: BCA Assay with RPMI Diluted with Water (W) or PBS (P) ................................ ................................ .. 89 31: BSA Standard Curve Diluted in PBS (1X) ................................ ................................ .............................. 90

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xiii LIST OF FIGURES Figure 1 Important Protein Adsorption Determinants 12 ................................ ................................ ............................. 5 2 Schematic of Major Adsorption Interaction 6,7 ................................ ................................ ............................ 9 3 Schematic of Surfactants Inhibiting Protein Binding ................................ ................................ ................ 11 4 Methanol Treatments to CEA SMPs ................................ ................................ ................................ ......... 12 5 Sterilization Treatments to SEM SMPs and Polystyrene ................................ ................................ ........... 13 6 Processing of SMP Particles ................................ ................................ ................................ ...................... 23 7 Adsorption Optimization Techniques ................................ ................................ ................................ ........ 24 8 Contact Angle and Surface Energy Measurements of CEA SMPs ................................ ............................ 31 9 pH Testing of CEA SMPs ................................ ................................ ................................ ......................... 33 10 DMA Results on CEA SMPs ................................ ................................ ................................ .................. 34 11 FTIR on CEA SMPs ................................ ................................ ................................ ................................ 35 12 Contact Angle and Surface Energy with SEM SMPs ................................ ................................ .............. 36 13 Water Equivalency Test with SEM SMPs ................................ ................................ ............................... 38 14 DMA Results on SEM SMPs ................................ ................................ ................................ .................. 41 15 Thermomechanical Reproducibility Analysis ................................ ................................ ......................... 42 16 FTIR on SEM SMPs ................................ ................................ ................................ ................................ 44 17 Experiment 1 MA ................................ ................................ ................................ ................................ .... 46 18 Experime nt 2 3. CEA ................................ ................................ ................................ .............................. 47 19 Surface Areas Implemented to Maximize Loading Capacity ................................ ................................ .. 48 20 Experiment 4. Surface Area 132mm 2 /ml ................................ ................................ ................................ 48 21 SMP Particles ................................ ................................ ................................ ................................ ........... 50 22 Nylon Particles ................................ ................................ ................................ ................................ ........ 51 23 Experime nt 5 Surface Area with SMP Particles ................................ ................................ ...................... 52 24 Experiment 6 Surface Area with SMP Particles Complete Submersion ................................ ................. 53 25 Experiment 7 Surface Area 648mm 2 /ml ................................ ................................ ................................ .. 54

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xiv 26 Experiment 8 Eosinophil Lysate. ECP Adsorption ................................ ................................ ................. 56 27 Experiment 8 Eosinophil Lysate. ED N Adsorption ................................ ................................ ................ 57 28 Experiment 8 Eosinophil Lysate. MBP1 Adsorption ................................ ................................ .............. 57 29 Experiment 9 Eosinophil Lysate. ECP Adsorption ................................ ................................ ................. 59 30 Experiment 9 Eosinophil Lysate. EDN Adsorption ................................ ................................ ................ 59 31 Experiment 9 Eosinophil Lysate. MBP1 Adsorption ................................ ................................ .............. 60 32 Experiment 10 Eosinophil Lysate. MBP1 Adsorption ................................ ................................ ............ 60 33 Experiment 10 Eosinophil Lysate. EDN Adsorption ................................ ................................ .............. 61 34 Experiment 11 Eosinophil Lysate. ECP Adsorption ................................ ................................ ............... 63 35 Experiment 12 CEA ................................ ................................ ................................ ................................ 64 36 Surface Area and Surfactant Optimization ................................ ................................ .............................. 65 37 Total Exclusion of Surfactants ................................ ................................ ................................ ................ 66 38 BSA Concentration Optimization ................................ ................................ ................................ ............ 67 39 Buffer Solution Optimization ................................ ................................ ................................ ................... 68 40 Reproducibility Testing ................................ ................................ ................................ ........................... 69 41 BSA Alexa Fluor 488 ................................ ................................ ................................ .............................. 70 42 Expe riments 1 7. Poly L Arginine ................................ ................................ ................................ .......... 7 1 43 Experiments 8 11. Eosinophil Lysate ................................ ................................ ................................ ...... 72 44 Experiments 12. BSA and Ionic Strength ................................ ................................ ................................ 73 45 SEM Adsorption of BSA ................................ ................................ ................................ ......................... 75 46 SE M Polymer after 3 Hour Incubation ................................ ................................ ................................ .... 77 47 Images of the SEM Polymer after 3 Hour Incubation ................................ ................................ ............. 78 48 Micro BCA Assay Performed on Experiment 12 CEA ................................ ................................ ........... 91 49 Bio Rad Assay Performed on Experiment 12 CEA ................................ ................................ ................. 91 50 Spectroscopy Performed on Experiment 12 CEA ................................ ................................ .................... 92 51 Spectroscopy Performed on Experiment 12 CEA ................................ ................................ ................... 93 52 Flamingo Fluorescent Gel Stain on BSA Standards, Exposure with a Fluorometer at 532nm ................. 94 53 Flamingo Fluorescent Gel Stain on BSA Standards, Exposure with UV Transilluminescence ............... 94

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xv 54 BSA Standard Curve from Fluorescent Gel Stain. ................................ ................................ ................... 95

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1 1. I ntroduction 1.1 S hape M emory P olymer : A Diagnostic A pproach for E osinophilic E sophagitis In developed countries, 15% of all medical cases are misdiagnosed 1 The Seattle Times reports third of the 2.7 trillion spent each year on healthcare in the U.S. are con sidered to be wasted The curren t diagnostics set in place for e osinophilic esophagitis allergi c disease of the esophagus, contributes to this heavy expense. In this disease, eosinophils invade the esophagus and we ar away the esophageal lining. Biopsies are the gold standard for detecting EoE and depend on measuring these e osinophils 2 Since the e osinophils are not evenly distributed within the esophagus, and the procedure is limited to acquiring less than a 0.7% sample size relative to the entire esophageal surface area, the diagnosis can be missed 3 One study reported a correlation between the quantity of these eosinophils and highly cationic eosino phil derived granule proteins ( EDGPs ) the y secrete in the luminal mucosa 4 Therefore, measuring mucosal inflammation can be one route to effectively diagnose EoE patients and is under investigation t hrough a novel esophageal string test 2,4 In this approach, a swallowable nylon string is used to capture these cationic prote ins in the lumen of the esophagus. The test drives down the diagnostic co sts and is minimally invasive. are an underlining issue due to small sample volume collected onto the string and the lack of specificity for the EDGPs. With these limitations of current detection methods, patients of EOE are needlessly suffering from symptoms that affect their quality of life. The initial aim of this study was to chemically tailor a polymeric surface for the specific capture o f EDGPs. This contribution can aid in the development of a minimally invasive, inexpensive an d reliable diagnostic for EoE. Incorporation of anionically charged groups to the surface of a shape memory polymer ( SMP ) can selectively adsorb these unique cat ionic proteins with additional leverage from the SMP to position itself near the lumen wall of the esophagus in Additionally, the shape memory effect of the polymer allows for substantial increase in recoverable surface area during deployment, correlating to capturing greater sample sizes. Adsorption mechanisms have been extensively studied and reported as a complex system, where the exact occurrences between the protein and surface interfaces are unknown 5 7 However, literature states the dominating factors that affect adsorption are electrostatic, hydrogen and hydrophobic interaction forces 5 9 Recent studies have found trends of

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2 adsorption throug h electrostatic and hydrophobic interactions. These groups found that adsorption through negative electrostatic interactions is higher relative to adsorption through hydrophobic interactions 5,7 We therefore hypothesized that the incorporation of negatively charged functional groups onto the surface of the SMP would specifically aid in adsorption of cationic EDGPs. The purpose of this study was to design a shape memory polymer ic surface to promote protein adsorption for the eventual binding of specific biomarker s of EoE. The results of the study show significant amounts of protein adsorption, specifically bovine serum albumin ( BSA ) after the incorporation of negatively charged sulfonic acid groups coupled with extensive adsorption system adjustments.

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3 2. B ackground 2.1 Eosinophilic E s ophagitis and the Esophageal String T est Eosinophilic e sophagitis is an emerging chronic inflammatory disease of the esophagus effecting thousands of Americans each year 10 The disease is charac terized by the infiltration of eosinophils to the esophagus. These cells are a gateway to esophageal inflammation as they have the capacity to initiate an inflammatory cascade causing a diverse set of symptoms. Serious complications include food impaction and str icture formation that could require urgent removal of the food or endoscopic balloon dilatations 10 The current requirement for diagnosing this disease is through es ophagogastroduodenoscopy ( EGD ) and histological examination of esophageal mucosal biopsies 10 These methods can be b oth inaccurate and unreliable. Biopsies represe nt 0.7% of the esophageal surface area, a small enough sample size to miss the disease 3 Endoscopies depend on abnormal physical features of the lumen that may not be present in all diseased patients 2 The Enterotest, performed originally to detect parasites in the intestine, is under experimentation for capturing highly cationic EDGPs biomarkers of EoE in the mucosa of the esophagus 4 This esophageal string test is a nylon string that is packed into a capsule. During the procedure, one end of the stri ng is taped to the cheek and the remaining string is carried through the GI tract, unraveling from the wei ghted capsule upon swallowing. The capsule is dislodged from the string and mucosal remnants a re captured onto the material. Further in vitro detection of EDGP other antibody driven tests. Although this is a great advancement in a non invasive diagnostic for EoE, there is room for improvement. In our application, we are altering the surface of the SMP to capture these cationic biomarkers of EoE in a simi lar fashion to the Enterotest. The shape memory effect of the polymer will aid in positioning the functionalized surface near the site of the biomarkers while the modified surface is designed to specifi cally adsorb the EDGPs of EoE. This can improve diagnosis considerably by obtaining greater sampling sizes and capturing specific proteins of the disease.

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4 2.2 Adsorption Principles Before the surface was modified, the principles of adsorptio n were defined and understood. Adsorption is the process of particulate or mol ecular binding onto a surface. Surfaces are not fully bound and consumed by their surrounding atoms as their bulk material; they have the capacity for binding atoms, an energy fa vorable mechanism that lowers their energy state. Thus, surfaces usually possess higher surface energy than their bulk material which we might identify with as a good adsorbent 8 Since the surfaces of polymers usually have low surface energy and are not as reactive as other surfaces such as metal because of their low chemical potential functional groups were incorporated to facilitate pro adsorption characteristics for EDGPs 11 Protein adsorption is a complex mechanism primarily because of the myriad of influential factors it encompasses, sur face energy being one of them. It' s a and the solution they are in. All of these entities have their own list of influencing characteristics displayed in figu re 1 The ones that overlap with each o ther were heavily implemented in the final experimental desi gn.

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5 Figure 1 Important Protein Adsorption D eterminants 12 The stages that comprise protein adsorption onto a polymeric surface are: 1 the transport of protein near the surface, 2 the attachment of protein onto the polymeric surface, 3 the rearrangement and re ori entation of the protein onto the surface, and 4 the desorption or permanent attachment of protei n onto the polymeric surface. The last stage is dependent on the extent of relaxation of the protein upon binding As the protein contacts the surface, interac tion forces increase, and if large enough to compete with the ouraging irreversible binding. If the residency time is maximal, the protein will have sufficient time to relax before des orption permits 7,12 Minimal residency time is due to few and weak interaction forces upon initial binding or the interference with other competing proteins. In the first step of adsorption, the transport of protein near the surface of the polymer is due mostly to coulombs in teractions (electrostatic interactions) with an interaction force of where r is the distance of

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6 As the protein is drawn closer, Vander Waals forces, with an interaction force of persists as the driving force for the initial stages of adsorption 12 The second stage, protein attachment, occurs if the polymer elicits active sites that compliment the amino acids o n the surface of the protein. Particularly functional groups such as COOH, OH, NH2, SH, SO3 and hydrophobic residues on both the surfaces of polymers and proteins, aid in the initial attachment onto a p olymeric bioma terial. Though this usually consists of multiple weak interactions, if more time is allowed, they become stronger and irreversible which affect the third stage, reorientation and relaxation onto the polymeric surface 8,12,13 The influential factors in figure 1 can determine if the protein will remain bound irreversibly or bind mo mentarily and desorb back into s ol ution, the 4 th stage in protein adsorption If the adsorbent competes with the intermolecular forces that keep the protein structurally sound, the protein is most likely to bind irreversibly due to perm anent conformational changes. For irreversible binding to occur, the protein must unveil its hydr ophobic core to the adsorbent. The more heterogeneous the intermolecular forces are on the adsorbent, the more likely it will be able to unfold the protein and permit maximal residency time for irreversible bindin g It is for this reason, the SMP was modified to have a negative charge and accrue the EDGPs to its surface, encouraging interaction with both electrostatic and hydr ophobic chemical constituents. 2.3 SMP s and Surface M odification Biological responses ca n be reprogrammed from the surface of biomaterials rendering polymeric surfaces important considerations to biomedical applications 14 Surface texture, surface potential and surface energy are mainstream surface features that can influence aggregation and/or binding to the surface and trigger such host response 15 Particularly, surface chemistry and topography dominate biological responses to biomaterials 16 By altering the surface profile, biological responses can be manipulated specifically to adsorb protein onto the surface, an important consideration in the field of biomedical applications 8 Concomitantly, bulk polymeric properties attribute to the core mechanical strength and agility of the device, playing an integral role in establishing durability, flexibility and c omfort. SMPs

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7 state upon the introduction of an external stimulus 17 19 In our appli cation, the SMP was utilized for the base component for this device because of this intrinsic ability t o memorize a predefined shape. An acr ylate based SMP was used, with the ability to be thermally stimulated to initiate shape transitions. Because the SMP can be stored in a temporary fixed state for elongated periods of time, it can be packed a nd stored in a small capsule. Upon the introduction of the polymer to the esophagus, the SMP is thermally stimulated and has the ability to undergo large recoverable deformations. This characteristic can be easily tailored such that the polymer returns to a site near the biomarkers and increases its surface area substantially. Together, these properties encourage adsorption by increasing the loading capacity of the polymer and placing them in close proximity to the biomarkers to shorten their transport path The union of SMPs and refined surface properties can pave the way for a myriad of biomedical devices. For this study, the focus is on designing an optimal SMP surface to promote binding of unique cationic, EoE biomarkers for the d evelopment and improveme nt of a diagnostic for this disease. The base formulation of the SMP is mostly hydrophobic, an encouraging factor of protein adsorption in general. Incorporation of negatively charged groups onto the surface can facilitate specific and preferential bindin g to eosin ophil derived granule proteins: major basic protein 1 ( MBP1 ), eosinophil cationic p rotein ( ECP ), eosinophil derived neurotoxin ( EDN ), and eosinophil peroxidase ( EPX )

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8 3. R ational for the E xperimental D esign and I mportant C onsiderations for Adsorption Studies 3.1 Prelude to R ationale In this study, the initial aim was to modify the surface of an acrylate based SMP to adsorb spec ific cationic proteins of EoE. Because protein adsorption is a complex mechanism, the initial experiments for this goal yielded no binding. System adjustments were iterated throughout a major portion of this study with a continued failure to adsorb proteins onto the SMP surface. The goals of the study broadened to modifying the surface of the SMP to eventu ally adsorb cationic proteins. Until the core principles of protein adsorption were understood, the specific adsorption of cationic proteins would be difficult to meet. A commercially avai lable adsorbent that served as a positive control was issued in the experimental process to check against previous exper iments and their environments. This analysis allowed us to see what particular factors are encouraging or discouraging for protein adsorption. From this, the degree of compl exity of adsorption was revealed as multiple factors can significantly inhibit or attenuate binding. After final system revisions were made from the positive control findings, significant amounts of BSA were adsorbed onto the modified polymer surface. Because of the numerous sys tem revisions that were implemented in this study, detailed below is the rationale for considering these important factors that contribute to the multifa ceted mechanism of adsorption. 3.2 Polymer 3. 2 1 Major Interactions Forces Involved in A dsorption Electrostatic, hydrophobic and hydrogen bonds are cited as the major interaction forces in protein adsorption 5,7,8,12 H owever, the dominating factor is still under controversy, as the literature remains inconsistent due to the complexity of adsorption. As such, all three in teraction forces were studied. The base formulation of our SMP is mostly hydrophobic, providing one of the major interaction forces. Tert Butyl Acrylate ( tBA ), the hydrophobic monomer in the SMP formula is displaced by the weight percent of the new fun ctional monomer being incorporated into the mixture, minimally decreasing the hydrophobicity but increasing the other interaction forces. For the initial goal of adsorbing cationic protein onto the surface of the polymer, negative electrostatic interactio ns were implemented. Methacrylic acid ( MA ), 2 carboxyethyl acrylate ( CEA ) and 2 sulfoethyl methacrylate ( SEM ) were chosen as monomers that would exhibit an electrostatic

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9 charge under physiological pH and were incorporated into the S MP at various weight percents. Below is a schematic of how the polymer surface would exhibit key int eraction forces of adsorption. At pH 7, the hydrogen on the carboxylic acid of MA and CEA would disassociate and leave the oxyg en species negatively charged The negatively charge surface of the polymer can provide interaction sites for the cationic proteins, encouraging adsorption through both electrostatic and hydrophobic (Van der Waals and London type) interactions. Figure 2 Sc hematic of M ajor Adsorption I nteraction 6,7 Methacrylic acid, 2 carboxyethyl acrylat e and 2 sulfoethyl methacrylate was incorporated into the SMP at increasing weight percentages to facilitate major interaction forces of adsorption. Because the incorporation of MA and CEA at pH 7 did not yield pr otein binding, the s ystem was adjusted such that the pH matched the isoelectric point of the study protein. At the new pH ( 4.5 ), the hydrogen atom on the carboxylic acid group should not disassociate, thus leaving the hydrogen intact to facilitate hydrogen bonding. After these polymeric alterations, binding still did not persist. The last modification was made with SEM We proposed this new monomer would work because it was a stronger acid than carboxylic acids used previously and may possess a larger int eract ion force for protein capture. Literature also verified using SO3 functional groups for protein adsorption 5

