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Diffusion controlled direct electron transfer of ferritin

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Title:
Diffusion controlled direct electron transfer of ferritin
Creator:
Halsey, James Martin
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
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English
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96 leaves : illustrations ; 28 cm

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Ferritan ( lcsh )
Voltammetry ( lcsh )
Electrochemistry ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 95-96).
Thesis:
Department of Chemistry
Statement of Responsibility:
by James Martin Halsey.

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|University of Colorado Denver
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|Auraria Library
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All applicable rights reserved by the source institution and holding location.
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ocm51782826
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LD1190.L46 2002m .H34 ( lcc )

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Full Text
DIFFUSION CONTROLLED DIRECT ELECTRON TRANSFER OF FERRITIN
by
James Martin Halsey
B.S. University of Nebraska at Omaha, 1979
B.A., University of Nebraska at Omaha, 1979
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry


This thesis for the Master of Science
degree by
James Martin Halsey
has been approved


Halsey, James Martin (M.S., Chemistry)
Diffusion-Controlled Direct Electron Transfer Of Ferritin
Thesis directed by Associate Professor Donald C. Zapien
ABSTRACT
Horse spleen ferritin is a protein whose functions are to sequester excess iron, to store
it in a soluble form, and to release the iron whenever needed. Electron transfer steps
voltammetry studies on ferritin have involved ferritin adsorbed at an electrode
surface. This thesis describes the effort to effect cyclic voltammetry on ferritin in
solution. It is hoped that this will give an indication as to whether or not the ferritin
adsorbed at electrodes behaves electrochemically as native ferritin.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
are known to be involved in uptake and release of iron by ferritin. To date, all cyclic
Signed
Donald C. Zapien
111


DEDICATION
I wish to acknowledge my lovely wife, Tina, for her patience and understanding
during this endeavor.


ACKNOWLEDGEMENTS
I wish to thank Dr. Donald C. Zapien for his guidance, support and perseverance to
assist me in this effort.
I would like to thank the members of my Masters committee: Dr. Donald C. Zapien,
Dr. Larry G. Anderson, and Dr. John C. Lanning.
I would also like to thank all the members of our research group for their support and
input.
I would like to thank Mr. Paul Miller for assembling the gold disk electrodes, Dr.
Larry G. Anderson and Mr. Jeff Boon for the loan of equipment. Dr. James
Chambers, University of Tennessee, provided some helpful suggestions on
polypyrrole.


CONTENTS
Figures......................................................................ix
1. Introduction...........................................................1
1.1 Research Objective.....................................................1
1.2 Ferritin Function......................................................2
1.3 Structure Of Ferritin..................................................2
1.4 Uptake And Release Of Iron By Ferritin.................................6
1.5 Electrochemistry Of Proteins...........................................7
1.5.1 Indirect Methods.......................................................7
1.5.2 Direct Electron Transfer Of Proteins...................................7
1.5.3 Direct Electron Transfer Of Ferritin..................................15
1.5.4 Diffusion-Controlled Direct Electron Transfer Of Ferritin.............15
1.5.5 Direct Electron Transfer Of Cytochrome c..............................18
1.5.6 Diffusion Controlled Direct Electron Transfer Of Ferritin.............20
1.6 Cyclic Voltammetry And Pseudocapacitance Of The
Electrolyte Double Layer..............................................21
1.7 Cyclic Voltammetry And Electroactive Species..........................22
vi


1.8 The Three Electrode Cell...........................................35
2. Experimental.......................................................40
2.1 Reagents And Materials.............................................40
2.2 Equipment..........................................................41
2.2.1 Plasma Reactor.....................................................41
2.2.2 Potentiostats And Recorder.........................................43
2.2.3 Cells..............................................................43
2.2.4 Electrodes.........................................................46
2.3 Procedures.........................................................52
2.3.1 Cleaning Of Apparatus..............................................52
2.3.2 Treatment Of Gold With 2-Mercaptoethylamine........................53
2.3.3 Plasma Modification Of HOPG Edge Plane Electrode...................53
2.3.4 Formation Of Films.................................................55
2.3.5 Formation And Conditioning Of Polypyrrole Layers...................55
2.3.6 Cyclic Voltammetry Of Ferritin Using Polypyrrole Electrodes........58
3. Results And Discussion.............................................59
3.1 Preliminary Electrode Modifications................................59
vii


3.2 Plasma-Modified HOPG Edge Plane Electrode........................61
3.3 Polypyrrole......................................................64
3.3.1 Observation Of Charging Current With Polypyrrole On A Gold
Electrode........................................................66
3.3.2 Diffusion-Controlled Direct Electron Transfer Of Electron
Ferritin On Polypyrrole..........................................69
3.3.3 Treatment With Hydrogen Peroxide.................................76
3.3.4 Troubleshooting Whether A Polypyrrole Film Will Give Cyclic
Voltammograms Of Ferrocyanide....................................82
3.3.5 Peak Current Dependence On Ferritin Concentration................88
3.4 Conclusions......................................................93
References..............................................................95
viii


FIGURES
Figure 1.1 Ribbon diagram of ferritin subunit...................................4
Figure 1.2 Quaternary structure of ferritin.....................................5
Figure 1.3 Methyl viologen mediated electron transfer...........................8
Figure 1.4 Direct electron transfer at an electrode.............................9
Figure 1.5 Formation of an insulating layer....................................11
Figure 1.6 Unfavorable orientation of a protein................................12
Figure 1.7 Plasma modification of an electrode surface.........................13
Figure 1.8 Formation of a chemisorbed layer on of an
electrode surface...................................................14
Figure 1.9 IR cell for determining structural changes in
oxidized and reduced ferritin.......................................16
Figure 1.10 Interaction between cytochrome c and adsorbed 4-
thiopyridine........................................................19
Figure 1.11 Potential versus time used in cyclic voltammetry....................23
Figure 1.12 Current response to changing potential, in the
absence of an electroactive species.................................24
Figure 1.13 Typical current response of an electroactive
species due to changing potential...................................25
IX


Figure 1.14 Electron transfer between electrode surface and
species in solution..............................................26
Figure 1.15 Current versus applied potential at start of cyclic
voltammogram.....................................................30
Figure 1.16 Current versus applied potential as current starts to
flow in a cyclic voltammogram....................................31
Figure 1.17 Current versus applied potential as diffusion
causes increase in current in a cyclic
voltammogram.....................................................32
Figure 1.18 Current versus applied potential as current reaches
a maximum value in a cyclic voltammogram.........................33
Figure 1.19 Diffusion of dye in a glass of water.............................34
Figure 1.20 Current versus applied potential as decrease in
diffusion causes decrease in current in a cyclic
voltammogram.....................................................36
Figure 1.21 Schematic of a three-electrode cell..............................37
Figure 1.22 Representation of our three-electrode cell.......................39
Figure 2.1 Plasma reactor...................................................42
Figure 2.2 Cell used for survey work........................................44
Figure 2.3 Low volume H-cell................................................45
Figure 2.4 Edge-plane pyrolytic graphite cell...............................47
Figure 2.5 Gold flag electrodes in glass sheaths............................48
x


Figure 2.6 Gold disk pressed into Kel-F rod................................50
Figure 2.7 Reference and auxiliary electrodes..............................51
Figure 2.8 Current versus time during formation of a
polypyrrole layer..............................................57
Figure 3.1 Cyclic voltammogram of ferritin at gold modified
with 2-mercaptoethylamine......................................60
Figure 3.2 Cyclic voltammogram of background with 2-
mercaptoethylamine modified gold, after scans in
ferritin.......................................................62
Figure 3.3 Cyclic voltammogram of ferritin at ammonia
plasma modified edge-plane pyrolytic graphite
electrode......................................................63
Figure 3.4 Cyclic voltammogram of background at ammonia
plasma modified edge-plane pyrolytic graphite
electrode......................................................65
Figure 3.5 Polypyrrole and counter ion.....................................67
Figure 3.6 Cyclic voltammogram of background and
ferrocyanide at polypyrrole-perchlorate modified
gold foil electrode............................................68
Figure 3.7 Cyclic voltammogram of ferritin at polypyrrole-
perchlorate modified gold foil electrode, electrode
area approximately 1 cm', scan rate 10 mV/s,
Ag/AgCl reference electrode....................................70
Figure 3.8 Cyclic voltammogram of background polypyrrole-
perchlorate modified gold foil electrode.......................72
xi


Figure 3.9 Cyclic voltammogram of ferritin at polypyirole-
perchlorate modified gold foil electrode.........................73
Figure 3.10 Peak cathodic current versus square root scan rate,
34 mg ferritin/mL, 1 M KC1, pH 7 buffer, 1 cm2
polypyrrole electrode............................................74
Figure 3.11 Cyclic voltammogram of background at
polypyrrole-perchlorate modified gold foil
electrode, after scans in ferritin...............................75
Figure 3.12 Cyclic voltammogram of background at
polypyrrole-perchlorate modified gold foil
electrode........................................................77
Figure 3.13 Cyclic voltammogram of ferritin at polypyrrole-
perchlorate modified gold foil electrode.........................78
Figure 3.14 Cyclic voltammogram of ferrocyanide at
polypyrrole-perchlorate modified gold foil
electrode........................................................80
Figure 3.15 Cyclic voltammogram of ferrocyanide at
polypyrrole-perchlorate modified gold flag
electrode, after destruction of polypyrrole
conductivity.....................................................81
Figure 3.16 Cyclic voltammogram on 4.1 mM ferrocyanide in
1 M potassium chloride, at gold disk electrode...................83
Figure 3.17 Background of 1 M potassium chloride, at
polypyrrole-perchlorate on gold disk electrode...................84
Figure 3.18 Cyclic voltammogram on 4.1 mM ferrocyanide in
1 M potassium chloride, at polypyrrole-
perchlorate on gold disk electrode...............................85
Xll


Figure 3.19 Background 1 M potassium chloride, 0.2 M
phosphate, pH 7 buffer at polypyrrole-perchlorate
on gold disk electrode..............................................86
Figure 3.20 Cyclic voltammogram of 4.4 mM ferrocyanide in
1 M potassium chloride, 0.2 M phosphate, pH 7
buffer at polypyrrole-perchlorate on gold disk
electrode...........................................................87
Figure 3.21 Cyclic voltammogram of 24 mg ferritin/mL 1 M
potassium chloride, 0.2 M phosphate, pH 7 buffer
at polypyrrole-perchlorate on gold disk electrode................................89
Figure 3.22 Cyclic voltammogram of 4.4 mM ferrocyanide in
1 M potassium chloride, 0.2 M phosphate, pH 7
buffer at polypyrrole-perchlorate on gold disk
electrode, after scans in ferritin..................................90
Figure 3.23 Peak current dependence on bulk concentration at
four scan rates, 0.08 cm2 polypyrrole electrode,
Ag/AgCl reference, Pt auxiliary, 1 M K.C1, 0.2 M
phosphate pH 7 buffer...............................................92
Xlll


1. Introduction
1.1 Research Objective
The aim of this effort is to effect the diffusion-controlled direct electron transfer of
ferritin. Direct electron transfer is defined as electron transfer between a species in
solution and an electrode. It is especially important if one wants to obtain kinetic
information or investigate chemical reactions that are coupled to electrochemical
reactions. To date, all direct electron transfer of ferritin has been due to ferritin
adsorbed at an electrode. As ferritin is not in an adsorbed state in organisms, it is
hoped that the diffusion-controlled reaction will more closely resemble the electron
transfer reactions of ferritin in vivo. Understanding the mechanisms of iron uptake
and release are important for the following reasons: understanding iron overload and
deficiencies, improving crop yields and controlling iron toxicity. Probing the role of
electron transfer will provide insight into the mechanisms of uptake and release of
iron by ferritin.
1


1.2 Ferritin Function
Ferritin is found in all known organisms, both aerobic and anaerobic. Ferritin has
been thought to have appeared in organisms that required iron, in response to the
presence of oxygen in the atmosphere. Oxygen oxidizes iron (II) to iron (III) with an
accompanying reduction in solubility. At physiological pH, the concentration of iron
(II) ion in equilibrium with iron (II) hydroxide is on the order of 1 O'2 M. At the same
pH, the concentration of iron (III) in equilibrium with iron (III) hydroxides is on the
order of 10 M. In addition to maintaining the iron in a soluble form, oxidizing iron
(II) to iron (III) and sequestering it within the protein shell, the formation of oxygen
radicals and superoxides is prevented through the reaction between oxygen and iron
(II). In anaerobes, ferritin is thought to consume and therefore detoxify oxygen1
1.3 Structure Of Ferritin
Ferritin is an assembly of 24 protein subunits, that form a hollow, nearly spherical
shell 12 nanometers in diameter. The interior volume of this hollow sphere is 256
cubic nanometers.2 This indicates an internal diameter of nearly eight nanometers
and shell thickness of two nanometers. Iron is stored as a mineral core within the
interior and in contact, at various points, with the interior of the shell. If the interior
2


of the protein is devoid of iron the shell is called apoferritin. The amount of iron
stored within the shell varies. Typically, the number of iron atoms per wild-type
ferritin ranges from 800 to 2500, but this number can be as high as 4500 iron atoms.
The molecular weight of the apoferritin shell is 450,000 grams per mole. The average
structure of the iron core is (FeOOH)8 FeO OPO3H2. Within the core, 20-33 % of the
iron present is in a different environment than the rest of the core's iron. These
differences are due to coordination of iron with differing amount of phosphates and
could account for differences in rate of iron release from ferritin in vitro. 4 Two types
of subunits are found in most vertebrate ferritins, being designated an H-subunit and
an L-subunit. The subunits vary in amino acid sequence, but in general are composed
of four long helices and two short helices. The four long helices are bundled
antiparallel and parallel to each other (figure 1.1). The 24 subunits self-assemble to
form the hollow sphere. The ends labeled as E of four subunits come together to
form six hydrophobic channels into the interior of the protein shell. The ends labeled
N, which is the N-terminus of the polypeptide, of three subunits come together to
form eight hydrophilic channels into the interior. It is through the hydrophilic
channels that iron enters and exits the shell, (figure 1.2)
3