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10 3.2.2 Shape Memory P olymer An acrylate based SMP wa s employed as the base polymer mainly because of its ability to self deploy in the esophagus near the site of the EoE biomarkers and its high specific surf ace area after shape recovery. Additional benefits are its ability to be packed into a small capsule in its temporary state fo r an elongated period of time. The SMP is also easily processed, cost efficient, non invasive and has great mechanical properties. 3.2.3 Surface A rea Surface a rea directly correl ates to the loading capaci ty of the polymer for protein. If the surface area is minimal, small samples sizes are retrieved, and make detection difficult. As such, a course of action was taken to increase surface area substantially and remained a focus for many experiments. 3.2.4 Impurities on the Polymeric S urface Careful processing of the SMP is crucial for binding experiments as the surface is highly reactive and cont amination is fairly easy. The polymer samples were handled with glov es and under clean conditions. The s amples were always stored in a Ziploc bag, away from air particulates, b efore the binding experiments. During the initial experiments, the shape memory coupo ns were washed with Sparkleen. Because this is an amphiphilic molecule, one concern was the hydrophobic portion of the molecule binding irreve rsibly to the polymer surface. Even if the bond was reversible, impurities such as this become part of the adsorption system and could interfere with binding by taking up site s on the protein or polymer that shield the interaction forces we were counting on and alt ering the adsorption kinetics. To prevent occupancy of the binding sites with such impurities, the polymers were switched to being washed with clean nano pure or dist illed (DI) water. The water source is important as well because organic and ionic species can bind to the polymer surface, altering the charge or taking vacancy on limited binding s ites. All surfaces and equipment the polymeric samples came into contact wi th were wiped down with methanol or ethanol and DI water

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11 Figure 3 Schematic of Surfactants Inhibiting Protein B inding 3.2. 5 Post P rocessing Initially, all polymers were post cured for 1 hour at 90C Since binding did not occur, one explanation was the possibility of free monomer that was not ev aporated during the post cure. As mentioned above, these impurities can take up valuable binding sites on the protein and polymer while shielding important interaction forces. Thus, methanol treatments were carried out to swell the polymer and eliminate leachable content However methanol treatments were swell ing the polymer to approximately 1/3 of its own size, which ultimately could irreversibly expand the pore sizes of the polymer networks and influence adsorption. Since our goal was to adsorb protein through el ectrostatic charge, the altered pore size added an additional avenue of protein adsorption Additionally, when methanol treatments w ere carried out with CEA at 5 and 15wt%, the samples cracked after vacuum drying them for 48 hour s. I n effect, met hanol treatments were stopped.

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12 Figure 4 Methanol T reatments to CEA SMPs 48 hour vacuum drying and methanol treatments on 5 and 15 wt% CEA causes polymeric cracks. Sterilization was examined to ensure riddance of impurities. Polystyrene and SEM samples (0, 0.5, 1 and 2.5 wt % ) were placed in an autoclave after DI washing. The 2.5 an d 1% SEM samples did not survive this process as they encountered numerous cracks Additionally the polystyrene samples became distorted so we reverted back to the original post cure of 1 hour at 90 C

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13 Figure 5 Sterilizati on T reatments to SEM SMPs and Polystyrene Polymeric breaks and distortion were created from autoclaving samples at 250F for 15minutes The first box shows 0 2.5 and 0. 5% SEM SMPs while the second encompasses polystyrene and 1% SEM SMP samples. 3.3 Protein 3.3.1 General C onsiderations for the Study P rotein The structural mobility, compactness, size, intramolecular forces and charge were all consider ed in picking a study protein. High structural mobility encourages irreversible binding onto the polymer surface by readily making conformational changes upon adso rption. This mobility allows for increased contact numbers onto the poly meric surface and thus heightens the affinity of the protein for the polymer Internal mobility also increases the rate of bin ding. The intramolecula r forces maintain structurally stability of the protein; hence, less internal motion occurs. Therefore, these intramolecular forces are what the adsorbent is competing against for final conformational changes to take place that le ad to irreversible binding. If the intramolecular forces are strong and plentiful, more energy is required from the adso rbent to denature the protein. Therefore, less intramolecular forces ar e better for this application. The number of disulfide bonds and apolar gro ups are

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14 key contributors to the proteins sta bility. A polar groups also contribute to the compactness of the protein an anti adsorption variable. The size of the protein is directly correlated to the number of i nteraction forces it can make. Since a larger protein can make more contacts onto the polymer surface, the strength of the total adsorption bond is stronger and will trend toward irreversible binding. A charged protein is structurally loose because of intramolecular repulsion between th e charged residues. This provides extra molecular m obility. Since the target proteins are EDGPs, a positively charged analog was used. Below are the proteins picked for this study and why T able 1 illustrates major protein characteristics that influence p rotein adsorption. Table 1 Protein Characterization Protein MW ( kDa ) PI % Hydrophobicity % Charged Sites # disulfide bonds Poly L Arginine 5 15 10.76 -----100 0 ECP 18.39 11.4 43.16 20.63 9 EDN 18.35 8.9 41.62 15.53 9 EPX 81.04 10.8 43.24 24.2 17 MBP 25.21 10.9 40.54 26.12 12 BSA 69.29 4.7 42.83 33.27 35 3.3. 2 Poly L Arginine Poly L a rginine was chosen because it is a cationic protein with a similar isoelectr ic point (10.76) to the EDGPs. 3.3.3 Eosinophil Derived Granule Proteins Poly L a rginine did not bind to the negativ ely charged polymeric surface. Since these synthetic analogs did not have hydrophobic domains to make irreversible interactions, in place. These are the actual target proteins for the diagnosis and will provide the correct intramolecular forces that we will be competing against. They were also chosen for the study protein after poly L arginine because of the high density of interaction forces these native proteins intrinsically have on their surface and core to promote irreversible binding.

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15 3.3. 4 Bovine Serum A lbumin BSA was primarily used because it is widely characterized and was the main study protein in other adsorption studies. Careful consideration of the experiments was taken because of the protein s tendency to oligomerize over time or during elevated temperature s Because oligomerization creates a bigger molecule, it may adsorb more readily (quickly and firmly) than single BSA molecules BSA also possesses pro adsorption characteristics because of its 1) high structural mobilit y, 2) hydrophobic cleft on its surface and 3) moderate size for more interactions (larger than most ) 3.4 Solution 3.4.1 Solution s Importance The solution is especially important in our experiments as it controls the ionization states of both the protein and polymeric surface s In essence, the n egative, electrostatic interactions incorporated on the polymeric surface are dependent on the pH and ioni c strength of the solution. Because the negative charges on the SMP specifically serve to bind the target, cationic proteins, it illustrates how cri tical the solution is to EDGP ad sorption. Similarly, if the pH and competing ions in solution displace the c ationic charge on the SMP device is eliminated. Initially, solutions mimicking physiological parameters were implemented but because of the high ionic strength, amino acid content and other interfering components other buffers were considered. After the process of elimination, ionic strength and the pH of the solution deemed the most important factors to consider and were adjusted appropriately. 3.4.2 Temperature All the adsorption studies were executed at phy siological temperature (37 C ). Increased temperatures accelerate the transport of the protein near the interfacial region of the polymer surface and contribute to internal mobility of the protein.

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16 3.4.3 pH The pH influences charged groups onto the surfa ce of the polymer and protein. If the pH is under the isoelectric point of the species, it a cquires a net positive charge. If the pH is above, the species has a net negative charge and if the pH is at the PI of the species, it is neutral. This is a central component to protein adsorption as it can impose repulsion forces between proteins and can change the acid/base nature of the polymer surface. 3.4.4 Ionic S trength Ionic s trength is crucial to protein adsorp tion as it defines the degree of interfe ring counter ions in solution. These ions can shield the charge that was designed for EDGP attachment or consume binding sites on the protein itself In this way, ions have the strength to weaken or persuade protein ad sorption one way or the other. The fi n al experiments were performed in low ionic strength conditions (0.01M). 3.4.5 Purity The cleanest reagents, including water must be used Ions and organic species compete with the target proteins to adsorb onto the polymer surface Not only do they consume valuable binding sites in lieu of the targeted protein but also they can alter the charges of both the protein and polymer in which the SMP device is reliant on for specific EDGP adsorption DI water was passed through a 0.2um filter and used to make up all solutions. All the beakers and materials that came into contact with the adsorbents were autoclaved or sterilized by m ethanol/ethanol prior to use.

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17 4. M aterials and Methods 4.1 Materials Tert butyl acryla te monomer, poly ( ethylene glycol) dimethacrylate (Mn=550) crosslinker CEA monomer (552348 50ML) MA monomer and the photo initiator 2 2 dimethoxy 2 phenylacetophenone we re ordered from Sigma Aldrich. SEM monomer was ordered from polysciences (cat 02597 50). Aliquots of poly L glutamic a cid (Sigma Aldrich MW 3,000 15,000, Stock p4636 25mg) and poly L a rginine (Sigma Aldrich MW 5,000 1500, Stock P4663) were prepared at 2,500ug/ ml in PBS (Invitrogen) diluent. Nylon ( ASTM D4066 PA 0114, White Amazon Supply) and polystyrene sheets (McMaster Carr 8734K39 1/16" Thick) were purchased to examine their adso rbent potential compared to SMPs. Nylon is the composite material of the esophageal string test and polystyrene is a known adsorbent that was u tilized as a positive control. ECP and E CP MBL International Corporation. The Micro BCA Protein Assay K it (Thermo Scientific / Pierce 23235) was the pri mary protein detection method. 4.2 Methods 4 2 1 Characterization 4.2.1.1 Contact A ngle and Surface Free E nergy Polymer surfaces were examined through a goniometer. A flat polymer was placed on a stage and a volume of DI water or Diiodomethane ( Sigma Aldrich 158429 25G) was sl owly dropped onto the surface. The droplet was photo documented and the angle betwe en the interfacial solid and liquid was calculated through the automated s oftware. The surface energies were calculated through F owkes equation depicted below. The surface energies of the associated liquids are listed as well. Note: Because surfaces are ac tive species, precaution was taken in handling and processing them to avoid contamination or destructs within its smooth surface.

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18 Fowkes Equation 12,20,21 : 4.2.1.2 pH T esting 12x59x 1mm CEA SMP samples were placed in 10mL of Acetate buffer at a pH of 4.45. Stir bars were added to each sample an d were placed on a stir plate. The pH was re corded every hour for 4 hours. The experiment was performed in duplicates. 4.2.1.3 Water Equiva lency T est SEM polymers, nylon and polystyrene were dehydrated in a vacuum oven for 48 hour s at 60 C and weighed for a baseline measurement. The polymers were incuba ted in excess DI water at 37C. The polymers were weighed and recorded every 12 hour s until they reached equilibrium, at 24 hour s. The experiments were performed in duplicate. 4.2.1.4 Dynamic Mechanical An alysis All CEA and SEM SMP samples were sized to 5x 30x1mm and their edges were s anded with 600 grit sand paper. The samples were cycled at 0 .1HZ, with a heating rate of 3 C /min with the testing temperature r anging from 0 100C The glass transition temperature was determined by the peak of the tan delta curves.

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19 4.2.1.5 Fourier Transform Infrared Analysis All CEA and SEM samples were polymerized between two glass slid es with a thin spacer < 0.5mm. Free radical polymerization was initiated a nd propagated with a UV source (black ray) at an intensity of ~ 11mW/cm 2 for 30 minutes. The samples were carefully removed and heat treated for 1 hour at 90 C Fourier transform infrared FTIR spectra were taken and the disappearance of the alkenes peak at 1610 1680cm 1 was used to determined convergence. 4.2 .2 Adsorption Testing 4.2.2.1 SMP S ynthesis SMP s were synthes ized using tBA monomer, PEGDMA crosslinker and DMPA p hotinitiator. MA CEA or SEM monomers were added to the base formulation listed initially at different mass fractions displacing tBA fractions. Solutions were made by mixing desirable weight percentages of monomers, tBA, PEGDMA and 0.1wt% of DMPA in a glass vial The solutions were i njected into a pre casted mold made from two glass slid es separated with 1mm spacers. A UV lamp (Black Ray) was used to polymerize the solutions at an intensity of ~11mW/cm 2 for 30 minutes. After polymerization, the polymer coupons were removed from its ca st and were heat treated at 90 C for 1 hour to evaporate unreacted monomers. The samples were sized and their edges sanded with 600 grit sand paper to even out t exture introduced from sizing. Afterwards, the samples were methodically washed with DI water. 4.2.2.2 Binding E xperiments SMP samples were incubated in protein solution for 1 hour at 37 C A protein only condition was incubated alone for quantification of i nitial protein concentrations. Media only (no SMP added) and SMP incubated in the media (no protein) are additional conditions that wer e used as background controls. All experimental conditions were performed in triplicate. Supernatant was collected in separate eppendorfs and frozen in a 20 C freezer until further use. Either the solution depletion method was used to quantitate protein bound indirectly to the samples or an extra elution step was performed to quan titate protein bound directly. With the elution step, the polymer sample wa s rinsed with PBS and dabbed with a Kim wipe befo re incubation with EST buffer. The samples were incubated for 30 minutes with a 30 second vortex st ep after ten minute intervals. The free solution was collected and frozen until further use. An alternative is

PAGE 35

20 the solution depletion method where the concentration of free protein after incubation with the SMP sample was subtracted from initial protein concentration. *Any deviances from these methods are noted in the experiment itself below. 4.2.2.3 Experiment 1. MA SMP coupons were prepared with 0, 5 and 15 wt% of MA (0:80:20, 5:75:20, 15:65:20 [MA : tBA: PEGDMA ]). A UV source was used for polymerization with the intensity ~20mW/cm 2 Circular discs were formed with a 6mm dye (McMaster Carr 3418A6) The discs w ere cleaned with methanol and DI water several times and dried at 90 C for 1hour. Samples were parafilmed in glass beakers overnight and used the next day for the binding experiment. SMP discs were incubated in 44ug/ ml of poly L arginine and p oly L glutamic acid for 5 minutes, 30minutes and 2hours at 37 C in 24 well pla tes. PBS was used because of its compatibility with the BCA assay and the surface to volume ratio was 37.68mm 2 The BCA assay was run on all samples after the free supernatant wa s collected and the solution depletion method was used for quantification of protein bound. 4.2.2.4 Experiment 2 3. CEA SMP coupons were prepared with 0, 5 and 15 wt% of CEA (0:80:20, 5:75:20, 15:65:20 [CEA : tBA: PEGDMA ]). A UV source was used for polymerization with the intensity ~20mW/cm 2 Circular discs were formed with a 10mm dye (McMaster Carr 3418A1) The discs were cleaned with Sparkleen and DI water several times and left out to dry overnight. Nylon was sized, sanded, washed and dried i n the same manner as the SMPs for experiment 3. SMP discs were incubated in 44ug/ ml of p o ly L arginine and p oly L glutamic acid for 1 hour at 37 C in 24 well plates. PBS was used because of its compatibility with the BCA assay and the surface to volume ra tio was 94.2mm 2 Six experimental repeats were performed per condition to help decrease the error bars. The BCA assay was run on all samples after the free supernatant was collected and the solution depletion method was used for quantification of protein b ound.

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21 4.2.2.5 Experiment 4. Surface A rea 132mm 2 / ml SMP coupons were prepared from the base formulation 80:20 (tBA : PEGDMA ). A UV source was used for polym erization with the intensity ~20 mW/cm 2 Square samples were formed with the dimensions of 20x20mm. The samples were cleaned with Sparkleen and DI water several times and left out to dry overnight. Nylon was sized, sanded, washed and dried in the same manner as the SMPs for experiment 3. SMP discs were incubated in 44ug/ ml of poly L arginine for 1 hour at 37 C in glass beakers. PBS was used because of its com patibility with the BCA assay. Three square samples were added per condition Because of the increased surface area, more protein solution had to be added (20mL) to cover the surfaces As such, the surface to volume ratio was 132mm 2 / ml. There were no experimental replicates because of the large polymer samples. The BCA assay was run on all samples after the free supernatant was collected and the solution de pletion method was used for quantification of protein bound. 4.2.2.6 Experiment 5 Surface A rea with SMP P articles SMP coupons were prepared from the base fo rmulation (80:20 tBA : PEGDMA ) in A UV source was used for polymerization with the intensity ~20mW/cm 2 The coupons were cleaned with Sparkleen and DI water several times and left out to dry overnight. Polymer and nylon particles were created from a dremel and collected into a vacuum device depicted below. Th e samples were imaged using an optical microscope at 6X magnification to s ee the range of sizes created. Small particles were disposed of through centrifugation steps (p articles were passed through a 230um sieve to rid small particles but this did not work well) so as not to interfere with the BSA assay and e licit a false positive result. One gram of SMP particles were measured in a polypropylene 50mL conical. Ultra pure H20 was added to the conical and spun at 1325 RCF at RT for 10min utes. The smaller part icles were f ound on top and were decanted. Three of these washes were performed as displayed below. The particles we re vacuum dried for 2.5 hours. SMP particles were weighed at 0.08, 0.04, 0.02, 0.01, 0.005 and 0.0025 grams and added to an ultra low attach ment ( ULA To determine the maximal surface to volume ratio, 1mL volumes of PBS were added to each sample u ntil 8mL was reached per well. With the findings, 0.06 and 0.04g were used for the binding experiment.