Figure 1.1 Ribbon diagram of ferritin subunit5
4


5


1.4 Uptake And Release Of Iron By Ferritin
The H-subunits catalyze the oxidation of iron (II) to iron (III) and the L-subunits
facilitate iron core formation.3 In the uptake of iron by ferritin, iron (II) first enters
one of the eight hydrophilic channels. As the iron moves towards the interior of the
shell glutamic acid residues E23, E58 and histidine residue H61 in an H-subunit
bind one iron (II). A second iron (II) is bound by glutamic acid residues E57,
E88, and E103. These binding sites are termed the ferroxidase center. There is one
ferroxidase center per H-subunit. The residues that form the ferroxidase center in the
H-subunit are not conserved in the L-subunits.6 The L-subunit contains an inner
surface that is negatively charged and these areas are thought to be nucleation sites
for the iron core.7 Before iron can be released from the core, it must first be
converted to iron (II). Thus, the release of iron from the core involves a reduction. It
has been suggested that there may be an unfolding of ferritin step involved in the
release of iron from ferritin.8
6


1.5 Electrochemistry Of Proteins
1.5.1 Indirect Methods
Indirect methods may be used to achieve voltammetry at electrodes. In the example
shown in figure 1.3, the oxidized form of methyl viologen (MV2+) approaches an
electrode, accepts an electron, and is reduced to MV+. The reduced form diffuses
away from the electrode and approaches the oxidized form of the species being
studied. The reduced form of MV gives up an electron to the oxidized form of the
protein, reducing the protein and being oxidized itself. The MV may now diffuse
back toward the electrode and repeat the process. Information obtained by this type
of experiment includes the value of n, and an estimate of E'. The principal
disadvantage of indirect electron transfer is that indirect electron transfer is not
specific. For example, if a protein has two or more electroactive centers, that react in
different potential windows, the mediator may react with all of centers at the same
potential.
1.5.2 Direct Electron Transfer Of Proteins
If one wishes to study the mechanisms for a reaction, one must effect direct electron
transfer between the species of interest and the electrode. Since it is the current that
7


oxidized
form
reduced
electrode
reduced
form
form
Figure 1.3 Methyl viologen mediated electron
transfer
8


reduced
form
oxidized
form
Figure 1.4 Direct electron transfer at an electrode


can be used to directly measure the rate of reaction (figure 1.4), the direct electron
transfer is required for one to obtain information on reaction kinetics, to detect
homogenous chemical reactions coupled to electrochemical reactions, and to study
proteins with more than one electroactive center. Some proteins do not exhibit
electron transfer at electrodes. Many factors may be responsible for this lack of
response. 1) The protein strongly adsorbs to the surface, denatures 9, and forms an
insulating layer (figure 1.5), or 2) the electroactive center of the protein is not
favorably oriented with respect to the electrode (figure 1.6). Direct electron transfer
may be effected by careful selection of electrode material or modifying the electrode
surface. Materials that often times serve as electrodes are bare metals, usually gold
and platinum. Other materials that may serve as electrodes are semiconductors,
various forms of carbon and conducting polymers. Electrode materials may be
modified by plasmas (figure 1.7) or chemical means. Chemical methods include
chemisorbed layers (figure 1.8); reactions that modify surface groups, such as the
silation of surface oxides, and coating the surface with conducting polymers. By
careful selection of electrode surface one controls the practical potential window
range for the system. For example, in aqueous systems, the working range is limited
to the potentials at which protons are reduced and water is oxidized.
10


Figure 1.5 Formation of an insulating layer
11


Figure 1.6
electroactive
center
Unfavorable orientation of a protein
12


1) Ar
plasma
2)NH3
plasma
Plasma modification of an electrode
surface
13


+
Gold
electrode
H-S/\/C02"
->
Gold
electrode
Figure 1.8 Formation of a chemisorbed layer on of
an electrode surface
14


1.5.3 Direct Electron Transfer Of Ferritin
Direct electron transfer of adsorbed ferritin has been accomplished by our
group. All proteins unfold, or deform, upon adsorption onto a solid surface.
This unfolding may impact the electrochemistry of the electroactive center by
changing its local environment and energy level. Changes in the energy level of the
electroactive center will in turn, cause changes in the E0' of the protein. If AE0'
(E'adsorbed E'native) < 25 mV, the protein has essentially retained its native properties.
If one desires to obtain structural information on the different redox states of ferritin
using techniques such as IR, the oxidation and reduction of ferritin in a solution
contained by an IR cell (figure 1.9), must be accomplished first.
1.5.4 Diffusion-Controlled Direct Electron
Transfer Of Ferritin
Redox reactions are known to be involved in the uptake and release of iron by ferritin.
The elucidation of the mechanism for the entry of iron into the core and the release of
iron from the core is of great interest to biochemists and physicians dealing with
conditions caused by iron overload or lack of iron. Electrochemical
15


IR source------>
> Detector
IR transparent
electrode
Figure 1.9 IR cell for determining structural
changes in oxidized and reduced ferritin
16


methods are well suited to studying electron transfer of proteins. Cyclic voltammetry,
in particular, is suited to the study of kinetics of homogenous processes that may
occur after the heterogeneous electron transfer process. Information obtained by
experiments on adsorbed ferritin includes a fair idea of the electron transfer kinetics,
and the formal potential for the redox pair. Controlled potential electrolysis of ferritin
adsorbed at a gold electrode modified with 3-mercaptopropionic acid revealed an n-
value of 1910 electrons per ferritin molecule. Varying the scan rate at the same
electrode and observing the increase in peak potential separation with increasing scan
rate indicated that the electron transfer was electrochemically irreversible.
Voltammetry of apoferritin in the absence of iron (II), and ferritin in the presence of
an iron complexing agent indicates that the currents observed when scanning ferritin
are due to the electrolysis of the iron core.11 Ferritin also exhibits electrochemical
activity when adsorbed at tin-doped indium oxide electrodes. It has been determined
that the packing density of the adsorbed ferritin varied proportionally to the ionic
strength of the solution from which it was adsorbed. Initially, the adsorbed ferritin
occurs in two independent states and upon reduction, the adsorbed protein
reconstructs to a new state. The ratio of ferritin adsorbed in the two states was shown
to be a function of ionic strength.12
17


1.5.5 Direct Electron Transfer Of Cytochrome c
Direct electron transfer of adsorbed cytochrome c has also been effected at tin-doped
indium oxide electrodes by Bowden. 13 Information gained from cyclic voltammetry
of the adsorbed species include AH0 and AS0 for the redox reaction. Direct electron
transfer of cytochrome c at a gold electrode was effected by treating the gold surface
with 4,4-dithiopyridyl by Hill.14 The disulfide bond of the 4,4-dithiopyridine
disassociate and the 4-thiopyridyl groups attach to the gold surface by the sulfide
groups, the aromatic amine groups being directed into the solution. These attached 4-
thiopyridyl groups do not themselves accept or give up electrons in the potential
window being scanned, but rather assist in the orientation of cytochrome c at the
electrode surface. The heme group in cytochrome c, containing the electroactive iron,
is located in a cleft in the protein. This cleft is surrounded by positively charged
lysine residues at neutral pH. These positively charged groups could interact with the
lone pair electrons on the nitrogen that is oriented into the solution. The electroactive
center may thus be drawn close to the electrode and in a favorable orientation (figure
1.10). Cooper etal15'16, treated various electrode materials with a layer of polypyrrole
carboxylic acid. At pH 7, the carboxylic acid groups will be deprotonated forming
negatively charged carboxylates. The isoelectric point (pi) of a protein is the pH at
which the overall charge on the protein is zero. At a pH value greater than its pi a
18


Gold
electrode
Figure 1.10 Interaction between cytochrome c and
adsorbed 4-thiopyridine
19


protein will have a net negative charge and at pHs less than its pi the overall charge
will be positive. The pi of cytochrome c is about 10, at pH 7 its overall charge is
negative. However, it was proposed that the partial positive charges on the lysine
groups around the electroactive heme center could favorably align with the negative
charges to allow for direct electron transfer.
1.5.6 Diffusion Controlled Direct Electron
Transfer Of Ferritin
The advantages of effecting direct electron transfer of dissolved ferritin over adsorbed
ferritin are 1) the ferritin-electrode surface reactions are minimized, 2) one will have a
better idea of the number of electrons transferred (n) and 3) some idea of the degree
of denaturation that occurs on adsorption will be known. It has been shown that
ferritin is deformed upon adsorption to an electrode surface. Following discussions
with Professor Katsumi Niki, it was decided that a positive surface may be the
electrode to achieve our goal. The pis of ferritins range from 4 to slightly over 6. The
pi of horse spleen ferritin is about 4.5. At pH 7, the overall surface is anionic.
Therefore, the approach to achieving diffusion-controlled cyclic voltammetry of horse
spleen ferritin was to modify the electrode surface with cationic groups. Three
approaches to modifying electrode surfaces were tried such that there was a positive
20


charge at the surface: treatment of a gold electrode with 2-mercaptoethylamine,
treatment of a edge-plane graphite electrode with an ammonia plasma, and formation
of a polypyrrole layer at a gold electrode
1.6 Cyclic Voltammetry And Pseudocapacitance Of
The Electrolyte Double Layer
The objective of this research was to modify an electrode surface, such that dissolved
ferritin would exhibit redox reactions at the electrode surface. To make the
interpretation of any observed ferritin voltammograms easier one of the first steps
was to form a layer that would only exhibit charging current in the potential window
for ferritin. This charging current is due to charging of the electrical double layer at
the electrode surface and is present even in the absence of an electroactive species.
This double layer acts as a capacitor in a circuit. The current in the circuit is given by
equation 1.1.17
i = i)xCd +((Ei/Rs-t) x Ca)exp(-t/(Rs x Cd (1.1)
x> is the scan rate, Cd is the capacitance of the double layer, E, is the initial
potential and Rs is the resistance of the solution.
The second term in the equation is transient and goes to zero as time increases. When
the potential sweep direction is changed, the current versus time trace is a mirror
21


image of the first part of the curve. Figure 1.11 shows the current response to the
changing potential as a function of time. Figure 1.12 shows the current response, in
the absence of an electroactive species, as a function of the applied potential. If the
layer on the electrode surface is not conductive, the charging current will be flat. The
preliminary goal of this work was to find a material which could be used to
reproducibly modify a gold electrode such that blank background scan will appear as
in figure 1.12, and a cyclic voltammograms of ferritin that are diffusion-controlled
and generally appear as figure 1.13.
1.7 Cyclic Voltammetry And Electroactive Species
When a sufficiently negative potential is applied to an electrode, the energy of the
electrons in the electrode is increased to a value where electrons will flow from the
electrode to the lowest unfilled energy level of the electroactive species in solution.
Conversely if a sufficient positive potential is applied, the energy will be lowered to a
value inducing electrons to flow from the highest energy level of the electroactive
species in solution into the electrode (figure 1.14). A change in charge per unit time
is a current.
22


potential, volts
Figure 1.11 Potential versus time used in cyclic
voltammetry
23


current, pA
0.2025
0.2020
0.2015
0.2010
0.2005
0.2000
0.1995 +
0
i i i--------1 i i i i
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
potential, volts
Figure 1.12 Current response to changing potential,
in the absence of an electroactive species
24


Figure 1.13 Typical current response of an
electroactive species due to changing potential
25


working energy
of electrons
on electrode
working energy
of electrons
on electrode

A
f
an oxidation has occurred
Figure 1.14 Electron transfer between electrode
surface and species in solution
26


i (current) : dO (coulombs)
dt (seconds)
(1.2)
Faradays Law, can be used to relate the amount of current to the reaction rate at an
electrode.
Q =N Faradays Law (1.3)
n x F
Where Q = amount of charged passed
n = number of moles of electrons passed per mole species of interest
F = Faradays constant
N = moles of electrolyzable species.
The derivative of Faradays Law with respect to time gives
dN = 1 x dO = Rate of electrochemical reaction (mole/sec) (1.4)
dt n x F dt
We must also look at the Nemst equation to understand the relationship between the
concentration of electroactive species at or near the electrode surface and the inherent
driving force of the reaction and applied potential.
27