PAGE 37

22

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23 Figure 6 Processing of SMP P articles The particles were weighed at 0.04 and 0.06 grams and added to a ULA 6 well plate (Costar Product 3471) 5mL of 44ug/ ml of poly L arginine was added and the samples were incubated for 1 hour at 37 C in a shaker at 115RMP. PBS was used because of its compatibility with the BCA assay After the incubations, 1mL of supernatant was aspirated from each condition into separate eppendorfs and spun at 13,000 g for 10 minutes at RT. The BCA assay was run on a ll samples after the free supernatant was collected and the solution depletion method was used for quantification of protein bound. 4.2.2.7 Experiment 6. Surfac e Area with SMP P articles Complete S ubmersion Refer to Experiment 5 1.22 grams of SMP particles were weighed and placed in glass cryovials. 1.7mL of 44ug/ ml of poly L arginine was added and the samples were incubated for 1 hour at 37 C in a shaker at 200RMP. PBS was used because of its com patibility with the BCA assay. After the incubat ions, 1mL of supernatant was aspirated from each condition into separate eppendorfs and spun at 13,000 g for 10 minutes at RT. The BCA assay was run on all samples after the free supernatant was collected and the solution depletion method was used for quan tification of protein bound.

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24 4.2.2.8 Experiment 7. Surface A rea 648mm 2 / ml SMP coupons were prepared from the base formulation 80:20 (tBA : PEGDMA ). A UV source was used for polymerization with the intensity ~20mW/cm 2 Square samples were sized to 20x20mm and cut further into ~5x 6mm samples. The samples were cleaned with Sparkleen and DI water several times and left out to dry overnight. Nylon was sized, sanded, washed and dried i n the same manner as the SMPs. Next, the samples were incubated in methanol f or 48 hour s to alleviate competing leachables that may still be prese nt after post heat treatments. Afterwards the samples were washed with DI water and dried in a vacuum oven at 60 C for 48 hours. SMP pieces were incubated in 44ug/ ml of poly L arginine fo r 1 hour at 37 C in glass vials. PBS was used because of its com patibility with the BCA assay. Three 20x20mm square samples each cut into 5X6mm samples were added per condition 5mL of protein solution was added per condition for a final surface to volume ratio of ~648mm 2 / ml. The samples were incubated at 37 C and shaking at 200RPMs. The samples were specifically oriented upright to limit them from floating to the top. The BCA assay was run on all samples after the free supernatant was collected and the sol ution depletion method was used for quantification of protein bound. Figure 7 Adsorption Optimization T echniques Orient the vials so they are standing up to limit polymers from floating to the top 4.2.2.9 Experiment 8. Eosinophil Lysate SMP coupons were prepared from the base formulation 80:20 (tBA : PEGDMA ). A UV source was used for polymerization with the intensity ~20mW/cm 2 Coupons were washed with Sparkleen and DI water and dried at room temperature. The coupons were cut into ~6X30mm wide strips and su bmerged in

PAGE 40

25 methanol for 48 hour s. The strips were vacuum dried for 48 hour s at 60 C Afterwards samples were sized to 5X12mm and their edges were sanded with 600 grit sand paper. Next, the samples were washed with DI w ater and dried once more for 1 hour at 60 C in a vacuum oven. Samples were stored in a Ziploc bag until further use. Nylon was treated in the sa me throughout all experiments. It was cleaned, cut and sized in the s ame manner as the SMP samples. No post treat ments were necessary except for 1hour incubation at 90 C after it was washed. Eosinophils ( E ) were i solated from peripheral blood. The cells were suspended in 0.025M of sodium acetate buffer (pH of 4.3) with 10% protease inhibitor (Roche) at a concent ration of 5X10 6 cells/ ml. The cells were sonicated to create EO lysate and spu n down at 300g for 10 minutes. The supernatant was collected and frozen at 80 C until use. SMP samples were incubated in 500k/ ml of blood eosinophil lysate per RPMI + 8% FBS buffer for 1 hour at 37 C final pH 7. The surface to volume ratio was 154mm 2 / ml. An elution step was carried out after the binding experiment. The supernatant was collected and ECP, EDN and MBP1 ELISAs were per fo rmed. 4.2.2.10 Experiment 9 10. Eosinophil Lysate The experimental parameters rema ined the same as experiment 8. The only exception is the polymer samples did no t undergo methanol treatments. The polymer samples were cleaned with Sparkleen and DI water. After this washing step, the polymer was vacuum dr ied at 90 C for 3.5 hours. The coupon was cut, sized to 5x12mm samples and sanded Next, they were washed with DI water and heat treated once more for 1 hour at 60 C in a vacuum oven to ensure the riddance of water. They samples were placed in a Ziploc bag for storage. 4.2.2.11 Experiment 11. Eosinophil Lysate To compare heat treated to methanol treated polymers, both post processing techniques were administered. See experiment 8 and 9 10 for polymer preparation. All parameters stayed the same with the EO prep except the low 300g spin was switched to a high speed spin at 13,000g to ensure not sonicated cellular debris was pulled down and exclude d from the lysate for binding.

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26 4.2.2.12 Experiment 12. CEA SMP coupons were prepared with 0, 5 and 15 wt% of CEA (0:80:20, 5:75:20, 15:65:20 [CEA : tBA: PEGDMA ]). After the standard polymerization and heat treatment, the samples were washed with Sparkleen and DI water. SMPs were sized t o 5X12mm dimensions and their edges were sanded with 600 grit sand paper. Finally, samples were washed with DI water several times and dried at 90 C for one hour. SMP samples were incubated in 1mg/ ml of BSA (Sigma) for 3 hours at 37 C in 1.5mL eppendorf tubes. Acetate buffer (40% of 0.1M acetic acid + 60% 0.1M sodium ace tate) was used at a pH of 4.5. The surface to volume ratio was 15mm 2 / ml. Three experimental repeats were performed per condition. The BCA assay was run on all samples a fter the free supernatant was collected and the solution depletion method was used for quantification of protein bound. 4.2 3 Pos itive Control Optimization of P olyst yrene M icrospheres 4.2.3.1 General procedure Polystyrene microspheres were purchased (Catalog code DS03V, 0.51um). The polystyrene microspheres were initially optimized for the appropriate quantity to surfactant ratio with resp ect to adsorption. Afterwards adsorption was tested against protein concentration, buffer solution, reproducibility and surfactant less solution. All the experimental groups remained the same from the previous binding experiments: protein only, media only (no microspheres added), microspheres incubated in media (no protein) and micros phe res incubated with protein. The groups were performed in triplicate. The samples were incubated in 120ug/ ml for 3 hours at 37 C unless indicated otherwise. After the incubation, the microspheres were spun at 9300 G for 15minutes at 10 C The supernatant wa s carefully collected in a separate eppendorf tube and spun again at 9300G for 15min at 10 C The supernatant was collecte d and the BCA assay was run. The solution depletion method was used for quantification.

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27 4.2.3.2 Surface Area and Surfactant O ptimiz ation M icrospheres were diluted 1:10, 1:100 and 1:1000 in acetate buffer (pH 4.45, Ionic strength 0.01M, reagent grade materials used with filtered DI H20) and incubated in BSA with a final concentration of 120ug/ ml for 3 hour s at 37 C *First 1:10 dilution was made by adding 300ul of microspheres into 2,700ul of acetate buffer 4.2.3.3 Total Exclusion of S urfactants 40ul of microspheres were placed in a centrifuge tube and diluted with acetate buffer. The tube was vortexed to wash the m icrospheres and spun at 14,000 g for 5 minutes at RT. The supernatant was discarded and one more wash was performed on the spheres. Next, the microspheres were diluted 1:100 in acetate buffer (pH 4.45, Ionic strength 0.01M, reagent grade materials used wit h filtered DI H20) and incubated in BSA with a final concentration of 120ug/ ml for 3 hour s at 37 C 4.2.3.4 BSA Concentration O ptimization The microspheres were diluted 1:100 in acetate buffer ( pH 4.45, Ionic strength 0.01M, reagent grade materials used w ith nano pure H20) and incubated in BSA with a final concentration of 120ug/ ml or 40ug/ ml for 3 hour s at 37 C 4.2.3.5 Buffer Solution O ptimization The microspheres were diluted 1:100 in either acetate buffer made from nano pure water or DI water p assed through a 0.2um filter. The diluted spheres were incubated in BSA with a final volume of 120ug/ ml for 3 hour s at 37 C 4.2.3.6 Reproducibility T esting The reproducibility of adsorption with polystyrene microspheres was tested with pipetting small versus large quantities of the microspheres when the micros phere dilutions were prepared. The microspheres were diluted 1:100 by two 1:10 serial dilutions. The first 1:10 dilution determined the error as the second dilution stayed the same for both conditions. of microspheres into 360ul of acetate buffer (pH 4.45, Ionic strength 0.01M, reagent grade materials used s prepared by adding 300ul into

PAGE 43

28 2700ul of acetate buffer for its first 1:10 dilution. The second dilution remained the same for both conditions; a 1:10 was performed by adding 300ul of the already prepared 1:10 dilution into 2700ul of acetate buffer. The f inal diluted microspheres (1:100) were incubated in BSA with a final concentration of 120ug/ ml for 3 hour s at 37 C 4.2.3.7 BSA Alexa Fluor 488 Polystyrene microspheres were diluted 1:100 in acetate buffer (pH 4.45, Ionic strength 0.01M, reagent grade materials used with filtered DI H20) and incubated in BSA alexa fluor 488 (Invitrogen A13100) at a final concentration of 120ug/ ml for 3 hours at 37 C 4.2 4 Positive Control Test Polystyrene Microspheres against Past E xperiments 4.2.4.1 General P rocedure The polystyrene microspheres we re treated the same as before. The microspheres were diluted 1:100 in the appropriate buffer specified and incubated with the study protein. The microspheres were spun at 9300 G for 15minutes at 10 C The supernatan t was carefully collected in a separate eppendorf tube and spun again at 9300G for 15min at 10 C The supernatant was colle cted and the BCA assay was run. The solution depletion method was used for quantification. 4.2.4.2 Experiments 1 7 Poly L Arginine Polystyrene microspheres were diluted 1:100 in PBS and incubated in 44ug/ ml of poly L arginine for 30 minutes, 1 hour, 2 hours and 3 hours at 37 C 4.2.4.3 Experiments 8 11 Eosinophil Lysate Polystyrene microspheres were diluted 1:100 in RPMI with 8% F BS and incubated in 500k of EO lysate (lysate was suspended in 0.25M sodium acetate buffer + 10% PI) for 1 hour at 37 C 4.2.4.4 Experiment 12 BSA and Ionic strength Polystyrene microspheres were diluted 1:100 in either high ionic strength acetate buffer (0.1M) or low ionic st rength acetate buffer (0.01M). The spheres were incubated in BSA at a final concentration of

PAGE 44

29 1mg/ ml for the high ionic strength condition and 120ug/ ml for the low ionic strength condition. The samples were incubated for 3 hours at 37 C 4.2 5 SEM Adsorption SEM polymers were synthesized by free radical polymerization using 0.4 wt% of DMPA photo initiator Mixtures of SEM monomer, the PEGDMA crosslinker and tBA monomer were injected between the glass mold mentioned previously with the exception of a 1.5mm spacer Polymerization was performed under a UV lamp source with an intensity of ~11mW/cm 2 for 30 minutes After polymerization, the polymer coupons were remov ed from their cast and were heat treated at 90 C for 1 hour to evaporate unreacted monomers The samples were sized to 5x19mm 2 and their edges sanded with 600 grit sand paper to even out texture introduced from sizing Polystyrene sheets (McMaster Carr) an d nylon samples were sized and sanded in the same manner for a comparative analysis against our SMPs ; Afterwards, all adsorbents were methodically washed with DI water. SMP samples were incubated in 120ug/ ml of BSA for 3 hours. Acetate buffer (pH 4.45, Ionic strength 0.01M and reagent grade materials used with filtered DI H20) was used as the media with 0.0048%SDS and 0.0002% NAN3 The samples had a surface to volume ratio of 262mm 2 / ml. The BCA assay was run on all samples after the free supernatant was collected and the solution depletion method was used for quantification of protein bound.

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30 5. R esults and Discussion 5.1 CEA Characterization 5.1.1 Contact Angle and Surface E nergy Water contact angle measurements in air were performed to test the incorporation of CEA, a hydrophilic monomer, onto the polymeric surface of the SMP Results show decreasing hydrophobicity with increasing CEA wt% In contrast, Diiodomethane, a nonpolar reagent, was used to m easure hydrophobicit y at the SMP surfaces Figure 8 shows increasing hydrophobicity with increasing CEA percentages, an opposite trend of what was shown with water Because surfaces are highly reactive, they tend to be labile, which may begin to explain th e adverse trend The surfaces may have adapted to their environment to stay at their most thermodynamically stable state. Surface free energy was calculated from Fowkes equation found in the methodology section By partitioning the components of interacti on forces between the solid and measuring liquid, both dispersive and polar interactions were derived although they are not completely divorced entities 20 The surface free energy results disclose the increase in both dispersive and polar f orces with increasing CEA wt%. These increases are advantageous to protein adsorption as electrostatic and hydrophobic domains are the dominating interactions.

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31 Figure 8 Contact A ngle and Surface Energy M easurements of CEA SMPs Water contact angles show decreasing hydrophobicity at the polymeric surfaces with increasing incorporation of the hydrophilic monomer, CEA. Table 2 Contact A ngle and Surface Energy Measurements: A verage of Ten T rials Sample Code 0% CEA 89.2 5.3 59.11 4.6 29.1 2.7 31.8 5% CEA 81.57 4.2 49.52 6.9 34.5 4.0 38.5 10% CEA 74.79 7.1 43.8 6.8 37.6 5.9 43.5 15% CEA 61.84 3.9 15.67 4.0 48.9 8.6 57.5 5.1.2 pH Testing physiological pH CEA SMPs were also studied at pH 4.5 to investigate hydrogen bond influences to protein adsorption because hydrogen bonds are another major interaction force driving adsorption 7 To ensure hydrogen atoms were not disassociating from carboxylic acid functional groups, and were available on the surface of the CEA SMPs, the pH was monitored over the course of 4 hours The decline of the 0 20 40 60 80 100 0% CEA 5% CEA 10% CEA 15% CEA Contact Angle ( ) Carboxyethyl Acrylate Influence on Contact Angle H2O Dioodomethane 0 10 20 30 40 50 60 0% CEA 5% CEA 10% CEA 15% CEA Surface Energy (mJ/m 2 ) Surface Free Energy Dispersive Polar

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32 Results show very little variance from the initial pH for all CEA SMP polymers Acetate buffer was used as a control to gauge general fl uctuations that may occur without the polymer influence The acetate buffer incubated with the polymer maintains a stable pH relative to that of the acetate buffer alone These results validate that the hydrogen atom does not disassociate from the carboxyl ic acid group, rendering it free for hydrogen bonding during adsorption testing.

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33 Figure 9 pH T esting of CEA SMP s The pH tests were carried out to measure the disassociation of hydrogen atoms from carboxylic acid functional groups on the surface of CEA SMPs Results show the preservation of the hydrogen atom onto the polymer, thus ability to form hydrogen bonds.

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34 5.1.3 Dynamic Mechanical Analysi s CEA SMPs were tested with DMA to show preservation of the shape memory effect after CEA incorporation All the polymers still retain their shape memory effect evidenced by the enormous temperature dependence illustrated by the 2 3 orders of magnitude drop in storage modulus at its glass transition temperature This indicates the recovery ability of the SMP The results display a 2 3 degree increase in the glass transition temperature per 5% CEA incorporation The slope of the storage modulus and width of the tan delta curve represents similar shape memory recovery to : PEGDMA These small deviances from the base polymer are insignificant and could still provide proper mechanical properties for packing the SMP into a small capsule and recover large deformations. Figure 10 DMA R esults on CEA SMPs Shape memory effect is preserved by CEA incorporation. 5.1.4 Fourier Transform Infrared Spectroscopy FTIR spectra of the CEA SMPs show the disappearance of the alkene peak at 1680 1610cm 1 This validates consumption of reactive alkene groups during free radical polymerization and concurrently, convergence to alkanes through the 2850 2970cm 1 peak

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35 Figure 11 FTIR on CEA SMPs The reactive alkene groups were consumed during polymerization of CEA SMPs 5.2 SEM SMP Characterization 5.2.1 Contact A ngle and Surface E nergy Water contact angle measurements in air display slight increases in hydrophobicity with 1% and 2.5% SEM incorporation Due to the low density of polymers, the flip flop of molecules on the surface is not uncommon 4 Atmospheric exposure and consequently adaptation, can lead to reversible changes on the surface to reduce high energy states As such, the O H of SEM could be e mbedded in the bulk of the polymer when exposed to air 0.5% SEM has a similar hydrophobicity to 0% SEM due to the low number density of functional groups introduced Both water and diiodomethane were used as measuring fluids to departmentalize the interfa cial interaction forces into dispersion or polar components From the contact angles alone, diiodomethane reveals the materials higher interactions with dispersive forces translating to their hydrophobic nature Nylon has substantial polar and dispersive f orces, which may be an artifact from poor manufacturing and thus surface impurities and imperfections that easily sway the results The hydrophobicity of a material extensively influences protein adsorption as it allows water to organize loosely on the sur face, granting a more energetically favorable displacement of water upon protein adsorption A hydrophilic surface binds water tightly and would require more energy to displace water for protein 5 From this, the SEM polymers would encourage protein adsorpt ion but because there are many other influential factors, contact angle measurements alone do not directly correlate to binding efficacy.

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36 Figure 12 Contact A ngle and Surface Energy with SEM SMPs Contact angle measurements of SEM SMPs do not display SEM functional groups at the surface Small percentages of SEM were incorporated and may fall outside of the sensitivity of contact angle measurements This method may not display detailed chemical constituents. 0 15 30 45 60 75 90 105 PS Nylon 0% SMP 0.5% SMP 1% SMP 2.5% SMP Contact Angle ( ) Contact Angle Measurements Batch1 DH20 Diiodomethane 0 15 30 45 60 75 90 105 PS Nylon 0% SMP 0.5% SMP 1% SMP 2.5% SMP Contact Angle ( ) Contact Angle Measurements Batch2 DH20 Diiodomethane 0 5 10 15 20 25 PS Nylon 0% SEM 0.5% SEM 1% SEM 2.5% SEM Surface Energy (mJ/m^2) Surface Energy. Batch 1 Dispersive Forces Polar Forces 0 5 10 15 20 25 PS Nylon 0% SEM 0.5% SEM 1% SEM 2.5% SEM Surface Energy (mJ/m^2) Surface Energy. Batch 2 Dispersive Forces Polar Forces

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37 Table 3 Contact A ngle and Surface E nergy Measurements: Average of Ten T rials Sample Code. Batch 1 Polystyrene 91.2 3.0 42.4 3.3 18.8 0.4 19.2 Nylon 57.3 4.4 46.2 2.7 19.6 11.8 31.4 0% SEM 74.5 3.5 53.4 7.0 17.2 5.4 22.6 0.5% SEM 73.6 4.6 58.3 4.2 16.2 6.3 22.5 1% SEM 77.8 2.5 55 3.6 16.9 4.3 21.2 2.5% SEM 85.5 2.8 52.3 7.3 17.4 1.8 19.2 Sample Code. Batch 2 Polystyrene 91.2 3.0 42.4 3.3 18.8 0.4 19.2 Nylon 57.3 4.4 46.2 2.7 19.6 11.8 31.4 0% SEM 82.4 2.1 61.7 5.1 15.6 3.4 18.9 0.5% SEM 79.2 3.3 60.2 5.3 15.8 4.3 20.2 1% SEM 83.1 1.9 58.8 2.6 16.1 2.9 19.0 2.5% SEM 83.9 3.8 60.8 2.6 15.7 2.8 18.6 Surface layers have more surface free energy than their bulk because their valence electrons are not shared by their neighboring atoms 1 As a result, surfaces are reactive and exist in an energetically unfavorable state In this way, surface free energy can represent the affinity of the surfaces for adsorption Fowkes partitioned the surface free energy into two components, dispersive and polar forces From tab le 3 batch 1 shows increasing dispersive or nonpolar surface free energy in the polymers with SEM incorporation However, 2.5% SEM in Batch 2 shows a decrease in dispersive surface free energy relative to the other SEM polymers Of the total surface free energies in the SEM polymers, the trend from greatest to lowest surface energy is 0.5% SEM< 1% SEM< 2.5% SEM Since the dispersive forces are pro adsorption, these may be the ones to consider for protein adsorption Strangely, n ylon has more dispersive and polar forces than all other materials Because non uniform surface heterogeneity and surface mobility can alter contact angle measurements representing the material, surface energies of nylon and between SEM batches are not representative of detailed chem ical compositions 1

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38 5.2.2 Reproducibility Between batches, the SMPs vary from approximately 0.5 8.5 degrees and can subsequently alter the trends seen within the SEM samples The variability can be attributed to topographical imperfections on the surface, non uniform chemical heterogeneity and/or surface mobility 1, 7 Performing more measurements on a greater surface area may contribute to lowering these differences. 5.2.3 Water Equivalency Test As evidenced with water equi valency tests, shown in figure 13 SEM was incorporated into the polymer, as the hydrophilicity increased with increasing wt % of SEM Nylon did not equilibrate at 24 hour s suggesting it could be more hydrophilic than the SEM sample s Due to its high physical and/or chemical crosslink density, diffusion of water into the polymer system is much slower 8 Since we performed a 3 hour time point for the binding studies, the trends before the 24 hour time point are more relevant. Figure 13 Water Equivalency T est with SEM SMPs Bulk absorption validates that the SEM monomer was incorporated into the SMP. 0 2 4 6 8 10 12 0 5 10 15 20 25 30 % Water Uptake Time (hrs) Water Equivilency Test. Batch 1 Nylon Polystyrene 0% SMP 0.5% SMP 1% SMP 2.5% SMP 0 2 4 6 8 10 12 0 5 10 15 20 25 30 % Water Uptake Time (hrs) Water Equivilency Test. Batch 2 Nylon Polystyrene 0% SMP 0.5% SMP 1% SMP 2.5% SMP

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39 Table 4 Water Equivalency Test: Average of 2 S amples Sample Code. Batch 1 % Water Uptake (12 HR) % Water Uptake (24 HR) Polystyrene 0.1 0.2 0.1 0.2 Nylon 3.3 0.0 5.1 0.0 0% SMP 1.4 0.1 1.4 0.0 0.5% SMP 4.8 0.0 5.3 0.0 1% SMP 7.8 0.2 8.2 0.3 2.5% SMP 10.6 0.5 10.1 0.6 Sample Code. Batch 2 % Water Uptake (12 HR) % Water Uptake (24 HR) Polystyrene 0.1 0.2 0.1 0.2 Nylon 3.3 0.0 5.1 0.0 0% SMP 1.3 0.1 1.4 0.2 0.5% SMP 4.2 0.2 4.7 0.0 1% SMP 7.4 0.0 7.8 0.0 2.5% SMP 11.1 0.2 11.0 0.1 5.2.4 Reproducibility of Water Equivalency Test Both batches f ollow the same trends and have percent differences less than 1 2.5% SEM consistently shows more variance between batch 1 and 2 in the water equivalency and contact angle tests The monomer mixture may have small difference s of SEM content with an error in the hu ndredths or thousandths place. 5.2.5 Dynamic Mechanical Analysis 5.2.5.1 Shape Memory E ffect Figure 14 shows the temperature dependence of the polymers by a 1 2 orders of magnitude decrease in storage modulus at their gl ass transition temperatures This transition or switching effect, allows the recovery of the polymer once it has been stored in its tempor ary state The elasticity or storage modulus gives the polymer its memory and allows it to return to its original shape 2 Above the Tg, about 60 100 C the SEM polymers should experience immediate elasticity as all the polymers are still in their rubbery phase The plateau of the storage modulus shown above the Tg can indicate physical cross linking or increased crystalline formations from secondary intermolecular interactions between chains 1, 2, 3 Since SEM introduces polarity to the SMP, more secondary intermolecular interactions between chains can take place in addition to chain realignment for crystalline/physical cross linkage. Incorporation of SEM shows

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40 kinetically different transitions or switching rates as their slopes are moderate compared to 0% S EM Thus, the ability of the former to recover to its memorized shape is slower Additionally, the weak mechanical properties of the SEM polymers may hinder specific deformations or programming to its temporary shape such that manufacturing processes would need to be carefully planned out. 5.2.5.2 Storage M odulus SEM incorporation leads to a reduced storage modulus below Tg Because chemical constituents have the largest effect on altering thermal mechanical properties, it is reason to believe the C O bon ds from SEM allow for increased chain/bond flexibility below the Tg 1 Tert Butyl Acrylate (tBA) is also displaced by the weight percent of SEM during monomer mixture preparations, contributing to lower Tg/storage modulus values. Less free volume from the b ulky tert Butyl groups of tBA restrict large molecular movements and as such, more energy requirements are needed for thermo mechanical transitions. 0.5 and 2.5% SEM have similar glass transition temperature slopes, regressing more gradually from their g lassy to rubbery phase relative to 0% SEM while 1% SEM has the slowest transition phase The storage modulus of 1% SEM is the highest of the SEM incorporated polymers, theoretically possessing better elastic properties for shape recovery 0.5 and 2.5% exhibit similar storage moduli and switching stages but in batch two, 2.5% SEM possesses better elastic recovery than 0.5% This may be a reproducibility error and should be repeated The trends within the SEM samples are less evident, but may be con tributed to the polymerization process itself as molecular weight, crystallinity and cross link density affect the storage modulus and Tg values as well as chemical composition 1, 3 5.2.5.3 Tan Delta P eak SEM incorporation into the SMP causes decreased crosslink density, increased crystallinity as seen by the lower amplitude, and a left shift of the tan delta peak relative to the 0% SEM polymer Thus, SEM polymers have less mechanical strength or damping abilities that may permit low impact breaks within the polymer system Additionally, the broad peaks of the SEM polymers dictate slower glass transition phases, in tune with the storage moduli slopes Two transitions are seen in 0.5% SEM that may be a caustic response from the annealing of polymeric chain s through increased molecular movement after its first glass transition phase The additional secondary intermolecular interactions yield crystallinity above

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41 the first glass transition temperature but can slip as the temperature increases further illustrat ing the second glass transition temperature 1 2.5% SEM has the greatest cross linking density and amorphous content compared to the other SEM incorporated polymers and thus has better damping abilities at its glass transition temperature The tan delta pea k of 1% SEM may indicate a more uniform increase in molecular weight during its polymerization process as it transitions slowly from its glassy to rubbery state with a tan delta peak spanning the largest temperature range. Figure 14 DMA R esults on SEM SMPs DMA storage modulus and tan delta graphs of SMPs with 0, 0.5, 1 and 2.5 wt% incorpo ration of SEM. The SEM samples have depreciated mechanical and shape memory properties.

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42 0 500 1000 1500 2000 2500 0% SEM 0.5% SEM 1% SEM 2.5% SEM Glassy Modulus (MPa) Glassy Modulus Batch 1 Batch 2 0 0.5 1 1.5 2 0% SEM 0.5% SEM 1% SEM 2.5% SEM Tan Delta Tan Delta Peak Batch 1 Batch 2 0 10 20 30 40 50 60 0% SEM 0.5% SEM 1% SEM 2.5% SEM Glass Transition Temperature (C ) Glass Transition Temperature Batch 1 Batch 2 0 1 2 3 4 5 6 7 0% SEM 0.5% SEM 1% SEM 2.5% SEM Rubbery Modulus (MPa) Rubbery Modulus Batch 1 Batch 2 Table 5 Thermal Mechanical Properties of SEM SMP s: Average of Two T rials Sample Code. Batch 1 Glassy Modulus (Mpa) Rubbery Modulus (Mpa) Tan Delta Peak Tg (C ) 0% SEM 1551.45 473.41 4.12 0.08 1.75 0.00 51.87 1.21 0.5% SEM 850.56 31.28 5.03 0.17 0.52 0.00 29.63 1.02 1% SEM 1214.05 264.81 5.27 0.59 0.52 0.02 40.6 0.59 2.5% SEM 650.33 9.61 4.47 0.08 0.78 0.16 32.39 5.42 Sample Code. Batch 2 Glassy Modulus (Mpa) Rubbery Modulus (Mpa) Tan Delta Peak Tg (C ) 0% SEM 1817.20 195.73 4.35 0.07 1.72 0.03 52.33 2.35 0.5% SEM 718.41 41.21 4.81 0.08 0.52 0.00 30.28 0.26 1% SEM 1212.45 71.63 5.32 0.10 0.48 0.01 42.29 0.42 2.5% SEM 1015.69 254.29 5.15 0.80 0.74 0.01 34.01 1.11 Figure 15 Thermomechanical Reproducibility A nalysis The stiffness between batches is the most var iable but the other properties (Tg and tan delta peak) have good reproducibility.

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43 5.2.5.4 Reproducibility of Thermomechanical P roperties Figure 15 illustrates reproducibility of thermo mechanical properties between batches The glassy modulus is most prone to fluctuations between batches Literature suggests these variations may be due to the delicate process of polymerization, including pre and post processing Variables such as aging, atmospheric exposure and post heat treatments after polymerization can contribute to molecular rearrangements and alter mechanical properties 1, 3 T he tan delta peak and Tg between both batches have similar trends with relatively low standard deviations However, the rubbery moduli of 2.5% SEM polymers potentially elicit different trends of stiffness in account of the standard deviations Since bindin g occurred at this rubbery state and stiffness is an influential factor in protein adsorption, this small trend change could directly affect binding abilities and could start to explain the variable binding trend between batches for 2.5% SEM If heat pocke ts were introduced in the polymer coupon during the polymerization process by the UV source, non uniform cross linkage, molecular rearrangements and stresses could have occurred in the 2.5% samples leading to different thermo mechanical properties Note th at the coupons used for DMA analysis were the same coupons used to prepare the samples for the binding experiment. An important distinction in the batches is the modulus switch between the 2.5% SEM and 0.5% SEM sample in batch 2 In batch one, the 2.5% an d 0.5% SEM samples are similar but the glassy and rubbery modulus of 2.5% is slightly lower than 0.5% SEM However, in batch 2, the glassy moduli and glass transition temperature phase of 2.5% SEM is higher than 0.5% Since stiffness could play a role in a dsorption, this may be a reason in a drop in adsorption at 2.5% SEM in batch 1. 5.2.6 Fourier Transform Infrared Spectroscopy Fourier Transform Infrared Spectroscopy was utilized to confirm complete polymerization The SEM polymers show the disappearanc e of a peak near 1610 1680cm 1 the alkene range. Additionally a large peak is shown in the 2850 2970cm 1 range, depicting convergence of alkene bonds to alkane bonds.

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44 Figure 16 FTIR on SEM SMPs The reactive alkene groups during SEM polymerization were consumed.

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45 5.3 Adsorption Testing 5.3.1 Experiment 1. MA Figure 17 shows the adsorption ability of MA at 5 and 15wt % compared to the non funct ionalized base polymer (0%) to p oly L amino acids poly L a rginine was used as an EDGP analog with a PI of 10.76 and poly L g lutamic acid was introduced as a negative control with a PI of approximately 3 Significant binding onto the polymeric surfaces did not occur Although the 15% MA SMP condition shows adsorption of poly L glutamic acid during the 2 hour time point, its binding is variable One explanation of why poly L glutamic acid may bind to the modified polymer is the presence of counter ions from PBS binding to the surface of the polymer first, en couraging poly L glutamic acid to bind secondary onto the new monolayer The large error bars and negative values indicate the levels of binding are beneath the sensitivity threshold of the assay 5 minute and 30minute incubations were inaccurate time poin ts as the post and pre incubations of SMP in protein solution were of the duration of the time point itself or exceeded beyond this point Since MA is a small monomer with its functional site near its vinyl group steric hindrance may attenuate binding In essence, because of the pure randomness and noise from the assay, a better monomer may contribute to higher levels of detectable binding coupled with an increase in surface area to provide more availa ble binding sites for binding.

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46 8 6 4 2 0 2 4 6 0 5 15 Protein Adsorbed (ug/m^2 Support) % Methacrylic Acid Poly L Amino Acid adsorbed. 30 min Incubation Poly L Arginine Poly L Glutamic Acid 10 5 0 5 10 15 0 5 15 Protein Adsorbed (ug/m 2 Support) % Methacrylic Acid Poly L Amino Acid adsorbed. 120 min Incubation Poly L Arginine Poly L Glutamic Acid Figure 17 Experiment 1 MA Time points of poly L a mino Acids adsorbed onto MA SMPs (Surface to volume ratio: 37.68mm 2 / ml poly L a rginine concentration: 44ug/ ml pH 7 and temperature: 37 C ). 5.3.2 Exp eriment 2 3. CEA Figure 18 shows Poly L amino acid adsor ption onto CEA SMP surfaces or n ylon Since the base material of the esophageal string test is made from nylon, it was introduced in the binding experiment for a comparative analysis against the modified SMP samples Experimen tal repeats were increased from 3 to 6 and the surface area to volume ratio was increased approximately 3 fold to decrease the error and to supply more binding support for detectable binding As such, the standard deviations and negative values still ex ist Two alternative approaches to have detectable binding are by: 1) picking a more sensitive detection method or 2) increasing the surface area further The data shows no appreciable amounts of binding with CEA incorporation Although the base formulatio n of the SMP (0%) mildly binds poly L arginine, the results of this are variable Nylon also does not show detectable amounts of binding suggesting the esophageal string test may work in capturing proteins through high specific surface area 25 20 15 10 5 0 5 0 5 15 Protein Adsorbed (ug/m 2 Support) % Methacrylic Acid Poly L Amino Acid adsorbed. 5 min Incubation Poly L Arginine Poly L Glutamic Acid

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47 40 30 20 10 0 10 20 0% CEA 5% CEA 15% CEA Nylon Protein Adsorbed (ug/m 2 Support) Amino Acid adsorbed. 1 HR Incubation Poly L Arginine Poly L Glutamic Acid Therefore, inc reasing the surface area further for all samples was employed in the next experiments to ensure enough support for detectable binding. Figure 18 Experim ent 2 3. CEA Time points of poly L a mi no a cids adsorbed onto CEA SMP s and/or n ylon ( s urface to volume ratio: 94.2mm 2 / ml poly L a rginine concentration: 44ug/ ml pH 7 and temperature: 37 C ). 5.3.3 Experiment 4 Surface A rea 132mm 2 / ml High specific surface area is important as it relates to the total loading capacity of the polymer for protein As such, figure 19 summarizes the trials of surface areas examined (some of which are outlined below) SMP particles were also created but exhibited no observable appreciation in poly L amino acid adsorption. 20 15 10 5 0 5 0 5 15 Protein Adsorbed (ug/m 2 Support) % 2 Carboxyethyl Acrylate Amino Acid adsorbed. 1 HR Incubation Poly L Arginine Poly L Glutamic Acid

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48 Figure 19 Surface Areas Implemented to Maximize Loading C apacity Figure 20 displays poly L arginine binding onto SMP (80:20 tBA : PEGDMA ) and nylon samples The surface area was increased to 2640mm 2 per condition from 188.4mm 2 per condition to absorb more protein onto the surface The surface to volume ratio only increased from 94.2mm 2 / ml to 132mm 2 / ml because more volume was needed to cover the larger samples Nonetheless, after increasing the surface area beyond previous exper iments, there are still n egative readings and large standard deviations The assay sensitivity threshold could be an avenue to consider but because the surface to volume ratio was not much more than the previous experiment, additional efforts were made to minimize the volume the adsorbents were incubated in and increase the surface area for the next experiments. Figure 20 Experiment 4. Surface A rea 132mm 2 / ml Poly L ami no acids adsorbed onto SMP and nylon (s urface to volume ratio: 132mm 2 / ml poly L a rginine concentration: 44ug/ ml pH 7 and temperature: 37 C ). 0.5 0 0.5 1 1.5 2 SMP Nylon Protein Adsorbed (ug/m 2 Support) Poly L Amino Acid adsorbed. 1 HR Incubation Poly L Arginine Poly L Glutamic Acid

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49 5.3.4 Experiment 5. Surface Area with SMP P articles Polymer particles were created to increase the loading capacity for the protein far beyond the surface areas introduced in the previous experiments High surface areas are usually necessary for implementing adsorption M any studies use particulate or beaded adsorbents for large surface ar ea exposure, f or instance, Sepharose 4B ( agarose beads) used for adsorption in chromatographic methods have a surface area of 8m 2 /ml 13 The largest surface to volume ratio achieved thus far is 0.132m 2 / ml reaching nowhere near this level. To a ttain a quantifiable amount of protein binding o nto our polymeric surface particulates may need to be created Even nylon has not adsorbed appreciable amounts of protein, and since this is the material the esophageal string test is made from, known to cap ture protein, the small surface area may be the underlying issue Therefore creating particles was the new concentration A dremel was used to create the adsorbent particles but in the process, various particle sizes were generated Figure 21 and 22 displa y the large range of the sizes created (.01 1.2mm SMP and 0.1 13.857mm nylon) Because n ylon sample sizes were significantly larger than the SMPs, they could not be used for comparison during the binding experiments These SMP particles were washed several time s and centrifuged to remove ex tremely small particles that could be difficult to remove after the binding procedure Centrifugation should pool the larger particles to the bottom, and the residing supernatant should contain the smaller sized particles This supernatant was decanted to remove these small particles After the particles were correctly processed and dried, the adsorption experiment was carried out and the results are displayed in figure 23.