For the reaction of iron (III) being reduced to iron (II), the Nemst equation is:
E = E'- 2.303 x RTx log
nF
[Fe2+]
(1.5)
E = cell potential
E0' = formal potential for the half reaction
R = ideal gas constant
n = number of mole of electrons per mole of electroactive species
T = temperature
F= Faradays constant
Since AG0' = -nFE0', the E' term reflects the inherent driving force for the reaction.
The second term reflects the effect of the concentrations of the oxidized and reduces
forms on the cell potential. As a more positive potential is applied to the electrode
surface, more of the iron (II) is oxidized to iron (III). When the cell potential equals
the formal potential, the concentrations of iron (II) and iron (III) are equal. When a
potential sweep is applied to a solution containing an electroactive species, a cyclic
voltammogram is the current response to the applied potential curve. In voltammetry,
the only place redox activity takes place is at or near the electrode surface. At point
1, (figure 1.15), the potential is at a sufficiently negative value so that no redox
activity takes place. At point 2, (figure 1.16), the positive potential sweep being
applied has decreased the energy of the electrons at the electrode surface sufficiently
that some of the reduced species at the surface become oxidized, and a current starts
28


to flow. Diffusion of reduced species to the electrode surface also begins to be driven
by a developing concentration gradient. The oxidized species are also undergoing
diffusion, but for the purpose of clarity are not shown. At point 3, (figure 1.17), the
potential is positive enough that the ratio of oxidized species to reduced species at the
electrode surface has increased, more reduced species diffuse to the surface and the
current increases. At point 4 (figure 1.18), the electrode potential is such that all of
the species at the surface are oxidized. At this point, the current reaches a maximum.
Recalling an example of a drop of dye being added to a glass of water (figure 1.19),
when the dye is first added, the concentration gradient is very high with the dye
occupying a very small volume. The dye quickly begins to diffuse toward the volume
of lesser concentration. As time passes, the dye becomes less concentrated, and flux
of material from a higher concentration to a lower continues, but at a slower rate. The
flux of material thus slows, as the concentration gradient has become less steep. The
diffusion, driven by the concentration gradient, will continue until the concentration
of dye is uniform throughout the solution. The same effect occurs past point 4. Point
5, (figure 1.20) is in the region of the voltammogram where the flux of reduced
species reaching the electrode surface is decreasing due to the increasing thickness of
the diffusion layer. As the flux of reduced species decreases, so does the current.
29



= Reduced species
= Oxidized species
Figure 1.15 Current versus applied potential at start
of cyclic voltammogram
30


Reduced species
Oxidized species
Figure 1.16 Current versus applied potential as
current starts to flow in a cyclic voltammogram
31



= Reduced species
- Oxidized species
Figure 1.17 Current versus applied potential as
diffusion causes increase in current in a cyclic
voltammogram
32



Reduced species
= Oxidized species
Figure 1.18 Current versus applied potential as
current reaches a maximum value in a cyclic
voltammogram
33


Figure 1.19 Diffusion of dye in a glass of water
34


1.8 The Three Electrode Cell
In the experiments conducted in this project, the three-electrode cell configuration
was used (figure 1.21). The working electrode is the electrode at which the oxidation
or reduction of our species of interest occurs. The auxiliary electrode is an electrode,
usually made of noble metal such as gold or platinum. When an oxidation occurs at
the working electrode, a reduction occurs at the auxiliary electrode. When a
sufficiently high positive potential, supplied by the power supply, is applied between
the working and auxiliary electrodes, an oxidation process will occur at the working
electrode. Electrons will be liberated from the species of interest to the working
electrode and flow through the external circuit. This electron flow is current, and is
measured by the ammeter. The potential of the working electrode is measured against
the constant potential of the reference electrode at all times. In this configuration, no
current flows through the reference electrode. A physical representation of our 3-
electrode cell is shown in figure 1.22. Our cell consists of an H-cell with a larger
main compartment and a smaller reference compartment. The working electrode is
placed in the main compartment, which contains a solution of our species of interest.
The reference and auxiliary electrodes are contained in the smaller compartment,
immersed in pure electrolyte. The compartments are connected by a glass frit to
35



(f | Reduced species
= Oxidized species
Figure 1.20 Current versus applied potential as
decrease in diffusion causes decrease in current
in a cyclic voltammogram
36


Figure 1.21 Schematic of a three-electrode cell
37


provide electrical contact, while, at the same time, to prevent the two solutions from
mixing. A provision is made for the deaeration of the sample solution in the working
arm; in this case, pressurized nitrogen is allowed to flow through the solution from
the bottom of the compartment. The power supply, ammeter, and voltmeter are all
contained in an instrument known as a potentiostat.
38


working
electrode
auxiliary
electrode
reference
electrode
'V*.-----
power suppley, voltmeter
& ammeter
potentiostat
Figure 1.22 Representation of our three-electrode
cell
39


2. Experimental
2.1 Reagents And Materials
Polypyrrole dispersed on polyurethane with a tosylate counter ion, polyanaline, gold
rods and Nation were purchased from Aldrich. Potassium chloride, sodium
dihydrogen phosphate, sodium hydroxide, hydrochloric acid (12 M), sulfuric acid (18
M), and acetonitrile, (HPLC grade) were purchased from Mallinckrodt. The
acetonitrile was used as received or distilled over phosphorus pentoxide and dried
over molecular sieves. Phosphorus pentoxide was obtained from MCB. Potassium
ferrocyanide was purchase from B & A. Pyrrole, HEPES, 2-mercaptoethylamine,
tetraethylammonium tosylate, tetrabutylammonium perchlorate,
tetramethylammonium perchlorate, poly-L-glutamic acid, poly-L-lysine
hydrobromide and ferritin were purchased from Sigma. The pyrrole was vacuum
distilled and stored over dried molecular sieves (4A). Nitrogen was purchased from
General Air, and gaseous ammonia was purchased from Matheson Tri-Gas.
Potassium dichromate, molecular sieves 4A, 8-12 mesh, hydrogen peroxide and
sodium perchlorate were obtained from Fisher Scientific Company. Ethanol (95%)
was purchased from Aaper Alcohol and Chemical Company. Diamond pastes were
40


purchased from Buehler and Elgin, gold foil and 0.01 mm gold wire from Alfa/Aesar.
Highly ordered pyrolytic graphite (HOPG) was kindly supplied by Dr. Arthur W.
Moore, Advanced Ceramics Corporation. All water used for electrochemical
experiments and rinsing of electrochemical equipment was pyrolytically distilled over
a heated platinum gauze catalyst in an oxygen atmosphere.
2.2 Equipment
2.2.1 Plasma Reactor
Glass and quartz tubes, glass tees and glass rods were purchased from Technical
Glass (Aurora, CO). A plasma reactor, (figure 2.1), was constructed using, a 12 mm
quartz tubing and various fittings. A manifold to control the concentrations of one or
more gases into the reactor was constructed from needle valves. The radio frequency
cavity was placed in the center of the quartz tube. Power was supplied by an Opthos
Microwave power generator, model MPG 4. The vacuum pressure was measured
using Granville-Phillips Series 275 pressure gauge. An 8 mm glass rod, with the ends
reduced to 4 mm diameter, was fitted to allow introduction of electrodes into the
plasma, by way of o-ring seals.
41


gases in
quartz tube
pusher rod
RF cavity
Figure 2.1 Plasma reactor
A
to vacuum
42


2.2.2 Potentiostats And Recorder
Cyclic voltammetry scans were performed using a Bioanalytical Systems CV27
Voltammograph (West Lafayette, IN) and a Cypress OMNI 90 Potentiostat. Data
were recorded on a Bioanalytical Systems RXY Recorder.
2.2.3 Cells
The H-Cells used for most work has been shown in figure 1.22. Some of the survey
scans were performed in low volume cells constructed by cutting the top end off
20 mL vials. Nitrogen was directed into these cells by fitting an Eppendorf pipette tip
onto polyethylene tubing and positioning the tip inside the vial, just below the lip
(figure 2.2). A low volume H-cell (Technical Glass, Aurora, CO) was constructed
from a 14 mm I.D. glass tube with a 24/40 standard taper ground glass fitting. A
small reference sidearm was attached to the side of the low volume, working arm by a
low-flow glass frit. A luer-lock capillary tube was fitted on the side of the working
arm, such that a stream of nitrogen could be directed at the surface of the liquid
(figure 2.3).
43


auxiliary
electrode
working
electrode
Figure 2.2 Cell used for survey work
44


24/40 ground glass taper
Figure 2.3 Low volume H-cell
45


2.2.4 Electrodes
A highly ordered plane graphite (HOPG) electrode was constructed by mounting the
HOPG with the edge planes exposed, in epoxy contained in a PVC tube (figure 2.4).
After the epoxy had cured, the working face of the electrode was polished in
successively finer emery paper, followed by crocus cloth, then successively finer
diamond pastes, ending with a paste of 0.25 pm diamond particles. The electrode was
then cleaned by sonication in a saturated solution of Alconox in ethanol. Shrink-wrap
tubing was attached to one end of the PVC tube. Electrical contact was made by
placing a small drop of mercury in contact with the graphite and inserting a wire into
the mercury. Gold foil was cut to size and fused to a gold wire. The wire and foil
flags were threaded through a 4 mm I.D. glass luer joint (Ace Glass). Flags whose
dimensions were too large for the inside diameters of the glass tubes were rolled into
cylinders and inserted inside the tubes, or were inserted into volumetric pipettes that
had been cut in half. Luer fittings were assembled on the tubes to allow for the
introduction of pressurized nitrogen (figure 2.5). A very simple electrode, with no
seams or other interfaces, was constructed by flattening one end of a gold rod, to
increase its surface area. This rod was then connected to the potentiostat via alligator
clips. Two gold disk electrodes were assembled by pressing 3 mm gold
46


Figure 2.4 Edge-plane pyrolytic graphite cell
47


H
L_
r
/

o
Figure 2.5 Gold flag electrodes in glass sheaths
48


rods into 9 mm outside diameter Kel-F (polychlorotrifluropolyethylene) rod that had
been bored and machined to accept the gold rods and conductor wires (figure 2.6).
The ends of the Kel-F rods opposite the gold rods were tapped to accept stainless
steel machine screws that pressed copper conductor wires firmly into the back of the
gold rods. Silver/silver chloride electrodes filled with 1 M potassium chloride were
used as reference electrodes in this work. Combination reference/auxiliary electrodes
were constructed using 1 mm I.D. luer joints (Ace Glass) and female lure-tipped
bulbs manufactured by Technical Glass. The luer-tipped tube was filled with 1 M
potassium chloride and fitted to the bulb. A silver wire was cleaned and inserted into
the tube until the inserted tip was in the bulb. A platinum wire was wrapped in a
loose spiral around the outside of the tube (figure 2.7). The last turn or so of the
platinum wire was wrapped around the flare on the bulb, thus securing the bulb in
place. Reference and auxiliary electrodes for use in the low volume cells have been
described by Zapien, D. C. and Johnson, M. A.18 Centricon tubes ultrafiltration tubes
were purchased from Millipore Corporation.
49


Figure 2.6 Gold disk pressed into Kel-F rod
50


II
(

Figure 2.7 Reference and auxiliary electrodes
51


2.3 Procedures
2.3.1 Cleaning Of Apparatus
All glassware used for electrochemical experiments was cleaned by soaking in a
chromic acid cleaning solution. After cleaning, the glassware was rinsed with water
and either allowed to air-dry or were rinsed with the solution being used. The gold
flag electrodes inside the glass sheaths were cleaned by soaking in chromic acid.
Prior to use, the cleaned gold electrodes were rinsed with water and then ethyl
alcohol. Alternately the gold flag electrodes were cleaned by removing the flags from
the protective glass sheaths and heated to incandescence in a fuel-rich, natural gas
flame. Graphite electrodes and gold disk electrodes were cleaned by polishing, first
on successively finer emery cloth, then crocus cloth, followed by successively finer
diamond polishing pastes. These electrodes were then rinsed with water followed by
ethyl alcohol and allowed to air-dry. Gold electrodes were further cleaned by
stepping between +1.50 V and -0.35 V versus a silver/silver chloride reference. If the
gold was protected by a sheath, the gold surface was purged with nitrogen, prior to
stepping to -0.35 V.
52


2.3.2 Treatment Of Gold With 2-
Mercaptoethylamine
A gold foil flag cleaned as described above. The flag was placed in a dilute solution
of 2-mercaptoethylamine in ethanol and left overnight. The electrode was then rinsed
with ethanol and air-dried.
2.3.3 Plasma Modification Of HOPG Edge Plane
Electrode
The plasma apparatus was assembled and checked for vacuum leaks. The pressure
was reduced to 0.25 torr using a vacuum pump and a liquid nitrogen cooled cold-
finger installed between the plasma apparatus and the pump. Valves were
successively opened, starting at the end of the apparatus nearest the pump to check
for leaks and the pressure observed after each valve was opened. After verifying that
there were no appreciable leaks in the system, helium was bled into the apparatus via
a needle valve. A helium plasma was ignited by applying the discharge from a Tesla
coil to the radio frequency (RF) cavity and slowly increasing the forward power on
the power supply. The helium plasma was then tuned using the adjustment knobs on
the RF cavity and the applied forward power until the plasma was uniform and the
reflected power minimized. The helium plasma was used to remove oxygen
functional groups at the graphite surface, prior to treatment with the ammonia plasma.
53