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50 Figure 21 SMP Partic les 80:20 ( tBA: PEGDMA ) SMP p articles imaged through an optical microscope Large ranges exist; some particles are below the detection limit of the scope The sizes ranged from 0.01 1.2mm Particles smaller than 0.01mm could not be measured using this method.

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51 Figure 22 Nylon Particles Nylon particles imaged through an optical microscope Nylon particles are larger than SMP; therefore, the two cannot be compared in further polyme r particle adsorption studies. The sizes ranged from 0.1 13.857mm. The exact surface area per condition is unknown due to the large variation of sizes created during the generation of the SMP samples As such, the weight of the particles was used to control for a fixed amount per cond ition The most saturated samples with the polymer particles were at 0.06g and a condition at 0.04g was also tested Poly L amino acids were the study protein at a pH of 7 in PBS buffer The results show that even though t he surface to volume ratio is maximal in this experiment relative to the others appreciable amounts of binding did not occur. The problems associated with creating SMP particles is the high variability of particulate size, the uncertainty of false negative results, the aggregation of the hydrophobic particles together in solution and the high buoyancy of the particles It can be speculated that the large error bars resulted from the large range of sizes produced w hen creating the SMP particles Initially, the particles were passed th rough a 230um mesh sieve to try to eliminate some of this variation but most of the sample sizes were so small that >80% of the particulate passed th ro ugh Thus, centrifugation was implemented to remove the particles but further optimization to create tigh ter particle sizes should be implemented There is uncertainty that all the small particles were in fact removed by this method This can lead to false negative results if the particles have bound protein on its surface and the

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52 10 0 10 20 30 0.04 g SMP 0.06g SMP Protein Adsorbed (ug/g Support) Amino Acid adsorbed. 1 HR Incubation Poly L Arginine Poly L Glutamic Acid solution depletion method is performed In this method, we only want to measure free bulk protein that was not bound to the polymer surface and total protein The difference of the two values yield the protein bound to the polymer surface If the polymer particle with protein bound is not properly removed from the free protein being measured, there is a large possibility of a false negative result The shape of the polymers also added variability to each condition Another concern is full exposure of the particles to the protein solu tion The purpose of creating SMP particles was to increase surface area, but because of the hydrophobicity of the polymers, they clumped together to reach a more thermodynamically favorable state during aqueous incubation The SMP particles also have a de nsity similar to water such that they tend to float in PBS Together, these findings show that the total surface areas of all the particles are not available for the protein to adsorb defeating the overall purpose in creating these particles One solution to this problem is forcing the solution to pass through all the particles This can be done by a rotary device or through vigor ous shaking in a closed vial bringing us to experiment 6 where this was carried out. Figure 23 Experiment 5 Surface A rea with SMP P articles Poly L amino acids adsorbed onto 0.04g and 0.06g of SMP (p oly L a rginine concentration: 44ug/ ml pH 7 and temperature: 37 C ). 5.3.5 Experiment 6. Surface Area with SMP P articl es Complete S ubmersion Figure 24 displays poly L amino acid adsorption onto SMP particles with an increase in surface to volume ratio The particles were incubated in a closed vial and were vigorously shaken throughout the incubation period to ensure full exposure of the polymeric pa rticles to the protein solution However, minimal adsorption took place with a high deviation for poly L arginine Because poly L glutamic acid has a negative value but a low standard deviation, this may be the limit of detection of the assay It is still not

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53 20 10 0 10 20 30 0.112 g SMP Protein Adsorbed (ug/g Support) Amino Acid adsorbed. 1 HR Incubation Poly L Arginine Poly L Glutamic Acid clear if the particles were removed before the BCA assay was performed which could interfere with the results as a false negative reading Therefore, we reverted back to creating square samples to increase the surface area. Figure 24 Experiment 6 S urface Area with SMP Particles Complete Submersion Poly L amino acid s adsorbed onto 0.122g of SMP (poly L a rginine concentration: 44ug/ ml pH 7 and temperature: 37 C ). 5.3.6 Experiment 7. Surface A rea 648mm 2 /ml Figure 25 displays protein adsorption after exposing 648mm 2 SMP surface area per 1mL of protein solution Poly L glutamic acid, the negative control binds to the polymeric surface better than poly L arginine Our aim is to specifically bind highly cationic proteins of EoE onto the SMP through anionic charged groups on the polymer surface Because the polymer used for this experiment was the base formulation with mostly hydrophobic bonds, it will bind protein more nonspecifically In addition glutamic acid was attac hed to sodium as purchased from Sigma the negative groups on the polymer surface negating its repulsion and serving as a negative control After performing several trials to increase the protein capacity of the polymer with little improvement, surface area considerations, although important, may not be the f actor causing minimal binding.

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54 0 1 2 SMP Nylon Protein Adsorbed (ug/m 2 Support) Poly L Amino Acid adsorbed. 1 HR Incubation Poly L Arginine Poly L Glutamic Acid Figure 25 Experiment 7 Surface A rea 648mm 2 / ml Poly L ami no acids adsorbed onto SMP and n ylon (Surface area to volume ratio: 648mm 2 / ml poly L a rginine concentration: 44ug/ ml pH 7 and temperature: 37 C ). After more consideration for maximizing surface to volume ratios, the best approach was submerging 5x12x 1mm SMP sample in 1mL volume, specifically in a 1.5mL eppendorf tube This combination allows the entire polymer surface to be submerged in solution and does not consume expensive processing time with creating numerous coupons The problem associated with incubating numerous SMP sample s in one condition is the uncertainty of a fully exposed surface The polymers lie on top of each other and can hide a large surface area One solid SMP support eliminates this issue and still exposes large surface area to the protein solution. Many stud ies refer to hydrophobic forces as the dominate interactions for binding proteins more firmly 5,7,8,12 Dehydration of the polymer and protein surfaces is a prerequisite for protein adsorption Water that resides on the surface of both the hydrophobic protein domains and hydrophobic polymer surfaces are highly ordered (loosely bound) and require less energy to displace than with hydrophilic surfaces where the water is tightly bound 12 In this way, hydrophobic domains increase polymer protein interactions and trend toward irreversible binding The hydrophobic forces were considered for the polymer surface but the protein needs c omplimentary hydrophobic domains for the interaction to take place Poly L arginine was chosen because of its net positive charge that resembles the unique cationic biomarkers of EoE However, the synthetic analog does not emulate physiological proteins; i t is just composed of one type of amino acid anionically charged polymer surface, it lacks a hydrophobic core to create long lasting, densely packed contacts Therefore, the stu dy protein was changed to native EDGPs for the next series of experiments The

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55 diversity of amino acids, particularly hydrophobic domains, and their increased interaction forces can facilitate irreversibly binding. 5.3.7 Experiment 8. Eosinophil Lysate Eosinophil cationic protein e os inophil derived neurotoxin and major basic rotein 1 were tested for We decided to optimize protein adsorption by using the base SMP formulation because of its abi lity to preferenc e cationic proteins because of its slight negative charge from the crosslinker, PEGDMA. The base SMP formulation also facilitate s hydrophobic interactions from tBA methyl groups (CH3). For interpretation of protein binding to our polymer s urface, amino acid analysis was performed for each protein P rotein pr operties listed below in table 6 can affect surface interactions during adsorption Since there are many protein adsorption factors it is hard to predict which ones are most influential in protein adsorption, thus 3 of the 4 proteins were quantified to determine binding For instance, Larger molecules tend to bind better because they have more sites of contact for the polymer surface 8 EC P, ED N and MBP1 have small molecular weights reducing the strength of the interaction to the polymeric surface ECP, EPX and MBP1 all share high cationic charges that creates a looser structural construct from the repulsion between adjacent charged residues Thus, the internal mobility needed upon adso rption and relaxation is greater for these proteins, enc ouraging protein adsorption EPX and MBP1 have the highest quantity of disulfide bonds that ultimately hold the protein in its tertiary structure and aid in its stability, a discouraging pr otein adsorption factor.

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56 0 20 40 60 80 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) ECP Adsorption Table 6 Protei n C haracterization Protein MW ( kDa ) PI % Hydrophobicity % Charged Sites # disulfide bonds ECP 18.39 11.4 43.16 20.63 9 EDN 18.35 8.9 41.62 15.53 9 EPX 81.04 10.8 43.24 24.2 17 MBP1 25.21 10.9 40.54 26.12 12 After performing adsorption testing, select EDGP binding onto the surface of the methanol treated SMP (80:20 tBA : PEGDMA ) was quantified. ECP binds both nylon and SMP polymers the best over EDN and MBP1. Two reason s why ECP may bind preferentially is its high isoelectric point and low disulfide bond count relative to the other proteins These factors place it in a looser construct with increased internal mobility To fully study this binding phenomenon, one variable should be eliminated that could cau se increased binding through pore size and not through chemical constituents on the surface The polymer was methanol treated for 48hours, which causes substantial swelling of the polymer This affect could stretch pore sizes beyond recovery through the de hydration step To eliminate pore size from interfering with binding, SMP samples were heat treated in lieu of methanol treatments to rid of u npolymerized monomers. Figure 26 Experiment 8 Eosinophil L ysate ECP Adsorption Eosinophil cationic protein (ECP) adsorption onto SMP methanol treated samples (Surface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 1).

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57 0 10 20 30 40 50 60 70 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) EDN Adsorption 285 300 315 330 345 360 375 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) MBP1 Adsorption Table 7 % Protein Bound Relative to T otal ECP Sample Code % Bound SMP + Lysate 63.51 20.35 Nylon + Lysate 50.23 11.85 Figure 27 Experiment 8 Eosinophil L ysate EDN Adsorption Eosinophil derived neurotoxin (EDN) adsorption onto SMP methanol treated samples (Surface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 1). Table 8 % Protein B ound Relative to T otal EDN Sample Code % Bound SMP + Lysate 2.89 0.04 Nylon + Lysate 2.89 0.02 Figure 28 Experiment 8 Eosinophil L ysate MBP1 Adsorption Major Basic Protein 1 (MBP1) adsorption onto SMP methanol treated samples (Surface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 1). Table 9 % Protein Bound Relative to T otal MBP1 Sample Code % Bound SMP + Lysate 1.91 0.07 Nylon + Lysate 1.96 0.12

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58 5.3.8 Experiment 9 10. Eosinophil Lysate After changing the post processing method of the SMP from methanol treatments to purely heat treatments, appreciable amounts of binding did not occur This suggests that if methanol did alter the increased surface area The adsorption mechanism is unknown but the results below imply it was not a function of purely non covalent binding of the equivalent surface area The % protein bound was made That said, after comparing nylon between experiments, it has variable binding which could be because of the adsorbent itself or reproducibility error of the assay. Nylon exhibited approximately 30% more ECP binding in experiment 9 than experiment 8 but dropped to nearly 58% comparing experiment 9 to 10 Nylon was purchased with surface scratches from the manufacturer, altering uni formity from sample to sample This alone could influence adsorption as the scratches could facilitate sites for physical entrapment of the protein and increased surface area EDN and MBP1 minimally bind to the polymer surface and do not need to be repeate d again To test the reproducibility of the ECP assay and if methanol treatments sincerely encourage binding, the experiment was performed once more with both heat treated metha nol treated and nylon samples.

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59 0 250 500 750 1000 1250 1500 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) ECP Adsorption 6.6 6.7 6.8 6.9 7 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) EDN Adsorption Figure 29 Experime nt 9 Eosinophil L ysate ECP Adsorption Eosinophil cationic protein (ECP) adsorption onto SMP heat treated samples (Surface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 1). Table 10 % Protein Bound Relative to T otal ECP Sample Code % Bound SMP + Lysate 8.61 1.92 Nylon + Lysate 86.21 8.49 Figure 30 Experiment 9 Eosinophil L ysate EDN Adsorption Eosinophil derived neurotoxin (EDN) adsorption onto SMP heat treated samples (surface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 1). Table 11 % Protein Bound Relative to T otal EDN Sample Code % Bound SMP + Lysate 0.43 0.01 Nylon + Lysate 0.43 0.00

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60 0 200 400 600 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) ECP Adsorption 285 300 315 330 345 360 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) MBP 1 Adsorption Figure 31 Experiment 9 Eosinophil L ysate. MBP1 Adsorption Major Basic Protein 1 (MBP1) adsorption onto SMP heat treated samples (s urface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 1) Table 12 % Protein Bound Relative to T otal MBP1 Sample Code % Bound SMP + Lysate 0.73 0.01 Nylon + Lysate 0.77 0.03 Figure 32 Experiment 10 Eosinophil L ysate MBP1 Adsorption Eosinophil cationic protein (ECP) adsorption onto SMP heat treated samples (s urface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 2). Table 13 % Protein Bound Relative to T otal ECP Sample Code % Bound SMP + Lysate 3.71 1.71 Nylon + Lysate 27.90 7.30

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61 5.5 6 6.5 7 7.5 SMP + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) EDN Adsorption Figure 33 Experiment 10 Eosinophil L ysate EDN Adsorption Eosinophil derived neurotoxin (EDN) adsorption onto SMP heat treated samples (s urface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 2). Table 14 % Protein Bound Relative to T otal EDN Sample Code % Bound SMP + Lysate 0.42 0.02 Nylon + Lysate 0.43 0.03 5.3.9 Experiment 11. Eosinophil Lysate Minimal ECP adsorption onto SMP was found for both heat treated and methanol treated samples ECP bound the n ylon samples at a higher degree but again, this could be because of slight imperfections that exist on the nylon surface The results for the methanol treated SMPs w ere not reproducible. This may be explained by delicate changes between the methanol treatments for the two experiments, as the smallest change in polymer processing could alter the surface severely Going back to the literature, adsorption is maximal if the pH of the solution is equal to or near the isoelectric point of the protein 5,7,8,12 This can be explained in a couple of different ways. The protein and polymeric surface should not be thought of in isolation rather protein protein interactions occur frequently in addition to polymer protein interactions. In our study, with a pH of 7, the eosinophil derived granule proteins are positively charged and can attribute lateral repulsion upon binding to adjacent b inding sites; the proteins may need to be uncharged to decrease protein protein interactions and promote polymer protein interactions In addition charged proteins take up more occupancy onto the polymer surface, reducing the amount of protein that can bi nd per meter squared of surface area. Studies have also shown that adsorbents introduced to multi component protein solutions tend to adsorb one protein or molecular species preferentially 13 This is dependent on the concentration and

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62 affinity of t he protein for the polymeric surface If the concentration of one protein is higher than the rest, it will have a higher probability of interacting with the surface and adsorbing over rare proteins in the mix Increased concentration also promotes stacking of proteins next to one another by decreasing the residency time of the protein onto the polymer surface, discouraging over relaxation states that could otherwise take up ample space 6 As such, the concentration of the study protein was increased to 1mg/ ml to decrease residency time on the polymer surface. For the affinity of a pr otein, if one protein has the correct surface amino acids that compliment the interaction forces produced by the polymer surface, that protein will bind preferentially. In the last set of experiments with the EO lysate, a pool of EO proteins were competing for the surface but the media itself was infused with 8% FBS that could have bound preferentially as well Thus, in the next experiment, only purified protein was introduced so we could study the mechanism involved in protein adsorption and control for co mpetition that may be taking place and altering adsorption of the EDGPs. The study protein was also changed to BSA The main justification is that BSA is frequently used as the study protein for a dsorptions studies. BSA is well characterized, has the high internal mobility, is a moderately sized protein that could bind irreversibly and is easily available in pure delipidated form. Below are its specific characteristics that aid in its pro adsorption characteristics. In correlation, the solution was switche d from PBS to acetate buffer with a pH of 4.5 to bring the net charge of BSA to zero.