The oxygen containing groups include carbonyls, hydroxyls, carboxyls, lactones, etc.,
which form at the surface upon exposure to air. A dummy electrode was constructed
using PVC tubing and epoxy. This dummy was polished in the same fashion as the
edge-plane HOPG electrode. This dummy was fitted to the 8 mm Pyrex rod with
shrink-wrap tubing. The dummy was inserted into the plasma reactor. The helium
plasma was reignited, and the dummy was slowly moved into the plasma. The
plasma melted the dummys PVC and epoxy. Several attempts were made, at
decreasing forward power levels, to introduce the dummy into the helium plasma
without damage to the PVC and epoxy. Through discussions with Dr. Larry G.
Anderson, it was suggested that there might be enough tenuous plasma outside the
plasma cavity to effectively treat the electrode. The apparatus was reconfigured to
allow the electrode to approach the plasma from the vacuum side of the plasma tube
with the belief that the gases, bled into the tube from the other end and swept towards
the vacuum, may cause a higher concentration of tenuous plasma on the vacuum
pump side of the RF cavity. The dummy electrode was brought to within 2.5
centimeters of the plasma and held there for five minutes, with no apparent damage.
After cleaning, an edge-plane HOPG electrode was fitted to one end of the eight-
millimeter rod with shrink-wrap tubing. The apparatus was reassembled and the
plasma ignited. The electrode was moved to within 2.5 cm of the plasma. The
electrode was treated for ten minutes with a helium plasma to remove surface oxygen
54


groups. The plasma gas was switched from helium to ammonia and the electrode was
treated for another ten minutes to apply amine groups to the surface. The plasma
power was turned off and the electrode was left in a low-pressure ammonia
atmosphere for another ten minutes, prior to venting the apparatus to the atmosphere
and removing the electrode. The treated electrode was removed from the glass rod
and assembled as described above (figure 2.4).
2.3.4 Formation Of Films
Nation was applied to a polished edge-pane HOPG electrode with a dropper and
allowed to air dry. Inspection of the dried Nation surface showed fractures in the
surface that exposed the graphite surface. Isopropyl alcohol (HPLC grade) was
applied to the Nation surface to redissolve and smooth the film.
2.3.5 Formation And Conditioning Of Polypyrrole
Layers
A pyrrole solution, 0.05 M, in acetonitrile containing 0.1 M counter ion was added to
the working arm of an H-cell. The counter ions were either perchlorate anions or
tosylate anions. A gold flag rolled into a cylinder and fitted inside a glass sheath, or a
gold disk was placed in an H-cell and coated with polypyrrole perchlorate. The
55


polypyrrole perchlorate was polymerized from a solution of acetonitrile, until no
more gold was visible and the surface appeared uniformly covered. The coated flag
was rinsed in acetonitrile to remove any perchlorate salt and then with water to
remove any acetonitrile. The solution in the working arm was deaerated by bubbling
nitrogen through the solution. After deaeration, the nitrogen flow was adjusted to
provide a nitrogen blanket above the solution in the working arm. The layers were
formed by both potentiostatic and potentiodynamic methods. Two potentiostatic
methods were used. The first method simply applied an oxidative potential to the
working electrode such that oxidation of pyrrole commenced immediately. The other
started at a potential where no oxidation took place and ramped anodically to the
potential where oxidation commenced, at which point the potential was held for the
duration of the film formation. The current was monitored versus time and the
amount of charge passed per unit area to form the film (figure 2.8) was used as an
estimate of the film thickness. In the potentiodynamic method, the potential was
scanned anodically to the point where oxidation occurred and then the potential
sweep rate was reversed. Several cyclic scans were performed until the polypyrrole
film covered the surface. Polypyrrole was formed from solutions of dry acetonitrile
and acetonitrile containing no added water, 1 % added water, and 2 % added water.
56


microamps
Figure 2.8 Current versus time during formation of
a polypyrrole layer.
57


2.3.6 Cyclic Voltammetry Of Ferritin Using
Polypyrrole Electrodes
Polypyrrole electrodes were conditioned by repeated cycling on buffered and non-
buffered electrolyte solutions, as well as in solutions of ferritin. The electrodes were
conditioned until no changes were observed in the voltammograms obtained by
subsequent cycles. The electrodes were then rinsed with fresh water and air-dried
prior to performing scans in ferritin. All ferritin solutions scanned, were adjusted to
pH 7 with buffer. The concentration of the buffer, sodium dihydrogen phosphate,
was varied from 0.1 M to 1 M, while that of the electrolyte, potassium chloride, was
varied from 0.1 M and 1 M.
58


3. Results And Discussion
3.1 Preliminary Electrode Modifications
The gold foil surface treated with the 2-mercaptoethylamine should have the thiol
groups adsorbed to the gold surface, and the amine groups at other end of the ethyl
tethers directed towards the solution. The directed amine groups were chosen since at
pH 7 they will be protonated, giving the electrode surface a positive charge. It was
hoped that this would interact favorably with the overall negative charge on the
ferritin shell at pH 7. Using the pKa of the conjugate acid of ethylamine, an estimate
of the ratio of conjugate acid to amine directed toward the solution should be around
6400 to 1. The short tether length adsorbates exhibit a more random orientation than
those with longer tethers. Longer chains adsorbates tend to self-align and form an
ordered layer, similar to the bristles on a toothbrush. Additionally, if the tethers
become too long they will form an insulating layer 19 and thus inhibit electron
transfer. A voltammogram of ferritin in a 0.1 M ionic strength phosphate buffer
(figure 3.1) is very similar to a voltammogram previously observed for adsorbed
ferritin, with a cathodic peak at approximately -0.4 V and an anodic peak at -0.1 V.20
In subsequent scans, the cathodic peak current at -0.4 V grew smaller
59


-0.60 V
+0.20 V
Figure 3.1 Cyclic voltammogram of ferritin at gold
modified with 2-mercaptoethylamine
60


and a small cathodic peak appeared at -0.2 V. After the scans in ferritin, the electrode
was rinsed with water and scans were performed in blank buffer. The voltammogram
for this scan (figure 3.2) still exhibited anodic and cathodic peaks, but at more
positive potentials, and was not similar to the background scan previously observed.
20. This result may be an indication that not all of the ferritin was strongly adsorbed
and there may be more than one type of adsorption. The first cycle in the clean buffer
exhibits a cathodic peak at -0.28 V and an anodic peak at -0.02 V. On subsequent
cycles, the peak at -0.28 V disappeared and a new cathodic peak at -0.20 V appears
and is persistent in additional sweeps. The anodic peak potential did not change with
repeated cycles. Gold electrodes treated with Nafion (a perfluoro polymer, with
perfluoro side-chains that are terminated with sulfonic acid groups) produced ferritin
voltammograms similar to those produced by the mercaptoethylamine-modified
electrode, and their use was discontinued.
3.2 Plasma-Modified HOPG Edge Plane Electrode
The background scans in blank buffer using the plasma-modified HOPG edge plane
electrode show the same charging background current as predicted by the equation
1.1. The voltammograms of ferritin (figure 3.3) looked like the scans of the
61


-0.6 V
+0.20 V
Figure 3.2 Cyclic voltammogram of background
with 2-mercaptoethylamine modified gold, after
scans in ferritin
62


-0.50 V
+0.80 V
Figure 3.3 Cyclic voltammogram of ferritin at
ammonia plasma modified edge-plane pyrolytic
graphite electrode
63


background (figure 3.4), indicating no activity at this electrode. It may have been
possible that the HOPG edge plane was not modified with amine groups by the
ammonia plasma apparatus used. As we did not perform an analysis of the electrode
surface with a surface-sensitive technique, such as x-ray photoelectron spectroscopy
before and after treatment in the plasma tube, the nature of the surface was not
characterized.
3.3 Polypyrrole
The majority of the work to modify the gold electrodes was performed using
polypyrrole. This polymer was chosen, as it is conductive in the potential range being
used to study ferritin. Additionally, in its conductive form, there is a positive charge
on every three to four repeating amine units. Dilute solutions of pyrrole were
electrochemically oxidized to form a polypyrrole layer on a gold substrate. The
counter ion is incorporated into the polypyrrole layer and contributes to the films
properties. The characteristics of the polymer film may also be controlled with
careful selection of the counter ion. The polymerization of pyrrole starts at a
potential that is high enough to oxidize pyrrole at an anode, to form a radical cation.
The concentration of radical cations is high at the electrode surface and dimerization
64


-0.50 V
+0.80 V
Figure 3.4 Cyclic voltammogram of background at
ammonia plasma modified edge-plane pyrolytic
graphite electrode
65


of the radicals occurs. The dication formed deprotonates to form a pyrrole dimer.
Oxidation of dimers occurs at a lower potential than monomers, oxidation of trimers
at a potential lower than dimers, and so forth. The dimer is oxidized to form a radical
cation and reacts with a pyrrole molecule forming a radical trication, which in turn, is
deprotonated to form a trimer. The trimer is oxidized and so forth until a polymer is
formed. Oxidation of the polymer and incorporation of counter ion lead to the
1
formation of a conductive polymer (Figure 3.5).
3.3.1 Observation Of Charging Current With
Polypyrrole On A Gold Electrode
After the gold/polypyrrole electrode was cycled three times in 1 M sulfuric acid and
then rinsed with water. Two scans were performed in 1 M potassium chloride, and a
charging current that had nearly parallel traces between the scan directions was
obtained. A few crystals of potassium ferrocyanide were added to the cell and the
solution stirred by bubbling nitrogen from the bottom. After the solution had been
mixed, a scan was obtained that showed the anodic and cathodic waves of
ferrocyanide superimposed on the charging current previously observed (figure 3.6).
66


Figure 3.5 Polypyrrole and counter ion
67


-0.15 V
+0.80 V
-0.20 V +0.60 V
Figure 3.6 Cyclic voltammogram of background
and ferrocyanide at polypyrrole-perchlorate
modified gold foil electrode.
68


Based upon these observations it is believed that this electrode is conductive and its
use to achieve direct electron transfer of ferritin is worth pursuing.
3.3.2 Diffusion-Controlled Direct Electron
Transfer Of Electron Ferritin On
Polypyrrole
A polypyrrole layer was formed on a gold electrode as described above, with the
exception of omitting the treatment in sulfuric acid, as subsequent attempts to treat
with sulfuric acid did not produce the charging current observed in the first
experiment. After rinsing to remove excess pyrrole, counter ion, and acetonitrile, the
electrode was placed in a solution of ferritin (5 mg per mL 0.1 M KC1, phosphate pH
7 buffer) and the cyclic voltammogram shown in figure 3.7 was obtained. The
voltammogram was obtained using a scan rate of 10 mV/s due to the slow theoretical
diffusion of ferritin, based upon calculations using the Einstein-Stokes equation. This
voltammogram possesses features suggesting diffusion-controlled electron transfer of
ferritin. The voltammogram is symmetrical and the peak separation is 480 millivolts
at 10 millivolts per second. The E0' estimated by the potential midway between the
cathodic peak potential and anodic peak potential was -0.06 volts, which matches
well with the E0' of -0.07 volts of ferritin adsorbed at a bare gold electrode. The
69


-1.00 V
+0.40 V
Figure 3.7 Cyclic voltammogram of ferritin at
polypyrrole-perchlorate modified gold foil
electrode, electrode area approximately 1 cm2,
scan rate 10 mV/s, Ag/AgCl reference electrode
70


similarity of the potentials suggests that ferritin does not unfold appreciably when it
adsorbs. This result suggests that absorbed ferritin probably retains the redox activity
of native ferritin. A systematic approach was begun to determine the conditions
needed to reproduce the original voltammogram. New cleaning solutions were
prepared and all glassware was cleaned. Acetonitrile was purified to remove any
water and new reagent solutions were made. Careful monitoring was made of the
amount of charge passed per unit area to form the polypyrrole layer and the amount
of water in the monomer solution was controlled. The polypyrrole layer was formed
at several potentials. Attempts to repeat this experiment were initially unsuccessful.
However, after continued trials, the voltammogram was reproduced. A polypyrrole
film was formed and transferred to a blank buffer solution and a background obtained
after three scans from +0.60 V to -0.75 V at 5 mV per second (figure 3.8). After the
background scan, the electrode was transferred to a solution of ferritin (34 mg/mL)
diffusion-controlled direct electron transfer of ferritin was observed (figure 3.9). A
plot of the peak cathodic current versus the square root of the scan rate produced a
linear fit, providing evidence of a diffusion controlled process (figure 3.10). After
rinsing the electrode, the background buffer was scanned and the blank was again
observed (figure 3.11), again indicating the voltammetric response in the ferritin
solution was indeed due to ferritin. Repeated attempts to reproduce the above
71


= 50 pA
-0.75 V
+0.60 V
Figure 3.8 Cyclic voltammogram of background
polypyrrole-perchlorate modified gold foil
electrode
72


-0.75 V
+0.60 V
Figure 3.9 Cyclic voltammogram of ferritin at
polypyrrole-perchlorate modified gold foil
electrode
73


peak cathodic current, p A
peak cathodic current vs square root of scan rate 08/23/01
60.0 y
50.0
40.0
30.0
20.0
10.0
0.0
0.750 0.950 1.150 1.350 1.550 1.750 1.950 2.150
square root scan rate, mV/s
sqrt rate vs Ipc Linear (sqrt rate vs Ipc)

I I I " I I I
Figure 3.10 Peak cathodic current versus square root
scan rate, 34 mg ferritin/mL, 1 M KC1, pH 7
buffer, 1 cm2 polypyrrole electrode
2.350
74


-0.75 V
+0.60 V
Figure 3.11 Cyclic voltammogram of background at
polypyrrole-perchlorate modified gold foil
electrode, after scans in ferritin
75


experiment again met with failure. The flattened gold rod was used instead of the
gold wire/gold foil flag assembly to determine if the problem was due to material
trapped between the gold foil and wire. No differences were observed. The lack of
differences between the flattened gold rod and the gold foil/wire assembly support the
hypothesis that the inability to reproduce the direct electron transfer of ferritin is not
due to contamination at the gold foil/wire interface. Additionally, as the flags were
surrounded by a glass sheath and the flattened rod was not, the implication was that
the presence of the glass sheath is not a factor in reproducing the voltammogram of
ferritin at gold/polypyrrole.
3.3.3 Treatment With Hydrogen Peroxide
An experiment was performed to verify whether non-uniformity of the polypyrrole
film was responsible for the non-reproducibility of the results. Polypyrrole-
perchlorate films are known to be porous and it may be possible for a species to
traverse the pores in the polypyrrole film and react at the gold surface. A background
(figure 3.12) was obtained in 1 M potassium chloride, pH 7, 0.1 M phosphate buffer
for a gold/polypyrrole-perchlorate film. A voltammogram of ferritin (51 mg/mL) in
the same buffer, is same as the background (figure 3.13) indicating the layer was not
76


-0.60 V
+0.55 V
Figure 3.12 Cyclic voltammogram of background at
polypyrrole-perchlorate modified gold foil
electrode
77


-0.60 V
+0.55 V
Figure 3.13 Cyclic voltammogram of ferritin at
polypyrrole-perchlorate modified gold foil
electrode
78


promoted direct electron transfer of fenitin. A few crystals of potassium ferrocyanide
were added to the background buffer, and it was verified that a voltammogram could
be obtained (figure 3.14). The film was air-dried overnight and a voltammogram of
8.9 mM ferrocyanide in the above electrolyte verified that the electrode film was still
conductive. Exposure to hydrogen peroxide is known to destroy the conductivity of
polypyrrole films. The electrode was soaked in 30 % hydrogen peroxide for fifteen
minutes, rinsed with water and allowed to air dry. After the conductivity of the
polypyrrole layer was destroyed, a scan in 8.9 mM ferrocyanide showed an attenuated
charging current superimposed by two new peaks, an anodic peak at +0.28V and a
cathodic peak at + 0.23 V (figure 3.15). These two new peaks appear to be a redox
couple. Scans in blank buffer and ferritin solutions revealed similar voltammograms.
From these observations, it is concluded that the observed voltammetric responses are
due to activity at the polypyrrole surface and not to diffusion into pores on the surface
and reaction at the gold surface.
79


-0.20 V
+0.60 V
Figure 3.14 Cyclic voltammogram of ferrocyanide at
polypyrrole-perchlorate modified gold foil
electrode
80


T=50 |xA
-0.20 V
+0.60 V
Figure 3.15 Cyclic voltammogram of ferrocyanide at
polypyrrole-perchlorate modified gold flag
electrode, after destruction of polypyrrole
conductivity
81


3.3.4 Troubleshooting Whether A Polypyrrole
Film Will Give Cyclic Voltammograms Of
Ferrocyanide
Unless otherwise noted, the scans were performed in an H-cell with a luer-lock
combination auxiliary and reference electrode. A gold disk electrode was immersed
in a 4.1 mM solution of ferrocyanide in 1 M potassium chloride and a voltammogram
obtained (figure 3.16). From the anodic peak current, the electrode area was
calculated and the value was in agreement with that calculated from the disk radius.
The electrode was then coated with a polypyrrole film and a background obtained in 1
M potassium chloride. The background was clean with parallel currents on the
anodic and cathodic sweeps (figure 3.17). A voltammogram was obtained in the 4.1
mM ferrocyanide solution and the voltammetric waves were observed to be
superimposed on the background charging current (figure 3.18). The disk was next
placed in a 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer and a new
background was obtained. This new background was very similar to the background
in 1 M potassium chloride, with the exception of increasing currents as the sweeps
approached the switching potentials (figure 3.19). A voltammogram obtained in 4.4
mM ferrocyanide in 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer showed
the expected waves superimposed on the new charging current (figure 3.20). To
82


-0.20 V
+0.60 V
Figure 3.16 Cyclic voltammogram on 4.1 mM
ferrocyanide in 1 M potassium chloride, at gold
disk electrode
83


-0.20 V +0.60 V
Figure 3.17 Background of 1 M potassium chloride,
at polypyrrole-perchlorate on gold disk
electrode
84


-5 nA
-0.20 V +0.60 V
Figure 3.18 Cyclic voltammogram on 4.1 raM
ferrocyanide in 1 M potassium chloride, at
polypyrrole-perchlorate on gold disk electrode
85


-0.20 V
+0.60 V
Figure 3.19 Background 1 M potassium chloride, 0.2
M phosphate, pH 7 buffer at polypyrrole-
perchlorate on gold disk electrode
86


-0.20 V
+0.60 V
Figure 3.20 Cyclic voltammogram of 4.4 mM
ferrocyanide in 1 M potassium chloride, 0.2 M
phosphate, pH 7 buffer at polypyrrole-
perchlorate on gold disk electrode
87


Full Text

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DIFFUSION CONTROLLED DIRECT ELECTRON TRANSFER OF FERRITIN by James Martin Halsey B.S. University ofNebraska at Omaha, 1979 B.A., University ofNebraska at Omaha, 1979 A thesis submitted to the University of Colorado at Denver in partial fulfillment of the requirements for the degree of Master of Science Chemistry 2002 r" --., I .. I iP \ .. .!... ( L..., ...... ..,.

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This thesis for the Master of Science degree by James Martin Halsey has been approved by Donald C. Zapien Larry G. Anderson ng tooz, Date

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Halsey, James Martin (M.S., Chemistry) Diffusion-Controlled Direct Electron Transfer Of Ferritin Thesis directed by Associate Professor Donald C. Zapien ABSTRACT Horse spleen ferritin is a protein whose functions are to sequester excess iron, to store it in a soluble form, and to release the iron whenever needed. Electron transfer steps are known to be involved in uptake and release of iron by ferritin. To date, all cyclic voltammetry studies on ferritin have involved ferritin adsorbed at an electrode sutface. This thesis describes the effort to effect cyclic voltammetry on ferritin in solution. It is hoped that this will give an indication as to whether or not the ferritin adsorbed at electrodes behaves electrochemically as native ferritin. This abstract accurately represents the content of the candidate's thesis. I recommend its publication. Donald C. Zapien lll

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DEDICATION I wish to acknowledge my lovely wife, Tina, for her patience and understanding during this endeavor.

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ACKNOWLEDGEMENTS I wish to thank Dr. Donald C. Zapien for his guidance, support and perseverance to assist me in this effort. I would like to thank the members of my Master's committee: Dr. Donald C. Zapien, Dr. Larry G. Anderson, and Dr. John C. Lanning. I would also like to thank all the members of our research group for their support and input. I would like to thank Mr. Paul Miller for assembling the gold disk electrodes, Dr. Larry G. Anderson and Mr. Jeff Boon for the loan of equipment. Dr. James Chambers, University of Tennessee, provided some helpful suggestions on polypyrrole.

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CONTENTS Figures .................................................................................................................... ix 1. Introduction .................................................................................................. 1 1.1 Research Objective ...................................................................................... 1 1.2 Fenitin Function .......................................................................................... 2 1.3 Structure OfF erritin ..................................................................................... 2 1.4 Uptake And Release Of Iron By Ferritin ..................................................... 6 1.5 Electrochemistry Of Proteins ....................................................................... 7 1. 5.1 Indirect Methods .......................................................................................... 7 1.5.2 Direct Electron Transfer Of Proteins ........................................................... 7 1.5.3 Direct Electron Transfer Of Ferritin .......................................................... 15 1.5.4 Diffusion-Controlled Direct Electron Transfer Of Ferritin ....................... 15 1.5 .5 Direct Electron Transfer Of Cytochrome c ................................................ 18 1.5.6 Diffusion Controlled Direct Electron Transfer OfFerritin ........................ 20 1.6 Cyclic Voltammetry And Pseudocapacitance Of The Electrolyte Double Layer ........................................................................... 21 1.7 Cyclic Voltammetry And Electroactive Species ........................................ 22 Vl

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1.8 The Three Electrode Cell ........................................................................... 35 2. Experimental .............................................................................................. 40 2.1 Reagents And Materials ............................................................................. 40 2.2 Equipment .................................................................................................. 41 2.2.1 Plasma Reactor ........................................................................................... 41 2.2.2 Potentiostats And Recorder ....................................................................... .43 2.2.3 Cells ........................................................................................................... 43 2.2.4 Electrodes ................................................................................................... 46 2.3 Procedures .................................................................................................. 52 2.3.1 Cleaning Of Apparatus .................................................. ........................... 52 2.3.2 Treatment Of Gold With 2-Mercaptoethylamine ...................................... 53 2.3.3 Plasma Modification Of HOPG Edge Plane Electrode .............................. 53 2.3.4 Formation Of Films ................................................................................... 55 2.3.5 Formation And Conditioning OfPolypyrrole Layers ................................ 55 2.3.6 Cyclic Voltammetry Of Ferritin Using Polypyrrole Electrodes ................ 58 3. Results And Discussion ............................... ............................................. 59 3.1 Preliminary Electrode Modifications ......................................................... 59 Vll

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3.2 Plasma-Modified HOPG Edge Plane Electrode ........................................ 61 3.3 Polypyrrole ................................................................................................. 64 3.3.1 Observation Of Charging Current With Polypyrrole On A Gold Electrode .................................................................................................... 66 3.3 .2 Diffusion-Controlled Direct Electron Transfer Of Electron Ferritin On Polypyrrole .............................................................................. 69 3.3.3 Treatment With Hydrogen Peroxide .......................................................... 76 3.3.4 Troubleshooting Whether A Polypyrrole Film Will Give Cyclic Voltammograms OfFerrocyanide ............................................................. 82 3.3.5 Peak Current Dependence On Ferritin Concentration ............................... 88 3.4 Conclusions ................................................................................................ 93 References ............................................................................................................. 95 Vlll

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FIGURES Figure 1.1 Ribbon diagram of ferritin subunit ........................................................... .4 Figure 1.2 Quaternary structure of ferritin .................................................................. 5 Figure 1.3 Methyl viologen mediated electron transfer. ............................................. 8 Figure 1.4 Direct electron transfer at an electrode ...................................................... 9 Figure 1.5 Formation of an insulating layer .............................................................. 11 Figure 1.6 Unfavorable orientation of a protein ....................................................... 12 Figure 1.7 Plasma modification of an electrode surface ........................................... 13 Figure 1.8 Formation of a chemisorbed layer on of an electrode surface ...................................................................................... 14 Figure 1.9 IR cell for determining structural changes in oxidized and reduced ferritin ................................................................... 16 Figure 1.10 Interaction between cytochrome c and adsorbed 4thiopyridine .............................................................................................. 19 Figure 1.11 Potential versus time used in cyclic voltammetry ................................... 23 Figure 1.12 Current response to changing potential, in the absence of an electroactive species ......................................................... 24 Figure 1.13 Typical current response of an electroactive species due to changing potential ............................................................ 25 IX

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Figure 1.14 Electron transfer between electrode surface and species in solution .................................................................................... 26 Figure 1.15 Current versus applied potential at start of cyclic voltanunogram ......................................................................................... 30 Figure 1.16 Current versus applied potential as current starts to flow in a cyclic voltanunogram ............................................................... 31 Figure 1.17 Current versus applied potential as diffusion causes increase in current in a cyclic voltanunogram ......................................................................................... 32 Figure 1.18 Current versus applied potential as current reaches a maximum value in a cyclic voltanunogram .......................................... 33 Figure 1.19 Diffusion of dye in a glass of water ......................................................... 34 Figure 1.20 Current versus applied potential as decrease in diffusion causes decrease in current in a cyclic voltanunogram ......................................................................................... 36 Figure 1.21 Schematic of a three-electrode ce11 ......................................................... .37 Figure 1.22 Representation of our three-electrode cel1.. ............................................. 39 Figure 2.1 Plasma reactor ......................................................................................... 42 Figure 2.2 Cell used for survey work. ....................................................................... 44 Figure 2.3 Low volume H-ce11 .................................................................................. 45 Figure 2.4 Edge-plane pyrolytic graphite cell.. ........................................................ .47 Figure 2.5 Gold flag electrodes in glass sheaths ...................................................... .48 X