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63 0 50 100 150 200 250 SMP+ Lysate SMP Methanol + Lysate Nylon + Lysate Protein Bound (ng/m 2 Support) ECP Adsorption Table 15 BSA Characterization Protein MW ( kDa ) PI % Hydrophobicity % Charged Sites # disulfide bonds BSA 69.29 4.7 42.83 33.27 35 Figure 34 Experiment 11 Eosinophil L ysate ECP Adsorption Eosinophil cationic protein (ECP) adsorption onto SMP heat treated samples (s urface area to volume ratio: 154mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and temperature: 37 C Donor 3). Table 16 % Protein Bound Relative to T otal ECP Sample Code % Bound SMP + Lysate 0.56 0.26 SMP Methanol + Lysate 2.04 1.41 Nylon + Lysate 27.00 2.29 5.3.10 Experi ment 12. CEA After incorporating COO functional groups to the polymeric surface, switching the study protein to BSA, increasing the concentration to 1mg/ ml and adjusting the pH of the solution buffer to the pI of the study protein, no protein was found bound to the CEA SMP s shown in figure 3 5 One key consideration of why the no binding occurred onto the CEA polymers is the high ionic strength of the solution The ionic strength of the acetate buffer used was at 0.1M This can have an enormous degree of control over protein adsorption as the counter ions in solution weaken electrostatic interactions both on the protein and polymer surfaces by competing with the same binding sites Since these molecules are small, they are transported to these sites on the protein and polymer faster an d can con sume critical sites ultimately influencing adsorption and adsorption kenetics 7 Therefore, for future experiments, the ionic strength was adjusted to 0.01M to correct for any competing counterions

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64 Since the mechanism of adsorption is complex, and there are a multitude of factors that could ultimately influence this mechanism, a positive control was needed to test if the correct experimental parameters were set in place for pro ad sorption Otherwise, the it erations of system adjustments coul d go beyond the period of this project. Figure 35 Experiment 12 CEA Bovine serum a lbumin (BSA) adsorption onto SMP samples (s urface area to volume ratio: 154mm 2 / ml conc. : 1mg/ ml pH 4.5 and temperature: 37 C ). 5.4 Posi tive Control Optimization of Polystyrene M icrospheres Polystyrene microspheres are an established adsorbent utilized in numerous studies to investigate protein adsorption 7 As such, 0.51um microspheres were purchased to validate experimental parameters set forth previously and serve as a positive control These spheres have a high specific surface area and are hy drophobic with a smooth surface to promote protein adsorption. 5.4.1 Surface Area and Surfactant O ptimization The surfactants in which the microspheres were suspended in were diluted several folds to find the best surface area to surfactant ratio for maximal adsorption. Results show 92.4% adsorption of BSA to polystyrene microspheres with a 1:100 dilution, the best choice for further adsorption studies with these spheres Though the 1:1000 dilutions yielded more protein bound per surface area, more error was associated with this condition. One reason for this may be because of the minimal amounts of SDS in solution SDS prevents the microspheres from clumping The hydrophobic end of the surfactant resides on the polymers surface while the flanking end is negatively charged creating a negatively charged polymeric surface The negative charges of the polymer particles cause repulsion from each other, thus controlling the 3 2 1 0 1 0% CEA 5% CEA 10% CEA 15% CEA Protein Adsorbed (ug/m 2 Support) BSA Adsorption

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65 10 0 10 20 30 40 Surfactant 1:10 Surfactant 1:100 Surfactant 1:1000 Protein Bound (ug/m 2 Support) Surfactant Influence on BSA Adsorption dispersion of the polymers in solution. The high standard deviation could be a result of non uniform clumping from sample to sample and thus more error in pipetting the spheres to the protein solution for the actual binding experiment Diluting the polymers ultimately leave more room on the polymer surface for protein adsorption but can also cause polymer polymer interactions as these spheres are free floating. A bsolute BSA adsorbed of the 1:10 and 1:1000 diluted microspheres amounted to only 0% and 3.06% The final SDS concentration of the 1:10 dilution was 0.048%, high enough to interfere with binding Because SDS is an anionic surfactant, it could occupy the binding sites of the po lymer or protein this illustrates that too much SDS is not good either for our purposes The 1:1000 dilutions reduce the surface area for protein to adsorb onto which may be the reason for such a low adsorption yield Here, only 5.57X10 8 microspheres were added per condition compared to the 1:100 dilutions where 5.5760X10 9 microspheres were added The 1:100 diluted microspheres seem to meet the perfec t balance between surface area and surfactant levels. Figure 36 Surface Area an d Surfactant O ptimization Optimal surface area to surfactant ratios for BSA adsorption onto polystyrene microspheres (s urface area to volume ratio 1:10: 185263mm 2 / ml s urface area to volume ratio 1:100:1852mm 2 / ml s urface area to volume ratio 1:1000:18.52mm 2 / ml BSA conc. : 120ug/ ml pH 4.5 and temperature: 37 C ).

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66 0 0.2 0.4 0.6 Washed polystrene microspheres Protein Bound (ug/m 2 Support) BSA Adsorbed to Washed Polystyrene Microspheres Table 17 % Protein Bound Relative to T otal BSA Sample Code % Bound % Bound Surfactant 1:10 10.23 0.25 % Bound Surfactant 1:100 92.40 1.47 % Bound Surfactant 1:1000 3.06 1.76 5.4.2 Total Exclusion of S urfactants To clarify if the same optimal surface area to volume ratio (1852mm 2 / ml ) without any interference of surfactants would bind even more, microspheres were washed once in acetate buffer to rid SDS from solution Interest ingly, adsorption decreased by approximately 85% This suggests a role surfactants may have in adsorption if the correct balance exists SDS, the anionic surfactant the microspheres were shipped in may have a role in attracting the BSA near the interfacial region of the polymer since the surfactants are utilized to charge the microspheres and prevent clumping Adversely, free SDS could weaken intramolecular forces of protein providing structural mob ility In any event, the microspheres were not washed but only diluted 1 :100 in future experiments. Figure 37 Total Exclusion of S urfactants Washed polystyrenes affect on BSA adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml BSA conc. : 120ug/ ml pH 4.5 and temperature: 37 C ). Table 18 % Protein Bound Relative to T otal BSA Sample Code % Bound Washed polystyrene beads 6.73 0.98

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67 0 1 2 3 4 5 High BSA Concentration (120ug/mL) Low BSA Concentration (40ug/mL) Protein Bound (ug/m 2 Support) Protein Concentration Influence on Protein Adsorption 5.4.3 BSA Concentration Optimization Next, the optimal concentration of BSA was tested One source discussed the optim al protein concentration range for adsorption studies to be 200 40ug/mL 12 Thus, the concentrations were chosen within this range As mentioned before, concentration can determine the residency time a protein spends on the polymer surface and as such, the degree to relaxation of the protein A more packed orientation could aid in more binding Results show higher binding per meter squared for high protein concentrations with ~52ug of protein bound where the low protein concentration had approximately 25ug bound From these results, the high protein concentration was used in the next experiments. It should be noted that the adsorption dropped in the 120ug/ ml BSA samples by nearly 35% relative to the surface area to surfactant experiment with the same experimental parameters The only minute alteration was the preparation of the acetate buffer wherein it was prepared from DI water passed through a 0.2um filter before, the acetate buffer was made from nano pure water in this experiment The next parameter of optimization tests these influences. Figure 38 BSA Concentration O ptimization Optimal bovine serum a lbumin (BSA) concentration for maximal adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml BSA conc. : 120ug/ ml pH 4.5 and temperature: 37 C ). Table 19 % Protein Bound Relative to T otal BSA Sample Code % Bound High Concentration 56.52 5.21 Low Concentration 63.47 1.98

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68 0 0.5 1 1.5 2 Nano Acetate Buffer Filtered Acetate Buffer Protein Bound (ug/m 2 Support) H20 Influence (in Solution) on Adsorption 5.4.4 Buffer Solution Optimization The buffer solution was tested to see if its water base composite effected adsorption Acetate buffer was made from nano pure water or filtered DI water Since the microspheres are highly reactive and are prone to adsorb nonspecifically to ions, organic species and microbes that could be contaminants of the water, the degree to this effect was examined The results show that the microspheres suspended in ac etate buffer made from filtered water does adsorb BSA better than the acetate buffer made from nano pure water Therefore, acetate made from filtered water was implemented in the future studies However, the degree to binding depreciated much further The variability amongst the positive control illustrates how sensitive adsorption is and the degree of detail that needs to be considered Numerous factors could have prevented maximal protein binding on either the solution, protein or polymer end For this re ason, the study was continued by examining one potential cause, pipetting error. Figure 39 Buffer Solution O ptimization Optimal purity for Bovine Serum Albumin (BSA) adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml BSA conc. : 120ug/ ml pH 4.5 and temperature: 37 C ). Table 20 % Protein Bound Relative to T otal BSA Sample Code % Bound Nano Acetate Buffer 8.67 2.31 Filtered Acetate Buffer 19.71 3.18

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69 5.5 6 6.5 7 7.5 Small Volumes Larger Volumes Protein Bound (ug/m 2 Support) BSA Adsorption onto Polystyrene Microspheres: Pipette Error Considerations 5.4.5 Reproducibility T esting The microspheres have a high initial concentration of 1.394X10 12 microspheres per ml. The total amount of microspheres after a 1:100 dilution, introduced to each experimental condition amounts to only 4ul of the undiluted stock Pipetting could contribute to enormous error if the spheres were clumped together or not vortexed properly to disperse the spheres and get a uniform amount each time The degree to pipetting error was measured t o see if this was problematic once methodical vortexing was controlled for The microspheres had an 8% difference of BSA adsorption when larger volumes were aspirated over smaller volumes Thus, larger sample dilutions were prepared for future experiments The exact reason why BSA adsorption decreased substantially previously is unknown, but meticulous precautions was taken to treat all the base parameters the same. Figure 40 Reproducibility T esting Reproducibility of Bovine Serum Albumin (BSA) adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml BSA conc. : 120ug/ ml pH 4.5 and temperature: 37 C ). Table 21 % Protein Bound Relative to T otal BSA Sample Code % Bound Small Volumes 80.47 0.51 Large Volumes 88.62 0.75 5.4.6 BSA Alexa Fluor 488 Polystyrene microspheres were optimized to bind abundant amounts of purified BSA in the previous experiments The spheres have an extremely high specific surface area and are hydrophobic to facilitate irreversible binding Since the surface area may still be an underlining issue for the SMP system, a more sensitive detection method was tested against the microspheres BSA conjugated to alexa f luor 488

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70 2 1.5 1 0.5 0 0.5 Polystyrene Micropheres Protein Bound (ug/m 2 Support) BSA Alexa Flour 488 Adsorption was purchased as the study protein with the ability to be quantified through its fluorescent tag up to nanogram levels The results show that the tagged surface interfered with adsorption significantly, as no protein was adsorbed onto the microspheres The modified surface of the protein may hide specific interacting amino acid residues that were before interacting with the polymeric surface The method was therefore not used in future SMP experiments. Figure 41 BSA Alexa Fluor 488 The affect of BSA 488 adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml BSA conc. : 120ug/ ml pH 4.5 and temperature: 37 C ). Table 22 % Protein Bound Relative to T otal BSA 488 Sample Code % Bound Polystyrene Microspheres 10.46 15.01 5.5 Positive Control Test Polystyrene Microspheres against Past E xperiments 5.5.1 Experiments 1 7 Poly L Arginine After the proper adsorption parameters were established to get ~90% binding, the positive control was used against previous experimental environments to justify if they were in fact, pro or anti adsorption systems The polystyrene beads were incubated in 44ug/ ml of poly L arginine in PBS buffer for 1 hour at 37 C Poly L arginine was proposed to discourage binding because of the lack of complimentary hydrophobic domains to facilitate irreversible binding However, the positive control unveiled prevalent amounts of binding to its surface Because the polystyrene spheres are a different polymeric system, adsorption mechanisms may not be completely translational This data shows that our polymer could have been the anti adsorption factor in the series of experiments with the amino acids.

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71 0 2 4 0.5hr 1hr 2hr 3hr Protein Bound (ug/m 2 Support) Poly L Arginine Bound Figure 42 Experiments 1 7. Poly L A rginine Poly L a rginine adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml poly L a rginine conc. : 44ug/ ml pH 7 and Temperature: 37 C ). Table 23 % P rotein Bound Relative to Total P oly L Arginine Sample Code % Bound 0.5 HR 91.79 3.84 1 HR 79.77 4.17 2 HR 96.07 1.82 3 HR 93.63 2.52 5.5.2 Experiments 8 11 Eosinophil Lysate The exa ct system from the previous EO l ysate experiments were tested with the polystyrene microspheres and the BCA assay was performed to quantify a total protein change The BCA absorbance values in figure 43, at 562nm show little difference s between each conditions, tions This is an indication that FBS saturates the solution and inhibits EDGP adsorption, by masking the binding sites on the polymer s urface before EDGPs can arrive at the polymer surface An additional consideration is that RPMI, the media, is flushed with amino acids, vitamins, glucose, inorganic salts and substances that could consume binding sites as well, or create a large backgroun d in colorimetric assays such that the protein signal is hidden. In this experiment, the Bio Rad protein assay was purposely chosen to have more leniencies with the buffer and to pick up more of a protein signal As a result, it un veiled the media complica tions.

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72 Sample No Dilution Absorbance (562nm ) 1: 10 Absorbance (562nm) 1: 50 Absorbance (562nm) 1: 100 Absorbance (562nm) 500k 2.115 1.854 1.04 0.796 2.12 1.776 0.984 0.703 2.152 1.661 0.989 0.752 500k+B 2.24 1.77 1.074 0.756 2.27 1.752 0.99 0.701 2.27 1.637 0.951 0.722 0 2.007 1.77 0.992 0.849 2.095 1.801 0.982 0.688 2.255 1.689 0.916 0.707 0+B 2.235 1.684 0.923 0.622 2.189 1.692 1.042 0.648 2.151 1.647 1.048 0.735 Figure 43 Experiments 8 11. Eosinophil L ysate Bio Rad protein assay results of EO Lysate adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml EO lysate conc. : 500k/ ml pH 7 and T emperature: 37 C ). 5.5.3 Experiment 12 BSA and Ionic strength Finally, polystyrene microspheres were incubated in low and high ionic strength solutions with BSA The results illustrate how important ionic strength is to adsorption In low ionic strength environments, 90% of BSA was bound to the microspheres Adversel y, a 10 fold increase in ionic strength leads to 0% adsorption In the next experiments to follow, ionic strength will be highly con sidered and adjusted to 0.01M.

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73 5 0 5 10 Low Ionic Strength High Ionic Strength Protein Bound (ug/m 2 Support) Ionic Strength influences on BSA Adsorption onto Microspheres Figure 4 4 Experiments 12. BSA and Ionic S trength Ionic strength influence on BSA adsorption onto polystyrene microspheres (s urface area to volume ratio 1:100:1852mm 2 / ml BSA conc. : 1mg/ ml or 120ug/ ml pH 4.5 and Temperature: 37 C ). Table 24 % Protein Bound Relative to T otal BSA Sample Code % Bound Low Ionic Strength 90.97 0.57 High Ionic Strength 3.74 0.14 5.6 SEM Adsorption Since protein adsorption is a complex mechanism, the positive control provided guidance toward a working system and insight into why the previous experiments did not work As such, the findings were implemented and adjusted on a new system to encourage protein binding 2 sulfoethyl methacrylate (SEM) was copolymerized with tBA, PEGDMA and DMPA at different weight percents to leave the SMP samples wi th negative functional groups on their surface s. B inding experiments were carried out with BSA at its isoelectric charge. Results show that SEM polymers bind more protein per surface area than the other adsorbent samples Polystyrene microspheres were use d as a positive control but because of its highly concentrated state, the surface to volume ratio for the spheres was 1852mm 2 / ml contrary to the other samples that were 262mm 2 / ml Thus, polystyrene sheets were purchased and sized as an additional control to eliminate surface area bias to binding and other variables introduced from free floating microspheres rather than a solid stationary adsorbent support Nonetheless, binding per surfac e area was normalized as seen below From the polymer characterization data, the 2.5% SEM samples between batches possess differences that could ultimately influence binding The surface of 2.5% from batch 1 has a larger dispersive (non polar) component th an that of batch 2 shown in table 3 Though contact angle in air can be misrepresentative of

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74 the same surface in aqueous solutions, the trend change in hydrophobicity exists with increasing wt % of SEM Thus, the hydrophobicity could potentially translate to the number density of SEM methyl groups at the surface in lieu of their OH groups If this is the case, the increase in polarity between adjacent binding sites of the polymer under aqueous solutions could lead to lateral repulsion upon protein binding a nd thus less binding The differences in the rubbery modulus between batches in 2.5% SEM could also affect protein binding as cells bind to stiffer materials over softer materials in cell culture The higher polar groups could alter the number of cross lin ks formed during the polymerization processes by repulsion Phase changes could ex i st within each polymer coupon. Nonetheless, f igure 45 shows that the SEM SMPs dominate BSA adsorption over the other adsorbents, including polystyrene, which is known to ads orb protein readily and is in fact used to develop binding assays used for biomedical applications. Nylon is the base component of the Enterotest, which is used in preliminary clinical tests to capture biomarkers of EoE. The results below suggest that our system may bind protein better than nylon, and with development, has the propensity to bind substantial amounts of EDGPs. Small differences of negatively charged functional groups on the SMP surface can greatly affect protein adsorption. From 0% SEM to 2.5 % SEM, there is >50% change in BSA adsorption. Figure 45 illustrates how hydrophobicity (polystyrene, gold SMP) or polarity alone (nylon) does not yield maximal BSA adsorption. The spacing of the functional groups relative to each other and the surface potential (electrostatic charge) along with hydrophobicity influence binding.