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Figure 2.6 Gold disk pressed into Kel-F rod ............................................................. 50 Figure 2.7 Reference and auxiliary electrodes .......................................................... 51 Figure 2.8 Current versus time during formation of a polypyrrole layer ..................................................................................... 51 Figure 3.1 Cyclic voltammogram offerritin at gold modified with 2-mercaptoethylamine ..................................................................... 60 Figure 3.2 Cyclic voltanunogram of background with 2mercaptoethylamine modified gold, after scans in ferritin ...................................................................................................... 62 Figure 3.3 Cyclic voltammogram of ferritin at ammonia plasma modified edge-plane pyrolytic graphite electrode .................................................................................................. 63 Figure 3.4 Cyclic voltammogram of background at ammonia plasma modified edge-plane pyrolytic graphite electrode .................................................................................................. 65 Figure 3.5 Polypyrrole and counter ion .................................................................... 67 Figure 3.6 Cyclic voltammogram of background and ferrocyanide at polypyrrole-perchlorate modified gold foil electrode .................................................................................... 68 Figure 3.7 Cyclic voltammogram of ferritin at polypyrrole perchlorate modified gold foil electrode, electrode area approximately 1 cm2 scan rate 10 mV/s, Ag/ AgCl reference electrode ................................................................... 70 Figure 3.8 Cyclic voltammogram of background polypyrroleperchlorate modified gold foil electrode ................................................. 72 XI

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Figure 3.9 Cyclic voltammogram of ferritin at polypyrroleperchlorate modified gold foil electrode ................................................. 73 Figure 3.10 Peak cathodic current versus square root scan rate, 34 mg ferritinlmL, 1 M KCl, pH 7 buffer, 1 cm2 polypyrrole electrode ............................................................................... 7 4 Figure 3.11 Cyclic voltammogram of background at polypyrrole-perchlorate modified gold foil electrode, after scans in ferritin ............................................................... 75 Figure 3.12 Cyclic voltammogram of background at polypyrrole-perchlorate modified gold foil electrode .................................................................................................. 77 Figure 3.13 Cyclic voltammogram of ferritin at polypyrroleperchlorate modified gold foil electrode ................................................. 78 Figure 3.14 Cyclic voltammogram offerrocyanide at polypyrrole-perchlorate modified gold foil electrode .................................................................................................. 80 Figure 3.15 Cyclic voltammogram offerrocyanide at polypyrrole-perchlorate modified gold flag electrode, after destruction of polypyrrole conductivity ............................................................................................. 81 Figure 3.16 Cyclic voltammogram on 4.1 mM ferrocyanide in 1 M potassium chloride, at gold disk electrode ....................................... 83 Figure 3.17 Background of 1 M potassium chloride, at polypyrrole-perch1orate on gold disk electrode ....................................... 84 Figure 3.18 Cyclic voltammogram on 4.1 mM ferrocyanide in 1 M potassium chloride, at polypyrroleperchlorate on gold disk electrode ........................................................... 85 Xll

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Figure 3.19 Background 1M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole-perchlorate on gold disk electrode .............................................................................. 86 Figure 3.20 Cyclic voltammogram of 4.4 mM ferrocyanide in 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole-perchlorate on gold disk electrode .................................................................................................. 87 Figure 3.21 Cyclic voltammogram of24 mg ferritinlmL 1M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole-perchlorate on gold disk electrode ................................... 89 Figure 3.22 Cyclic voltammogram of 4.4 mM ferrocyanide in 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole-perchlorate on gold disk Figure 3.23 electrode, after scans in ferritin ............................................................... 90 Peak current dependence on bulk concentration at four scan rates, 0.08 cm2 polypyrrole electrode, Ag/ AgCl reference, Pt auxiliary, 1 M KCl, 0.2 M phosphate pH 7 buffer ............................................................................. 92 Xlll

PAGE 14

1. Introduction 1.1 Research Objective The aim of this effort is to effect the diffusion-controlled direct electron transfer of ferritin. Direct electron transfer is defined as electron transfer between a species in solution and an electrode. It is especially important if one wants to obtain kinetic information or investigate chemical reactions that are coupled to electrochemical reactions. To date, all direct electron transfer of ferritin has been due to ferritin adsorbed at an electrode. As ferritin is not in an adsorbed state in organisms, it is hoped that the diffusion-controlled reaction will more closely resemble the electron transfer reactions of ferritin in vivo. Understanding the mechanisms of iron uptake and release are important for the following reasons: understanding iron overload and deficiencies, improving crop yields and controlling iron toxicity. Probing the role of electron transfer will provide insight into the mechanisms of uptake and release of iron by ferritin. 1

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1.2 Ferritin Function Ferritin is found in all known organisms, both aerobic and anaerobic. Ferritin has been thought to have appeared in organisms that required iron, in response to the presence of oxygen in the atmosphere. Oxygen oxidizes iron (II) to iron (III) with an accompanying reduction in solubility. At physiological pH, the concentration of iron (II) ion in equilibrium with iron (II) hydroxide is on the order of I o-2 M. At the same pH, the concentration of iron (III) in equilibrium with iron (III) hydroxides is on the order of 10 -I& M. In addition to maintaining the iron in a soluble form, oxidizing iron (II) to iron (III) and sequestering it within the protein shell, the formation of oxygen radicals and superoxides is prevented through the reaction between oxygen and iron (II). In anaerobes, ferritin is thought to consume and therefore detoxify oxygen 1 1.3 Structure Of Ferritin Ferritin is an assembly of24 protein subunits, that form a hollow, nearly spherical shell 12 nanometers in diameter. The interior volume of this hollow sphere is 256 cubic nanometers.2 This indicates an internal diameter of nearly eight nanometers and shell thickness of two nanometers. Iron is stored as a mineral core within the interior and in contact, at various points, with the interior of the shell. If the interior 2

PAGE 16

of the protein is devoid of iron the shell is called apoferritin. The amount of iron stored within the shell varies. Typically, the number of iron atoms per wild-type ferritin ranges from 800 to 2500, but this number can be as high as 4500 iron atoms.3 The molecular weight of the apoferritin shell is 450,000 grams per mole. The average structure of the iron core is (FeOOH)s FeO OP03H2 Within the core, 20-33% of the iron present is in a different environment than the rest of the core's iron. These differences are due to coordination of iron with differing amount of phosphates and could account for differences in rate of iron release from ferritin in vitro. 4 Two types of subunits are found in most vertebrate ferritins, being designated an H -subunit and an L-subunit. The subunits vary in amino acid sequence, but in general are composed of four long helices and two short helices. The four long helices are bundled antiparallel and parallel to each other (figure 1.1). The 24 subunits self-assemble to form the hollow sphere. The ends labeled as "E" of four subunits come together to form six hydrophobic channels into the interior of the protein shell. The ends labeled "N", which is theN-terminus of the polypeptide, of three subunits come together to form eight hydrophilic channels into the interior. It is through the hydrophilic channels that iron enters and exits the shell. (figure 1.2) 3

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Figure 1.1 Ribbon diagram of ferritin subunit5 4

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Figure 1.2 Quaternary structure of ferritin 5

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1.4 Uptake And Release Of Iron By Ferritin The H-subunits catalyze the oxidation of iron (II) to iron (Ill) and the L-subunits facilitate iron core formation. 3 In the uptake of iron by ferritin, iron (II) first enters one of the eight hydrophilic channels. As the iron moves towards the interior of the shell glutamic acid residues E23, E58 and histidine residue H61 in an H-subunit "bind" one iron (II). A second iron (II) is "bound" by glutamic acid residues E57, E88, and El03. These "binding sites" are termed the ferroxidase center. There is one ferroxidase center per H-subunit. The residues that form the ferroxidase center in the H-subunit are not conserved in the L-subunits.6 The L-subunit contains an inner surface that is negatively charged and these areas are thought to be nucleation sites for the iron core. 7 Before iron can be released from the core, it must first be converted to iron (II). Thus, the release of iron from the core involves a reduction. It has been suggested that there may be an unfolding of ferritin step involved in the release of iron from ferritin. 8 6

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1.5 Electrochemistry Of Proteins 1.5.1 Indirect Methods Indirect methods may be used to achieve voltammetry at electrodes. In the example shown in figure 1.3, the oxidized form of methyl viologen (MV2+) approaches an electrode, accepts an electron, and is reduced to MV+ .. The reduced form diffuses away from the electrode and approaches the oxidized form of the species being studied. The reduced form of MV gives up an electron to the oxidized form of the protein, reducing the protein and being oxidized itself. The MV may now diffuse back toward the electrode and repeat the process. Information obtained by this type of experiment includes the value of n, and an estimate of '. The principal disadvantage of indirect electron transfer is that indirect electron transfer is not specific. For example, if a protein has two or more electroactive centers, that react in different potential windows, the mediator may react with all of centers at the same potential. 1.5.2 Direct Electron Transfer Of Proteins If one wishes to study the mechanisms for a reaction, one must effect direct electron transfer between the species of interest and the electrode. Since it is the current that 7

PAGE 21

electrode oxidized form e\:0 reduced form reduced form 0 e0 oxidized form Figure 1.3 Methyl viologen mediated electron transfer 8

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e-electrode reduced form e-oxidized form Figure 1.4 Direct electron transfer at an electrode 9

PAGE 23

can be used to directly measure the rate of reaction (figure 1.4), the direct electron transfer is required for one to obtain information on reaction kinetics, to detect homogenous chemical reactions coupled to electrochemical reactions, and to study proteins with more than one electroactive center. Some proteins do not exhibit electron transfer at electrodes. Many factors may be responsible for this lack of response. 1) The protein strongly adsorbs to the surface, denatures 9 and forms an insulating layer (figure 1.5), or 2) the electroactive center of the protein is not favorably oriented with respect to the electrode (figure 1.6). Direct electron transfer may be effected by careful selection of electrode material or modifying the electrode surface. Materials that often times serve as electrodes are bare metals, usually gold and platinum. Other materials that may serve as electrodes are semiconductors, various forms of carbon and conducting polymers. Electrode materials may be modified by plasmas (figure 1.7) or chemical means. Chemical methods include chemisorbed layers (figure 1.8); reactions that modify surface groups, such as the silation of surface oxides, and coating the surface with conducting polymers. By careful selection of electrode surface one controls the practical potential window range for the system. For example, in aqueous systems, the working range is limited to the potentials at which protons are reduced and water is oxidized. 10

PAGE 24

Figure 1.5 Formation of an insulating layer 11

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Figure 1.6 electroactive center Unfavorable orientation of a protein 12

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0 NH2 0 l)Ar NH2 OH plasma NH2 /OH 2)NH3 NH2 -c plasma \\ 0 NH2 Figure I. 7 Plasma modification of an electrode surface 13

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Gold electrode + H-s/\/CO; Bls/\/co; Wh0m-s/\/co; Figure 1.8 > s/\/co; Gold electrode Formation of a chemisorbed layer on of an electrode surface 14

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1.5.3 Direct Electron Transfer Of Ferritin Direct electron transfer of adsorbed ferritin has been accomplished by our group. 10 1112.18 20 All proteins unfold, or deform, upon adsorption onto a solid surface. This unfolding may impact the electrochemistry of the electroactive center by changing its local environment and energy level. Changes in the energy level of the electroactive center will in tum, cause changes in the E0 of the protein. If (E0'adsorbed-E0'native) < 25 mV, the protein has essentially retained its native properties. If one desires to obtain structural information on the different redox states of ferritin using techniques such as IR, the oxidation and reduction of ferritin in a solution contained by an IR cell (figure 1.9), must be accomplished first. 1.5.4 Diffusion-Controlled Direct Electron Transfer Of Ferritin Redox reactions are known to be involved in the uptake and release of iron by ferritin. The elucidation of the mechanism for the entry of iron into the core and the release of iron from the core is of great interest to biochemists and physicians dealing with conditions caused by iron overload or lack of iron. Electrochemical 15

PAGE 29

IR source I I I I I > I I I I I I I IR transparent electrode Detector Figure 1.9 IR cell for determining structural changes in oxidized and reduced ferritin 16

PAGE 30

methods are well suited to studying electron transfer of proteins. Cyclic voltammetry, in particular, is suited to the study of kinetics of homogenous processes that may occur after the heterogeneous electron transfer process. Information obtained by experiments on adsorbed ferritin includes a fair idea of the electron transfer kinetics, and the formal potential for the redox pair. Controlled potential electrolysis of ferritin adsorbed at a gold electrode modified with 3-mercaptopropionic acid revealed an n value of 1910 electrons per ferritin molecule. Varying the scan rate at the same electrode and observing the increase in peak potential separation with increasing scan rate indicated that the electron transfer was electrochemically irreversible. Voltammetry of apoferritin in the absence of iron (II), and ferritin in the presence of an iron complexing agent indicates that the currents observed when scanning ferritin are due to the electrolysis of the iron core. 11 Ferritin also exhibits electrochemical activity when adsorbed at tin-doped indium oxide electrodes. It has been determined that the packing density of the adsorbed ferritin varied proportionally to the ionic strength of the solution from which it was adsorbed. Initially, the adsorbed ferritin occurs in two independent states and upon reduction, the adsorbed protein reconstructs to a new state. The ratio of ferritin adsorbed in the two states was shown to be a function of ionic strength.12 17