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75 0 5 10 15 20 25 30 35 40 45 Polystyrene Microspheres Polystyrene Nylon 0% SEM 0.5% SEM 1% SEM 2.5% SEM BSA Adsorbed (ug/m 2 Support) BSA Adsorption. Batch 2 0 5 10 15 20 25 30 35 40 45 Polystyrene Microspheres Polystyrene Nylon Gold 0% SEM 0.5% SEM 1% SEM 2.5% SEM BSA Adsorbed (ug/m 2 Support) BSA Adsorption. Batch 1 Figure 45 SEM A dsorption of BSA Adsorbents from two different batches were tested for adsorption of BSA Comparisons were made against polystyrene microspheres with *P< 0.01 * * * *

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76 Table 25 % Protein Bound Relative to T otal BSA After the binding experiment, the SEM SMP samples were collected, washed and visualized for further evidence of protein bound to their surfaces Figure 46 shows BSA bound to the polymer surface after its incubation step compared to a polymer incubated in media alone Figure 47 shows a closer look of the p olymer surfaces under a 10X magnification captured on an optical bench microscope Visually and through the values obtained by the colorimetric assay, the saturation point of the polymers may be near the 1% SEM SMP sample as there is not much change betwee n 1% and 2.5% In addition it should be noted that little SEM monomer was incorporated into the SMP system but drastic changes occurred between sample s The 0% SEM bound <10% of the total protein in solution where the mere 1% addition of the SEM monomer y ielded >65% adsorption of BSA In the previous experiments, CEA was incorporated at 5%, 10% and 15 %, which could have over populated the polymer surface The distances of the functional groups from each other are an important consideration in adsorption studies, as proteins interact not only with the polymer surface, but also with each other If two proteins are binding to adjacent binding sites, repulsion can occur if they are too close to one another This event is reduced by neutralizing the protein through the pH of the media but this only adjusts the net charge, such that repulsion is minimized but can still occur. Sample Code. Batch 2 % Protein Bound Microspheres 83.31 Polystyrene 11.18 Nylon 2.72 0% SEM 7.26 0.5% SEM 18.77 1% SEM 71.47 2.5% SEM 78.73 Sample Code. Batch 1 % Protein Bound Microspheres 39.89 Polystyrene 11.82 Nylon 2.25 Gold 6.10 0% SEM 9.44 0.5% SEM 29.32 1% SEM 65.62 2.5% SEM 61.54

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77 Figure 46 SEM Polymer after 3 Hour I ncubation SEM polymer incubated in media alone (Left) compared to SEM polymer incubated in BSA (Right)

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78 Figure 47 Images of the SEM Polymer a fter 3 Hour I ncubation BSA adsorption onto the SEM SMP imaged with an optical microscope at 10X magnification.

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79 6. C onclusion The results of the present study demonstrate that altering the surface chemistry of an acrylate based SMP can encourage protein binding onto its surface with additional system adjustments SEM SMP s had a higher yield of protein adsorption than known adsorbents used currently in biomedical applications such as polystyrene Additionally our 1% and 2.5% SEM polymers bound BSA at least 60 % better than nylon, the base material of the esophageal string test with a known capacity to capture proteins and cells in vivo This suggests the SEM polymer s have potential to improve current diagnostics of EoE by adsorbing substantial quantities of protein in a minimally invasive way with development Further tests and o ptimization of t he S EM SMP system for specific adsorption to biomarker s of EoE would increase the sensitivity of the eventual device. During the developmental process, difficulties in improving the current diagnostics by depending purely on electrostatic and hydrophobic interactions for adsorption of EoE biomarkers onto our SMP became evident The complexity of this aim originates from the vast collection of dominating factors for adsorption that compete with the surface c hemistry of our biomaterial and the relatively weak non covalent interactions we are basing specific adsorption on The combination of surface chemistry and high surface area could strengthen these weak interactions and facilitate more adsorption Chemica l modification of the SMP through copolymerization was performed rather than other surface alteration techniques such as texturing, ligand attachment or thin film deposition B ecause of the eventual desire to adsorb proteins specifically, and the complexit y that arises from processing, validation of the modified surface and additional multi procedural stages associated with these other techniques which would ultimately reside outside the time frame for this study chemical modificatio n was pursued and optimized Since EoE is a chronic inflammatory disease of the esophagus with a high rapport for misdiagnosis through the invasive procedure of endoscopy the progression toward a better diagnostic for this disease is well justified While the esophageal s tring test ( EST ) is a vast improvement to a minimally invasive diagnostic for this disease, the surface of the SEM polymer already binds substantially more protein than nylon, the ba se material of the EST T he reason for EST capturing protein in vivo may be due to its large surface area Together, with the surface chemistry of our functionalized SMP and the addition of

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80 large surface area, our SMP system could be an even greater improvement to the diagnostics in place Through the developmental stages of adsorption testing, electrostatic interactions were highly influenced by the media Since these are the interactions we are depending on for specific interactions, optimization and more development of the polymer surface under physiological mimicking syste ms should be implemented Since the SEM SMP was tested under a pH that matched the isoelectric point of BSA, the study protein, it would be beneficial to test i f the SEM polymers adsorb cationic proteins of EoE at a pH of 7 This would unveil if the active functional groups on the surface of the polymer encourage specific binding with the electrostatic interactions in play on the protein surface It was also found that competing proteins, ions and molecular species could bind preferentially to our polymer s ystem and consume binding sites originally meant for the target protein Additional optimization and tests could be performed to correct for this event at a high deg ree along with other esophageal proteins If the surface area of our polymer system were increased substantially or specific texture or nano pockets were incor porated for capture of the EDGP s in combination of the tailored SEM SMP, the polymer could be cor rected for competing proteins and molecules. The SEM polymer would consequently be tested under pH 7 wit h a mixture of proteins including EDGPs to find the optimal loading capacity of our polymer system The SEM SMP could also be valuable for in vitro medical research. We have shown SEM SMP binds more effectively than polystyrene, a common polymer used in biomedical applications for attaching cells to the bottom of cell culture plates, affinity chromatography or immunoprecipitation to purify and study biological samples and microspheres used to optimize multiplex assays Our SEM SMPs can pave the way for similar applications with development. In summary, the results of the present study illustrate how the right balance of electrostatic and hydrophobic functional groups on the SMP could influence protein adsorption for the eventual development of a diagnostic for EoE Th rough the experimental process, the dependencies of protein adsorption were discovered The mechanism is based on weak non covalent int eractions As such, chemical modification of SMPs coupled with high surface area may provide better specificity strength and greater binding of the target proteins In future work, the SMP can be altered for the capture of EDGPs through physical entrapment between micro crevasses, which match the size of the target protein as

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81 described briefly above Another route could be incorporating active biological species into the polymer such as antibodies or amino acid peptide sequences to facilitate a specific res ponse in vivo

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82 R EFERENCES 1. Berner, E. S. & Graber, M. L. Overconfidence as a cause of diagnostic error in medicine. The American journal of medicine 121, S2 23 (2008). 2. Lieberman, J. a & Chehade, M. Eosinophilic esophagitis: diagnosis and management. Immunology and allergy clinics of North America 32, 67 81 (2012). 3. Saffari, H. et al. Patchy eosinophil distributions in an esophagectomy specimen from a patient with eosin ophilic esophagitis: Implications for endoscopic biopsy. The Journal of allergy and clinical immunology 130, 798 800 (2012). 4. Furuta, G. T. et al. The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis. Gut (2012). doi:10.1136/gutjnl 2012 303171 5. Hu, J., Li, S. & Liu, B. Adsorption of BSA onto sulfonated microspheres. Biochemical Engineering Journal 23, 259 263 (2005). 6. Kim, J. Protein adsorption on polymer particles. 4373 4381 (2002). 7. Yoon, J., U, J. K. & Kim, W. The relationship of interaction forces in the protein adsorption onto polymeric microspheres 413 419 (1999). 8. Temenoff, J. S. & Mikos, A. G. Biomaterials The intersection of Biology and Materials Science 1 478 (Pearson, 2008). 9. Andrade, J. & Hlady, V. Protein adsorption and materials biocompatibility: a tutorial review and suggested hypotheses. Biopolymers/Non Exclusion HPLC (1986). at 10. Gupta K, S., Chehade, M. & Sampson A, H. Gastrointestinal Endoscopy Clinics of North America 157 167, 33 43 (Saunders, 2008). 11. or, K., Moses, R. L. & Jacobs, J. J. Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion. Tissue engineering 7, 55 71 (2001). 12. Tengvall, P. Comprehensive Biomaterials Biocompat ibility, surface engineering, and delivery of drugs, Genes and other molecules 63 73 (Elsevier, 2011). 13. Hlady, V., Buijs, J. & Jennissen P., H. Methods for studying Protein Adsorption. Methods Enzymol. 402 429 (1999). 14. Castner, D. G. & Ratner, B. D. Biomedical surface science: Foundations to frontiers Surface Science 500, 28 60 (Elsevier, 2002). 15. Mani, G., Feldman, M. D., Patel, D. & Agrawal, C. M. Coronary stents: a materials perspective. Biomaterials 28, 1689 710 (2007). 16. Chen, H., Yuan, L., Song, W., Wu, Z. & Li, D. Biocompatible polymer materials: Role of protein surface interactions. Progress in Polymer Science 33, 1059 1087 (2008).

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83 17. Behl, M. & Lendlein, A. Shape memory polymers are an emerging class of active polymers that. 10, 20 28 ( 2007). 18. Lendlein, A. & Langer, R. Biodegradable, elastic shape memory polymers for potential biomedical applications. Science (New York, N.Y.) 296, 1673 6 (2002). 19. Lendlein, A. & Kelch, S. Shape Memory Effect From permanent shape. 20. Methods for the calculation of surface free energy of solids. 24, 137 145 (2007). 21. Surface free energy Background calculation and examples by using contact angle measurements. at < http://www.attension.com/404?aspxerrorpath=/$2/attensionan5 surfacfreeenergy 250810.pdf> 22. High Sensitivity ELISA For Human and Mouse. at 23. Western Blotting Detect ion Reagents. at 24. Thermo Scientific Pierce Protein Assay Technical Handbook Version 2. at 25. NanoOrange Protein Qua ntitation Kit. 1 6 (2008). at 26. Protein Measurements. at 27. Jones, L. J., Haugland, R. P., Singer V. L. & Probes, M. Development and Characterization of the Based Assay of Proteins in Solution. Biotechniques 34, 850 861 (2003). 28. CBQCA Protein Quantitation Kit ( C 6667 ). (2001). at 29. Quant iT TM Protein Assay Kit. (2007). at 30. Quant iT TM Linearity Reproducibility and Sensitivity. 7707 at 31. Flamingo TM Fluorescent Gel Stain. at 32. Micro BCA TM Protein Assay Kit. 0747, 1 6 at < http://www.piercenet.com/instructions/2160412.pdf >

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84 Appendix A : D etection A.1 Overview of Det e ction M ethods Many protein quantification methods were considered in the initial stages of the experimental design illustrated in t ables 26 28 Based on these findings and the micro BCA assay w ere mainly used b ecause of their high sensitivity and reproducibility ELISA w ere performed when quantifying one protein species from a pool of proteins in solution and t he micro BCA assay was used when quantifying purified protein Although there are various other methods for protein detection onto polymer surfaces such as Fourier transform attenuated total reflection infrared spectroscopy ( ATR FTIR ) ellipsometry total internal reflection fluorescence spectroscopy (TIRF) or using radioisotope labeled proteins they were not implemented in the experimental design because of the ir availability and the limited time frame of this project 9,13 Instead, UV absorption, fluorescence colorimetric assays and antibody based assays were utilized as our choices to measure protein concentrations These choices are suitable for both elution or solution depletion methods but the critical determinants for these assays are their level of sensitivity because of our low polymeric surface area At these low protein levels, the assay is re quired to be sensitive enough to discern low protein concentrations and have reproducible results at these small scales. 8 a Bio Rad protein assay was used, analogous to the Bradford assay Because of media interference with the micro BCA assay and the high costs associated with the multiple ELISAs, the Bio Rad protein assay was substituted.

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85 Table 26 : Antibody Based A ssays. Antibody technology grants high specificity and sensitivity but can be expensive. Antibody based assays Sensitivity Comments ELISA 22 $$$ Multi step procedure (time expensive) Western Blot Mini Gels 23 $$$ High variability from gel to gel Multi step procedure (time expensive)

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86 Table 27 : Absorbance Based A ssays C olor i metric assays or absorbance of pure protein ha ve a large linear range are less time consuming and complex than antibody based assays and are cost efficient These are non specific assays where pure protein is usually detected. Absorbance based assays Sensitivity Comments Coomassie Plus (Bradford) micro assay 24,25 1 g/ml to 25 g/ml Proteins precipitate over time High protein to protein signal variability Interference with detergent Modified Lowry 24,25 1 g/ml to 1.5 m g/ml Protein to protein variati on Lengthy, multistep procedure (additional variability) Interference with detergents, disulfides, copper chelating agents, carbohydrates, glycerol and other substances Micro BCA method 24 0.5 g/ml to 40 g/ml Not compatible with reducing agents Susceptible to interference with thiols, copper chelating agents, tyrosine, cysteine, tryptophan and other substances Protein to protein variability Absorbance at 280 nm 25 50 g/ml to 2 mg/ml High protein to protein variability Detection influenced by nucleic acids and other contaminants Nano Drop 26,27 A280 0.1 mg /ml to 100 mg/ml BSA Quick Reads aromatic amino acids

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87 Table 28 : Fluorescence Based A ssays Fluorescence based assays Sensitivity Comments NanoOrange assay 25 10 n g/ml to 10 g/ml Samples can be read up to six hours later Low protein to protein signal variability Detection not influenced by reducing agents or nucleic acids Sensitive to salts and detergents Toxicity levels unknown CBQCA 28 10 n g/ml t o 150 g/ml Multistep procedure (additional variability) Not compatible with buffers containing amines or thiols Highly toxic Quant Kit 29,30 0.25 g/ml to 4 g/ml Samples can be read up to 3 hours later Low sample volumes Highly basic proteins behave aberrantly Flamingo Fluorescent Gel Stain 31 Sensitivity limit of 0.25ng 5ng L inear range over 3 orders of magnitude UV and Mass Spectroscopy compatible Multistep procedure Variability from gel to gel with electrophoresis Fluorescent assays can be as sensitive as antibody based assays but are non specific (unless the protein of interest has a fluorescent tag itself) These are more cost efficient than antibody technology but can be tox ic

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88 A.2 Micro BCA Assay The micro BCA assay is a colorimetric assay with a bicinchoninic acid formulation developed to quantitate total protein 32 T his assay was used in the project to quantitate pure protein after perfo rming the binding experiments. Because our initial study protein was poly L arginine a synthetic protein the BCA assay was chosen because of its compatibility sensitivity with low toxicity and good reproduci bility for this protein While the assay caters to the study protein, it is susceptible to reducing agents and other interfering substances that may exist in media solutions. As such, media solutions were tested to see if they elicited a false positive signal The buffers initially chosen were Hanks balanced salt solution (HBSS) and RPMI because they both are used in cell culture to mimic physiological environments These buffer s were diluted with DI water and/or PBS and the BCA assay was performed Tables 29 and 30 show the net absorbance values at 562nm Results show that bot h buffers elicit a st ro ng signal at the same absorbance used to detect protein that would ultimately interfere with the quantification of the protein and as such cannot be used in the binding experiments. These false positive results decrease the sensitivity of the assay for the study protein and contribute to high background noise BSA was diluted in PBS to gene rate a standard curve as shown in Table 31 The result of the standard curve clearly shows the absorbance values reside within the linear range of the assay 2 40ug/ml, with little interference of the buffer which could otherwise hide the signal of the pro tein The high end of the standard curve has an absorbance of approximately 0.7nm would be necessary to be within the high end of this linear range and a dilution of about 1:32 with the RPMI buffer These baseline dilutions would dilute out the protein to a n undetectable level by this assay rendering it unpractical for our application Dialysis ly o philization or use of a rotary evaporator could be p erformed after the binding experiment to displace the interfering media but these processes are lengthy, and add both inter and intra experim e ntal variability As such, PBS or acetate buffer was used in place with careful consideration of contents that ma y elicit a false positive signal.

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89 Table 29 : BCA A ssay with Hanks Balanced Salt Solution (HBSS) Dilutions of HBSS Net A(562nm) Repeat 1 Net A(562nm) Repeat 2 Net A(562nm) Repeat 3 NO DILUTION OVRFLW OVRFLW OVRFLW 1: 2 3.549 3.533 3.585 1: 4 1.903 1.925 1.893 1: 8 1.024 0.979 0.99 1: 16 0.577 0.556 0.582 1: 64 0.238 0.242 0.23 1: 128 0.178 0.173 0.172 Table 30 : BCA Assay with RPMI Diluted with W ater (W) or PBS (P) Dilutions of RPMI Net A(562nm) Repeat 1 Net A(562nm) Repeat 2 Net A(562nm) Repeat 3 RPMI: NO DILUTION OVRFLW OVRFLW OVRFLW 1: 2 W OVRFLW OVRFLW OVRFLW 1: 4 W OVRFLW OVRFLW OVRFLW 1: 8 W 2.633 2.449 2.512 1: 16 W 1.446 1.388 1.393 1: 32 W 0.811 0.795 0.796 1: 64 W 0.461 0.45 0.453 1: 128 W 0.284 0.276 0.332 1: 256 W 0.153 0.143 0.164 1: 2 P OVRFLW OVRFLW OVRFLW 1: 4 P OVRFLW OVRFLW 3.501 1: 8 P 2.354 2.463 2.413 1: 16 p 1.28 1.326 1.366 1: 32 P 0.738 0.757 0.777 1: 64 P 0.422 0.435 0.452 1: 128 P 0.271 0.273 0.29

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90 Table 31 : BSA Standard Curve D iluted in PBS (1X) BSA concentration (ug/ ml ) Net A(562nm) Repeat 1 Net A(562nm) Repeat 2 Net A(562nm) Repeat 3 200 2.78 2.61 2.739 40 0.746 0.751 0.767 20 0.423 0.421 0.424 10 0.252 0.238 0.234 5 0.146 0.15 0.148 2.5 0.122 0.121 0.121 1 0.116 0.117 0.117 0.5 0.115 0.111 0.113 0 0.113 0.088 0.124 Several adsorption experiments were performed but minimal amounts of binding occurred and the detection limit of the micro BCA assay was challenged Figure 48displays experiment 12 with BSA adsorbed onto CEA SMP s Negative values and large error bars illustrates the amount of protein bound is within the noise of the assay The solution depletion method was used as an indirect met hod to quantitate protein bound to the adsorbents (where the change in protein concentration was calculated) and requires the assay to discern similar values if minimal adsorption is taking place thus the limit of detection is crucial E xposing large surf ace areas of the polymer to the protein solution did not improve adsorption and so the detection method was reconsidered I f the polymers have monomers that did not completely polymerize the free species may be released during the binding experiment into the media, and in this way, could alter the reaction of the colorimetric micro BCA assay Since CEA is a reducing agent, further detection methods were tried to eliminate this possibility.

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91 3 2 1 0 1 0% CEA 5% CEA 10% CEA 15% CEA Protein Adsorbed (ug/m 2 support) BSA Adsorption: Micro BCA Assay Figure 48 Micro BCA Assay P erformed on Exper iment 12 CEA Micro BCA Assay linear range from 2 40ug/ ml A.3 Bio Rad Protein Assay The Bio Rad assay is a total protein assay based off the Bradford assay with a linear range of 1.2ug/ ml to 10ug/ ml using the micro assay procedure This assay is less sensitive to reducing reagents and other interfering substances compared to the micro BCA assay Therefore, it was used to measure protein from experiment 12 Results in figure 49 show the presence of negati ve values and large error bars even still To eliminate the possibility of interference of a false positive or negative result altogether from media with free monomer species, a non colorimetric assay was performed. Figure 49 Bio Rad Assay P erformed on Exper iment 12 CEA The linear range is 1.2 ug/ml 10ug/ml 5 4 3 2 1 0 1 2 0% 5% 10% 15% Protein Adsorbed (ug/m 2 support) CEA BSA Adsorption: Bio Rad Protein Assay

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92 A.4 Spectroscopy Aromatic amino acids of prote i ns elicit an absorbance at 280nm Even though this is a less sensitive method of detection, it was thought to be useful to try this method incase an adverse reactio n was occurring between the colorimetric working reagents and the media solution encompassing small quantities of unpolymerized monomer content after a 3 hour incubation period A spectroscopic reading at 280nm was used on the samples from experiment 12 Two micro liters of each sample was placed on the Biotek Take 3 micro volume plate 16 times to account for error in the spectroscopy readings and pipetting Figure 50 displays no binding T he large deviations show that we are still within the noise of the assay Low amounts of binding may just be occurring Since l ow sample volumes could account for so me of this error, one more measurement was taken using higher sample volumes. Figure 50 Spectro scopy P erformed on Exper iment 12 CEA A biotek synergy machine was used with the Take 3 micro volume plate to read protein levels at 280nm 16 repeats per condition were performed A280nm linear range is 0.1mg/ml 100mg/ml. A sa m ple volume of 100ul was placed in a microcuvette and measured by a biophotometer Each sample was performed in triplicate Figure 50 shows tighter standard deviations but negative values still persists Oxidation and reduction reactions should not affect the results of this method as the protein samples are read without additions of other reagents and nucleic acids and aromatic amino acids are read The buffer should not have any contaminants that would interfere even after incubation with the polymers These results suggest the sensitivity of th e assay needs to be addressed. 0.06 0.04 0.02 0 0.02 0.04 0.06 0.08 0% 5% 10% 15% Protein Adsorbed (ug/m 2 support) % CEA BSA Adsorption: Spectrosocpy with micro volume plate

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93 Figure 51 Spectroscopy P erformed on Exper iment 12 CEA A biophotometer was used to read protein levels at 280nm. 3 repeats per condition were performed A280nm linear range is 0.1mg/ml 100mg/ml. A.5 Flamingo F luoresc ent Gel S tain Flamingo stain is a non specific or total protein fluorescent stain that is performed on SDS PAGE gels 31 Its linea r range spans 3 orders of magnitude and its sensitiv ity limit approaches 0.25 5ng. This new detection method was used to see if we could remove oursel ves from the noise of the assay and detect reproducible amounts of binding A destaining step was carried out with 0.1% (w/v) Tween 20 for 10 minutes because the signal of the BSA bands was high Figure 52 shows the BSA standard curves on each gel measured with a Typhoon f luoro meter at 532nm The first t hree lanes in each gel were over saturated and could not be used for quantification using densitometry Since this stain is compatible with UV transilluminescence, the gels were exposed to UV and were quantified with densitometry using Quantity One software shown in figure 53 The l inear range using this method is 960ng 60ng Once densitometry was performed, the standard curve was generated shown in figure 54 The values from both gels per condition were averaged and plotted against the densitometry results Because the standard devi ations are extremely high, representing poor reproducibility between gels the flamingo stain would not be a better detection method t han the micro BCA assay for our binding experiments The variability may be a contribution from the gels, the multistage p rocedure or loading error Regardless, two gels should not be compared to one another beca use of such large differences. 200 150 100 50 0 50 0% 5% 10% 15% Protein Adsorbed (ug/m 2 support) CEA BSA Adsorption: Spectroscopy with biophotometer

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94 Figure 52 Flamingo Fluorescent Gel Stain on BSA S tandards Exposure with a Fluoro meter at 532nm The f irst three lanes were over saturated and could not be used for quantification. Figure 53 Flamingo Fluorescent Gel S tain on BSA Standards, E xposure with UV T ransilluminescence The BSA bands at 67kD were quantified using Quantity One software by densitometry The linear range for these results is 960ng 60ng.

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95 y = 1102.8x + 485478 R = 0.9502 0.00E+00 5.00E+05 1.00E+06 1.50E+06 2.00E+06 0 200 400 600 800 1000 1200 Density (INT/mm 2 ) Concentration (ng) BSA Standard Curve Figure 54 BSA Standard Curve from Fluorescent Gel S tain. Standard curve was generated from averaging densitometry values of the two gels per condition and plotted against the known concentration This detection approach is not suitable for our binding studies because of the large standard deviations which is associated with poor reproducibility from gel to g el. From these series of experiments, the micro BCA assay seems to be the best approach in terms of time, complexity, compatibility, sensitivity and reproducibility Overall, t hese results suggest that the CEA SMP system does not bind above 2ug/ml of protein a nd that more system adjustments needed to be made to improve adsorption An additional method could be executed with the BCA assay to detect the protein directly on the solid support (polymer) This may help remove negative values from the graphs and can facilitate as a direct method for detection without elution The polymer should be delicately washed and dabbed with a Kim wipe to rid of any collection of protein that is not bound onto the polymer system Working reagent of the BCA assay could be added to the solid support until completely submerged The incubation would proceed as normal Afterwards, a small quantity of the reacted solution can be transferred to a micro plate and read at 562nm. In future work, ATR FTIR, ellipsometry, TIRF and radio lab eling may be useful detection methods to consider in low surface area experiments The direct detection methods may give a more accurate way to determine protein bound onto surfaces without an elution step to strip the protein from the polymer surface Cau tion should be taken with radio labeled or fluorescently labeled protein as these changes to the study prote in could influence adsorption. Labeling may alter the conformation of the true protein structure and ultimately create a false adsorption scenario Protein structure of such alterations should be studied before going down this route For instance, fluorescent tags can be hydrophobic, facilitating adsorption onto the hydrophobic surface or consume vital amino acid groups needed for adsorption.

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96 Appendix B: Protocols B.1 Acetate Buffer Materials : Sodium acetate (Sigma) Acetic a cid (reagent grade from Sigma) DI water passed through a 0.22um filter pH meter 70% ethanol 2 autoclaved glass beaker Procedure : 1. Dilute 1N a c etic acid to 0.1M with DI water. 2. Dilute 1N of sodium acetate to 0.1M with DI water 3. Add 20mL of 0.1M acetic acid and 30mL of 0.1M sodium acetate to 950mL of DI H20 4. Mix the solution well before using the pH meter 5. Place ethanol in one of the glass beakers and DI H20 in the other 6. Place the pH meter probe in the ethanol to clean off particulate or debris and place in DI water before placing it in the acetate buffer 7. Adjust the pH to 4.5 with1N HCL or 1N NaOH

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97 B.2 Polystyrene Microspheres Materials: Acetate b uffer (I=0.01M, pH=4.45) 1.5mL eppendorf tubes Pure BSA (Sigma delipidated) 15mL polypropylene conical tubes Glass flow cytometer tubes (5mL) Procedure: 1. Prepare 200ug/ml of BSA in acetate buffer to add to the protein conditions listed below 2. Label eppendorf tubes as follows (in bold) : *Conditions performed in triplicate A. 120ug/ ml (p rotein) B. 0ug/ ml (a cetate buffer) C. 120ug/ ml + B (protein + m icrospheres) D. 0ug/ ml + B (acetate buffer + m icrospheres) 3. Add 600ul of BSA prepared in step 1 to all protein conditions listed above (6 total tubes) In condition C, proteins will bind to the adsorbent C ondition A is the p rotein only sample where no adsorbent is added for a total protein measurement 4. Add 600ul of acetate buffer to all other conditions These conditions are to measure background noise 5. Vortex the polystyrene microspheres for 30sec and dilute to a 0.1% solid (1:100 dilution) with acetate buffer Note: To minimize pipetting e rror, larger serial dilutions of 1:10 (300ul spheres into 2.7mL acetate buffer) can be prepared for a final 1:100 dilution This was performed in the glass flow cytometer tubes. 6. Add 400ul of the diluted polystyrene microspheres to conditions C and D pipe tte up and down three times to mix well 7. Add 400ul of acetate buffer to conditions A and B to match the final 1ml volume to the other samples pipette up and down three times to mix well 8. Incubate samples at 37 C in 5% CO2 incubator for 3hours 9. Remove samples from the incubator and centrifuge for 15 minutes at 9300G force at 10C 10. C ollect 700ul of supernatant being mindful not to perturb the microsphere pellet on the bottom of the eppendorf tube, and place in a separately labeled eppendorf 11. Spin the sup ernatant collected in step 10 once more at 9300G force for 15 minutes at 10 C 12. Collect 600ul of supernatant being mindful not to perturb the microsphere pellet on the bottom of the eppendorf tube, and place in a separately labeled eppendorf 13. Store samples at 20 C until further use or continue with the micro BCA assay

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98 B.3 SEM Binding Experiment Materials: 6 SMP samples (5x19x 1.5mm 2 ) 6 p olystyrene samples (5x19x 1.5mm 2 ) 6 n ylon samples (5x19x 1.5mm 2 ) Acetate b uffer (I=0.01M, pH=4.45) DI H20 passed through a 0.2um filter with 1.2% SDS and 0.05% NAN3 Pure BSA (Sigma delipidated) 1.5mL e ppendorf tubes 3 g lass beakers (autoclaved) Tweezers 70% e thanol Procedure: 1. Prepare 200ug/ml of BSA in acetate buffer to add to the protein conditions listed below 2. Label eppendorf tubes as follows (in bold) : *Conditions performed in triplicate A. 120ug/ ml (p rotein ) B. 0ug/ ml (a cetate buffer) C. 120ug/ ml + B (protein + m icrospheres) D. 0ug/ ml + B (acetate buffer + m icrospheres) E. 120ug/ ml + 0% SMP ( p rotein + 0%SMP) F. 0ug/ ml + 0% SMP (a cetate buffer + 0% SMP) G. 120ug/ ml + 0.5% SMP (p rotein + 0.5%SMP) H. 0ug/ ml + 0.5% SMP (a cetate buffer + 0.5% SMP) I. 120ug/ ml + 1% SMP (p rotein + 1%SMP) J. 0ug/ ml + 1% SMP (a cetate buffer + 1% SMP) K. 120ug/ ml + 2.5% SMP (p rotein + 2.5%SMP) L. 0ug/ ml + 2.5% SMP (a cetate buffer + 2.5% SMP) M. 120ug/ ml + PS (protein + p olystyrene) N. 0ug/ ml + PS (acetate buffer + p olystyrene) O. 120ug/ ml + N (protein + n ylon) P. 0ug/ ml + N (a cetate buffer + n ylon ) 3. Add 600ul of BSA prepared in step 1 to all protein conditions listed above. (24 total tubes) These will be the conditions where the proteins will bind to the adsorbents The only exception is c ondition A the protein only sample, where no adsorbent is added for a total protein measurement 4. Add 600ul of ace tate buffer to all other conditions These conditions are to measure background noise. 5. Perform a 1:100 dilution of the DI H20 with 1.2%SDS and 0.05% NaN3 in acetate buffer Note: Polystyrene microspheres are shipped in DI H20 with 1.2%SDS and 0.05% NaN3 T o keep consistency between conditions, all are treated the same way. Thus, this solution is diluted in the same manner as the spheres. 6. Add 400ul of the final 1:100 DI H20 solution prepared in step 5 to all the conditions excep t the polystyrene microsphere conditions, C and D This is because the microsphere conditions are already suspended in this solution.

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99 7. Vortex the polystyrene microspheres for 30sec and dilute to a 0 .1% solid (1:100 dilution) with acetate buffer Note: To minimize pipetting error, larger serial dilutions of 1:10 (300ul spheres into 2.7mL acetate buffer) can be prepared for a final 1:100 dilution. 8. Add 400ul of the diluted polystyrene microspheres to conditions M and N 9. With tweezers, add the other adsorbent samples in the ir appropriate epp endorf tube s being careful not to contaminate the tweezers Make sure there is only one adsorbent per eppendorf tube and that it is fully submerged in the solution. 10. Incubate samples for 3 hour s in a 37 C incubator with 5% CO2 11. After the time point, remove a ll the samples from the incubator 12. For all conditions EXCEPT the polystyrene microspheres, carefully and slowly aspirate 700ul of supernatant into a separately labeled eppendorf tube to measure the protein concentration Do not touch the polymer with the pipette tip. Store at 20 C until further use or continue with the micro BCA assay 13. For the polystyrene micro sphere conditions, centrifuge the samples for 15 minutes at 9300G force at 10 C to pull down the spheres from the free protein solution 14. Collect 700ul of the supernatant from the polystyrene samples being mindful not to perturb the microsphere pellet on the bottom of the eppendorf tube, and place in a separately labeled eppendorf 15. Spin the supernatant collected in step 14 once more at 9300G force for 15 minutes at 10 C 16. Collect 600ul of the supernatant from the polystyrene samples once more, being mindful not to perturb the microsphere pellet on the bottom of the eppendorf tube, and place in a separately labeled eppendorf Store these sample s at 20 C or continue with the micro BCA assay 17. Once all the free protein in solution is collected for each sample prepare a stati on to proce ss the SMP s for microscopy 18. Take three AUTOCLAVED glass containers and wash them thoroughly with DI water and ethanol Suspend 70% ethanol in one of them Suspend DI water in another The beaker that is left will serve as a waste dispensary Place a layer of Kim wipes on the bench to dab the polymer samples on. 19. Take CLEAN tweezers and soak them in the ethanol bea ker for 10minutes 20. Remove the tweezers from the ethanol and place in DI water to rinse 21. Take a polymer sample and wash it with 1ml of PBS 22. Dab the polymer sides on CLEAN Kim wipes and place the polymer sample in a 6well plate keeping note which polymer i t is. 23. Capture an image of the surface of the polymer under an optical microscope at 10X magnification and discard the polymer 24. Repeat steps 19 23 for each polymer piece The tweezers d o not have to be submerge d for 10min every time, just suspend in ethanol and rinse in DI water.

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100 B.4 Eosinophil Lysate Materials: 0.025M Sodium Acetate buffer ( brought to pH 4.3 with Acetic Acid) 10X p rotease inhibitor cocktail: (Roche, Catalog #1 836 170): dissolve one tablet i n 1mL of DI water and make appropriate aliquots to avoid freeze thaw cycles Sonicator with probe Ice and ice bucket 15mL, 50mL p olypropylene conical tubes Purified blood e osinophils (EOs) Note: Keep everything COLD (4 C ) to attenuate de granulation of Procedure: 1. Calculate the volume of buffer needed to suspend the cells i n for a final concentration of 5x 10 6 cells/ ml Ex: For 10x 10 6 Cells, 2mL of buffer is needed. 2. Prepare the volume calculated above of sodium acetate buffer with 10% protease inhi bitor cocktail Add 10X protease inhibitor cocktail at a 1:10 dilution into the appropriate amount of sodium acetate Place the protease inhibitor cocktail in an ice bucket during use Chill prepared buffer at 4 C before use. 3. Suspend the eosinophils in the sodium acetate buffer + PI solution made abov e at a final concentration of 5x 10 6 cells/ ml Keep the suspension on ice 4. Clean the sonicator before use by wiping down the probe with 70% ethanol and DI water 5. Introduce the sonicator probe to the sample solut ion making certain the sample is placed on ice Do not touch the edges or bottom of the tube with the probe 6. Increase the amplitude knob such that the power is at 15% continuously for 10 seconds. Do not exceed 20% power proteins may denature by the heat p roduction Also, do not lift the probe out of solution while the sonicator is on this creates bubbles 7. Turn the knob back to zero and allow the sample to chill on ice for 30sec to alleviate any heat generation 8. Swirl the sample and repeat step 6 once mor e 9. Centrifuge this lysate at 300g, 4 C for 10min to get rid of insoluble cellular contents. 10. Place the centrifuged sample in an ice bucket and gently collect the supernatant in a separate chilled 15mL conical avoiding the pellet on the bottom The pellet r etrieved after centrifugation can be thrown away. 11. Aliquot 110ul of lysate into separate eppendorf tubes with the concentration of the cells/volume ratio recorded (5x10 6 cells/ ml ). 12. Freeze samples at 80 C