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1.5.5 Direct Electron Transfer Of Cytochrome c Direct electron transfer of adsorbed cytochrome c has also been effected at tin-doped indium oxide electrodes by Bowden. 13 Information gained from cyclic voltammetry of the adsorbed species include and for the redox reaction. Direct electron transfer of cytochrome c at a gold electrode was effected by treating the gold surface with 4,4' -dithiopyridyl by Hil1.14 The disulfide bond of the 4,4' -dithiopyridine disassociate and the 4-thiopyridyl groups attach to the gold surface by the sulfide groups, the aromatic amine groups being directed into the solution. These attached 4thiopyridyl groups do not themselves accept or give up electrons in the potential window being scanned, but rather assist in the orientation of cytochrome c at the electrode surface. The heme group in cytochrome c, containing the electroactive iron, is located in a "cleft" in the protein. This "cleft" is surrounded by positively charged lysine residues at neutral pH. These positively charged groups could interact with the lone pair electrons on the nitrogen that is oriented into the solution. The electroactive center may thus be drawn close to the electrode and in a favorable orientation (figure 1.1 0). Cooper et a/15.16 treated various electrode materials with a layer of polypyrrole carboxylic acid. At pH 7, the carboxylic acid groups will be deprotonated forming negatively charged carboxy1ates. The isoelectric point (pi) of a protein is the pH at which the overall charge on the protein is zero. At a pH value greater than its pi a 18

PAGE 32

Gold electrode Figure 1.10 Interaction between cytochrome c and adsorbed 4-thiopyridine 19

PAGE 33

protein will have a net negative charge and at pHs less than its pi the overall charge will be positive. The pi of cytochrome c is about 10, at pH 7 its overall charge is negative. However, it was proposed that the partial positive charges on the lysine groups around the electroactive heme center could favorably align with the negative charges to allow for direct electron transfer. 1.5.6 Diffusion Controlled Direct Electron Transfer Of Ferritin The advantages of effecting direct electron transfer of dissolved ferritin over adsorbed ferritin are 1) the ferritin-electrode surface reactions are minimized, 2) one will have a better idea of the number of electrons transferred (n) and 3) some idea of the degree of denaturation that occurs on adsorption will be known. It has been shown that ferritin is deformed upon adsorption to an electrode surface. Following discussions with Professor Katsumi Niki, it was decided that a positive surface may be the electrode to achieve our goal. The pis of ferritins range from 4 to slightly over 6. The pi of horse spleen ferritin is about 4.5. At pH 7, the overall surface is anionic. Therefore, the approach to achieving diffusion-controlled cyclic voltammetry of horse spleen ferritin was to modify the electrode surface with cationic groups. Three approaches to modifying electrode surfaces were tried such that there was a positive 20

PAGE 34

charge at the surface: treatment of a gold electrode with 2-mercaptoethylamine, treatment of a edge-plane graphite electrode with an ammonia plasma, and formation of a polypyrrole layer at a gold electrode 1.6 Cyclic Voltammetry And Pseudocapacitance Of The Electrolyte Double Layer The objective of this research was to modify an electrode surface, such that dissolved fenitin would exhibit redox reactions at the electrode surface. To make the interpretation of any observed fenitin voltammograms easier one of the first steps was to form a layer that would only exhibit charging current in the potential window for fenitin. This charging current is due to charging of the electrical double layer at the electrode surface and is present even in the absence of an electroactive species. This double layer acts as a capacitor in a circuit. The current in the circuit is given by equation 1.1.17 u is the scan rate, Cd is the capacitance of the double layer, Ei is the initial potential and Rs is the resistance of the solution. (1.1) The second term in the equation is transient and goes to zero as time increases. When the potential sweep direction is changed, the current versus time trace is a mirror 21

PAGE 35

image of the first part of the curve. Figure 1.11 shows the current response to the changing potential as a function of time. Figure 1.12 shows the current response, in the absence of an electroactive species, as a function of the applied potential. If the layer on the electrode surface is not conductive, the charging current will be flat. The preliminary goal of this work was to find a material which could be used to reproducibly modify a gold electrode such that blank background scan will appear as in figure 1.12, and a cyclic voltammograms of ferritin that are diffusion-controlled and generally appear as figure 1.13. 1.7 Cyclic Voltammetry And Electroactive Species When a sufficiently negative potential is applied to an electrode, the energy of the electrons in the electrode is increased to a value where electrons will flow from the electrode to the lowest unfilled energy level of the electroactive species in solution. Conversely if a sufficient positive potential is applied, the energy will be lowered to a value inducing electrons to flow from the highest energy level of the electroactive species in solution into the electrode (figure 1.14). A change in charge per unit time is a current. 22

PAGE 36

0.8 0.7 0.6 ., !:: O.S 0 :> a 0.4 I 0.3 0.2 0.1 0 0 so 100 Figure 1.11 150 time, seconds 200 2SO Potential versus time used in cyclic voltammetry 23 300

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0.2025 0.2020 0.2015 1 l 0.2010 () 0.2005 0.2000 0.1995 ..,.. ___ ,...... __ --'T ___ ""T"" ___ T""" __ __,r----""'T'----r----.. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 potential, volts Figure 1.12 Current response to changing potential, in the absence of an electroactive species 24

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Figure 1.13 Typical current response of an electroactive species due to changing potential 25

PAGE 39

,_ unoc:cupied enerr;y level c# 1 /......----speaeain IIOiution 1 l bigllest occupied merzylewlm species in IIOiution 1 1 l tqhest occupied enerr;y level m species in solution a reduction has occurred ............ higbesl occupied I-'ll eocrr;y leYel m 1 species in solutiCIII-1 1 l Figure 1.14 1 l an oxidation has occurred Electron transfer between electrode surface and species in solution 26

PAGE 40

i (current) = dO (coulombs) dt (seconds) (1.2) Faraday's Law, can be used to relate the amount of current to the reaction rate at an electrode. 0 =N Faraday's Law (1.3) nxF Where Q = amount of charged passed n = number of moles of electrons passed per mole species of interest F =Faraday's constant N = moles of electrolyzable species. The derivative of Faraday's Law with respect to time gives dN = 1 x _QQ =Rate of electrochemical reaction (mole/sec) (1.4) dt n x F dt We must also look at the Nemst equation to understand the relationship between the concentration of electroactive species at or near the electrode surface and the inherent driving force of the reaction and applied potential. 27

PAGE 41

For the reaction of iron (Ill) being reduced to iron (II), the Nemst equation is: E = cell potential E0 = formal potential for the half reaction R = ideal gas constant (1.5) n = number of mole of electrons per mole of electroactive species T = temperature F= Faraday's constant Since 8G0 = -nFE0', the P' term reflects the inherent driving force for the reaction. The second term reflects the effect of the concentrations of the oxidized and reduces forms on the cell potential. As a more positive potential is applied to the electrode surface, more of the iron (II) is oxidized to iron (Ill). When the cell potential equals the formal potential, the concentrations of iron (II) and iron (III) are equal. When a potential sweep is applied to a solution containing an electroactive species, a cyclic voltammogram is the current response to the applied potential curve. In voltammetry, the only place redox activity takes place is at or near the electrode surface. At point 1, (figure 1.15), the potential is at a sufficiently negative value so that no redox activity takes place. At point 2, (figure 1.16), the positive potential sweep being applied has decreased the energy of the electrons at the electrode surface sufficiently that some of the reduced species at the surface become oxidized, and a current starts 28

PAGE 42

to flow. Diffusion of reduced species to the electrode surface also begins to be driven by a developing concentration gradient. The oxidized species are also undergoing diffusion, but for the purpose of clarity are not shown. At point 3, (figure 1.17), the potential is positive enough that the ratio of oxidized species to reduced species at the electrode surface has increased, more reduced species diffuse to the surface and the current increases. At point 4 (figure 1.18), the electrode potential is such that all of the species at the surface are oxidized. At this point, the current reaches a maximum. Recalling an example of a drop of dye being added to a glass of water (figure 1.19), when the dye is first added, the concentration gradient is very high with the dye occupying a very small volume. The dye quickly begins to diffuse toward the volume of lesser concentration. As time passes, the dye becomes less concentrated, and flux of material from a higher concentration to a lower continues, but at a slower rate. The flux of material thus slows, as the concentration gradient has become less steep. The diffusion, driven by the concentration gradient, will continue until the concentration of dye is uniform throughout the solution. The same effect occurs past point 4. Point 5, (figure 1.20) is in the region of the voltammogram where the flux of reduced species reaching the electrode surface is decreasing due to the increasing thickness of the diffusion layer. As the flux of reduced species decreases, so does the current. 29

PAGE 43

CD 1 == Reduced species Q == Oxidized species Figure 1.15 Current versus applied potential at start of cyclic voltamrnogram 30

PAGE 44

@t == Reduced species Q == Oxidized species Figure 1.16 Current versus applied potential as current starts to flow in a cyclic voltanunogram 31

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== Reduced species Q == Oxidized species Figure 1.17 Current versus applied potential as diffusion causes increase in current in a cyclic voltanunogram 32

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8 == Reduced species Q == Oxidized species Figure 1.18 Current versus applied potential as current reaches a maximum value in a cyclic voltammogram 33

PAGE 47

\ Figure 1.19 Diffusion of dye in a glass of water 34

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1.8 The Three Electrode Cell In the experiments conducted in this project, the three-electrode cell configuration was used (figure 1.21 ). The working electrode is the electrode at which the oxidation or reduction of our species of interest occurs. The auxiliary electrode is an electrode, usually made of noble metal such as gold or platinum. When an oxidation occurs at the working electrode, a reduction occurs at the auxiliary electrode. When a sufficiently high positive potential, supplied by the power supply, is applied between the working and auxiliary electrodes, an oxidation process will occur at the working electrode. Electrons will be liberated from the species of interest to the working electrode and flow through the external circuit. This electron flow is current, and is measured by the ammeter. The potential of the working electrode is measured against the constant potential of the electrode at all times. In this configuration, no current flows through the reference electrode. A physical representation of our 3electrode cell is shown in figure 1.22. Our cell consists of an H -cell with a larger main compartment and a smaller reference compartment. The working electrode is placed in the main compartment, which contains a solution of our species of interest. The reference and auxiliary electrodes are contained in the smaller compartment, immersed in pure electrolyte. The compartments are connected by a glass frit to 35

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@t === Reduced species Q === Oxidized species Figure 1.20 Current versus applied potential as decrease in diffusion causes decrease in current in a cyclic voltammogram 36

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power suppley ammeter working electrode 0 voltmeter auxiliary electrode reference electrode Figure 1.21 Schematic of a three-electrode cell 37

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provide electrical contact, while, at the same time, to prevent the two solutions from mixing. A provision is made for the deaeration of the sample solution in the working arm; in this case, pressurized nitrogen is allowed to flow through the solution from the bottom of the compartment. The power supply, anuneter, and voltmeter are all contained in an instrument known as a potentiostat. 38

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'MJrkiDg power suppley, voltmeter mWllary elecbodc electmde & ammeter nleraoe elec:tnJde ""' potentiostat ,.... I--I--::J( '----" Figure 1.22 Representation of our three-electrode cell 39

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2. Experimental 2.1 Reagents And Materials Polypyrrole dispersed on polyurethane with a tosylate counter ion, polyanaline, gold rods and Nation were purchased from Aldrich. Potassium chloride, sodium dihydrogen phosphate, sodium hydroxide, hydrochloric acid (12M), sulfuric acid (18 M), and acetonitrile, (HPLC grade) were purchased from Mallinckrodt. The acetonitrile was used as received or distilled over phosphorus pentoxide and dried over molecular sieves. Phosphorus pentoxide was obtained from MCB. Potassium ferrocyanide was purchase from B & A. Pyrrole, HEPES, 2-mercaptoethylamine, tetraethylanunonium tosylate, tetrabutylanunonium perchlorate, tetramethylanunonium perchlorate, poly-L-glutamic acid, poly-L-lysine hydrobromide and ferritin were purchased from Sigma. The pyrrole was vacuum distilled and stored over dried molecular sieves (4A). Nitrogen was purchased from General Air, and gaseous anunonia was purchased from Matheson Tri-Gas. Potassium dichromate, molecular sieves 4A, 8-12 mesh, hydrogen peroxide and sodium perchlorate were obtained from Fisher Scientific Company. Ethanol (95%) was purchased from Aaper Alcohol and Chemical Company. Diamond pastes were 40

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purchased from Buehler and Elgin, gold foil and 0.01 mm gold wire from Alfa/Aesar. Highly ordered pyrolytic graphite (HOPG) was kindly supplied by Dr. Arthur W. Moore, Advanced Ceramics Corporation. All water used for electrochemical experiments and rinsing of electrochemical equipment was pyrolytically distilled over a heated platinum gauze catalyst in an oxygen atmosphere. 2.2 Equipment 2.2.1 Plasma Reactor Glass and quartz tubes, glass tees and glass rods were purchased from Technical Glass (Aurora, CO). A plasma reactor, (figure 2.1), was constructed using, a 12 mm quartz tubing and various fittings. A manifold to control the concentrations of one or more gases into the reactor was constructed from needle valves. The radio frequency cavity was placed in the center of the quartz tube. Power was supplied by an Opthos Microwave power generator, model MPG 4. The vacuum pressure was measured using Granville-Phillips Series 275 pressure gauge. An 8 mm glass rod, with the ends reduced to 4 mm diameter, was fitted to allow introduction of electrodes into the plasma, by way of o-ring seals. 41

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gasesm 1 quartz tube 1 pusher rod i RF cavity l to vacuum Figure 2.1 Plasma reactor 42

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2.2.2 Potentiostats And Recorder Cyclic voltammetry scans were performed using a Bioanalytical Systems CV27 Voltammograph (West Lafayette, IN) and a Cypress OMNI 90 Potentiostat. Data were recorded on a Bioanalytical Systems RXY Recorder. 2.2.3 Cells The H-Cells used for most work has been shown in figure 1.22. Some of the survey scans were performed in low volume cells constructed by cutting the top end off 20 mL vials. Nitrogen was directed into these cells by fitting an Eppendorf pipette tip onto polyethylene tubing and positioning the tip inside the vial, just below the lip (figure 2.2). A low volume H-cell (Technical Glass, Aurora, CO) was constructed from a 14 mm I.D. glass tube with a 24/40 standard taper ground glass fitting. A small reference sidearm was attached to the side of the low volume, working arm by a low-flow glass frit. A luer-lock capillary tube was fitted on the side of the working arm, such that a stream of nitrogen could be directed at the surface of the liquid (figure 2.3). 43

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reference electrode nitrogen auxiliary electrode I I I j 0 working electrode Figure 2.2 Cell used for survey work 44

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working ann reference > auxuliary arm Figure 2.3 24/40 ground glass taper / nitrogen .:-<-inlet glass frit Low volume H-cell 45

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2.2.4 Electrodes A highly ordered plane graphite (HOPG) electrode was constructed by mounting the HOPG with the edge planes exposed, in epoxy contained in a PVC tube (figure 2.4). After the epoxy had cured, the working face of the electrode was polished in successively finer emery paper, followed by crocus cloth, then successively finer diamond pastes, ending with a paste of0.25 J.tm diamond particles. The electrode was then cleaned by sonication in a saturated solution of Alconox in ethanol. Shrink-wrap tubing was attached to one end of the PVC tube. Electrical contact was made by placing a small drop of mercury in contact with the graphite and inserting a wire into the mercury. Gold foil was cut to size and fused to a gold wire. The wire and foil flags were threaded through a 4 nun I.D. glass luer joint (Ace Glass). Flags whose dimensions were too large for the inside diameters of the glass tubes were rolled into cylinders and inserted inside the tubes, or were inserted into volumetric pipettes that had been cut in half. Luer fittings were assembled on the tubes to allow for the introduction of pressurized nitrogen (figure 2.5). A very simple electrode, with no seams or other interfaces, was constructed by flattening one end of a gold rod, to increase its surface area. This rod was then connected to the potentiostat via alligator clips. Two gold disk electrodes were assembled by pressing 3 nun gold 46

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shrink-wrap tube mercury pool gold wrre sheath plug Figure 2.4 Edge-plane pyrolytic graphite cell 47

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v 0 I I Figure 2.5 Gold flag electrodes in glass sheaths 48

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rods into 9 nun outside diameter Kel-F (polychlorotrifluropolyethylene) rod that had been bored and machined to accept the gold rods and conductor wires (figure 2.6). The ends of the Kel-F rods opposite the gold rods were tapped to accept stainless steel machine screws that pressed copper conductor wires firmly into the back of the gold rods. Silver/silver chloride electrodes filled with 1 M potassium chloride were used as reference electrodes in this work. Combination reference/auxiliary electrodes were constructed using 1 mm I.D. luer joints (Ace Glass) and female lure-tipped bulbs manufactured by Technical Glass. The luer-tipped tube was filled with 1 M potassium chloride and fitted to the bulb. A silver wire was cleaned and inserted into the tube until the inserted tip was in the bulb. A platinum wire was wrapped in a loose spiral around the outside of the tube (figure 2.7). The last turn or so ofthe platinum wire was wrapped around the flare on the bulb, thus securing the bulb in place. Reference and auxiliary electrodes for use in the low volume cells have been described by Zapien, D. C. and Johnson, M. A.18 Centricon tubes ultrafiltration tubes were purchased from Millipore Corporation. 49

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Figure 2.6 Gold disk pressed into Kel-F rod 50

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Figure 2.7 Reference and auxiliary electrodes 51

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2.3 Procedures 2.3.1 Cleaning Of Apparatus All glassware used for electrochemical experiments was cleaned by soaking in a chromic acid cleaning solution. After cleaning, the glassware was rinsed with water and either allowed to air-dry or were rinsed with the solution being used. The gold flag electrodes inside the glass sheaths were cleaned by soaking in chromic acid. Prior to use, the cleaned gold electrodes were rinsed with water and then ethyl alcohol. Alternately the gold flag electrodes were cleaned by removing the flags from the protective glass sheaths and heated to incandescence in a fuel-rich, natural gas flame. Graphite electrodes and gold disk electrodes were cleaned by polishing, first on successively finer emery cloth, then crocus cloth, followed by successively finer diamond polishing pastes. These electrodes were then rinsed with water followed by ethyl alcohol and allowed to air-dry. Gold electrodes were further cleaned by stepping between + 1.50 V and -0.35 V versus a silver/silver chloride reference. If the gold was protected by a sheath, the gold surface was purged with nitrogen, prior to stepping to -0.35 V. 52

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2.3.2 Treatment Of Gold With 2Mercaptoethylamine A gold foil flag cleaned as described above. The flag was placed in a dilute solution of 2-mercaptoethylamine in ethanol and left overnight. The electrode was then rinsed with ethanol and air-dried. 2.3.3 Plasma Modification Of HOPG Edge Plane Electrode The plasma apparatus was assembled and checked for vacuum leaks. The pressure was reduced to 0.25 torr using a vacuum pump and a liquid nitrogen cooled coldfinger installed between the plasma apparatus and the pump. Valves were successively opened, starting at the end of the apparatus nearest the pump to check for leaks and the pressure observed after each valve was opened. After verifying that there were no appreciable leaks in the system, helium was bled into the apparatus via a needle valve. A helium plasma was ignited by applying the discharge from a Tesla coil to the radio frequency (RF) cavity and slowly increasing the forward power on the power supply. The helium plasma was then tuned using the adjustment knobs on the RF cavity and the applied forward power until the plasma was uniform and the reflected power minimized. The helium plasma was used to remove oxygen functional groups at the graphite surface, prior to treatment with the ammonia plasma. 53

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The oxygen containing groups include carbonyls, hydroxyls, carboxyls, lactones, etc., which form at the surface upon exposure to air. A dummy electrode was constructed using PVC tubing and epoxy. This dummy was polished in the same fashion as the edge-plane HOPG electrode. This dummy was fitted to the 8 mm Pyrex rod with shrink-wrap tubing. The dummy was inserted into the plasma reactor. The helium plasma was reignited, and the dummy was slowly moved into the plasma. The plasma melted the dummy's PVC and epoxy. Several attempts were made, at decreasing forward power levels, to introduce the dummy into the helium plasma without damage to the PVC and epoxy. Through discussions with Dr. Larry G. Anderson, it was suggested that there might be enough tenuous plasma outside the plasma cavity to effectively treat the electrode. The apparatus was reconfigured to allow the electrode to approach the plasma from the vacuum side of the plasma tube with the belief that the gases, bled into the tube from the other end and swept towards the vacuum, may cause a higher concentration of tenuous plasma on the vacuum pump side of the RF cavity. The dummy electrode was brought to within 2.5 centimeters of the plasma and held there for five minutes, with no apparent damage. After cleaning, an edge-plane HOPG electrode was fitted to one end of the eight millimeter rod with shrink-wrap tubing. The apparatus was reassembled and the plasma ignited. The electrode was moved to within 2.5 em of the plasma. The electrode was treated for ten minutes with a helium plasma to remove surface oxygen 54

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groups. The plasma gas was switched from helium to ammonia and the electrode was treated for another ten minutes to apply amine groups to the smface. The plasma power was turned off and the electrode was left in a low-pressure ammonia atmosphere for another ten minutes, prior to venting the apparatus to the atmosphere and removing the electrode. The treated electrode was removed from the glass rod and assembled as described above (figure 2.4). 2.3.4 Formation Of Films Nafion was applied to a polished edge-pane HOPG electrode with a dropper and allowed to air dry. Inspection of the dried Nafion surface showed fractures in the surface that exposed the graphite surface. Isopropyl alcohol (HPLC grade) was applied to the Nafion surface to redissolve and smooth the film. 2.3.5 Formation And Conditioning Of Polypyrrole Layers A pyrrole solution, 0.05 M, in acetonitrile containing 0.1 M counter ion was added to the working arm of an H-cell. The counter ions were either perchlorate anions or tosylate anions. A gold flag rolled into a cylinder and fitted inside a glass sheath, or a gold disk was placed in an H-cell and coated with polypyrrole perchlorate. The 55

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polypyrrole perchlorate was polymerized from a solution of acetonitrile, until no more gold was visible and the surface appeared uniformly covered. The coated flag was rinsed in acetonitrile to remove any perchlorate salt and then with water to remove any acetonitrile. The solution in the working arm was deaerated by bubbling nitrogen through the solution. After deaeration, the nitrogen flow was adjusted to provide a nitrogen blanket above the solution in the working arm. The layers were formed by both potentiostatic and potentiodynamic methods. Two potentiostatic methods were used. The first method simply applied an oxidative potential to the working electrode such that oxidation of pyrrole commenced immediately. The other started at a potential where no oxidation took place and ramped anodically to the potential where oxidation commenced, at which point the potential was held for the duration of the film formation. The current was monitored versus time and the amount of charge passed per unit area to form the film (figure 2.8) was used as an estimate of the film thickness. In the potentiodynamic method, the potential was scanned anodically to the point where oxidation occurred and then the potential sweep rate was reversed. Several cyclic scans were performed until the polypyrrole film covered the surface. Polypyrrole was formed from solutions of dry acetonitrile and acetonitrile containing no added water, 1 %added water, and 2% added water. 56

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14.0 12.0 10.0 8.0 ., i 6.0 '8 4.0 2.0 0.0 -2.0 0 100 Figure 2.8 200 300 400 500 seconds Current versus time during formation of a polypyrrole layer. 57 600

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2.3.6 Cyclic Voltammetry Of Ferritin Using Polypyrrole Electrodes Polypyrrole electrodes were conditioned by repeated cycling on buffered and nonbuffered electrolyte solutions, as well as in solutions of ferritin. The electrodes were conditioned until no changes were observed in the voltammograms obtained by subsequent cycles. The electrodes were then rinsed with fresh water and air-dried prior to performing scans in ferritin. All ferritin solutions scanned, were adjusted to pH 7 with buffer. The concentration of the buffer, sodium dihydrogen phosphate, was varied from 0.1 M to 1 M, while that of the electrolyte, potassium chloride, was varied from 0.1 M and 1 M. 58

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3. Results And Discussion 3.1 Preliminary Electrode Modifications The gold foil surface treated with the 2-mercaptoethylamine should have the thiol groups adsorbed to the gold surface, and the amine groups at other end of the ethyl tethers directed towards the solution. The directed amine groups were chosen since at pH 7 they will be protonated, giving the electrode surface a positive charge. It was hoped that this would interact favorably with the overall negative charge on the ferritin shell at pH 7. Using the pKa of the conjugate acid of ethylamine, an estimate of the ratio of conjugate acid to amine directed toward the solution should be around 6400 to 1. The short tether length adsorbates exhibit a more random orientation than those with longer tethers. Longer chains adsorbates tend to self-align and form an ordered layer, similar to the bristles on a toothbrush. Additionally, if the tethers become too long they will form an insulating layer 19 and thus inhibit electron transfer. A voltammogram of ferritin in a 0.1 M ionic strength phosphate buffer (figure 3.1) is very similar to a voltammogram previously observed for adsorbed ferritin, with a cathodic peak at approximately -0.4 V and an anodic peak at -0.1 V.20 In subsequent scans, the cathodic peak current at -0.4 V grew smaller 59

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I =1.25 JiA -0.60 v +0.20 v Figure 3.1 Cyclic voltarnmogram of ferritin at gold modified with 2-mercaptoethylamine 60

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and a small cathodic peak appeared at -0.2 V. After the scans in ferritin, the electrode was rinsed with water and scans were performed in blank buffer. The voltammogram for this scan (figure 3.2) still exhibited anodic and cathodic peaks, but at more positive potentials, and was not similar to the background scan previously observed. 20 This result may be an indication that not all of the ferritin was strongly adsorbed and there may be more than one type of adsorption. The first cycle in the clean buffer exhibits a cathodic peak at -0.28 V and an anodic peak at -0.02 V. On subsequent cycles, the peak at -0.28 V disappeared and a new cathodic peak at -0.20 V appears and is persistent in additional sweeps. The anodic peak potential did not change with repeated cycles. Gold electrodes treated with Nation (a perlluoro polymer, with perlluoro side-chains that are terminated with sulfonic acid groups) produced ferritin voltammograms similar to those produced by the mercaptoethylamine-modified electrode, and their use was discontinued. 3.2 Plasma-Modified HOPG Edge Plane Electrode The background scans in blank buffer using the plasma-modified HOPG edge plane electrode show the same charging background current as predicted by the equation 1.1. The voltammograms of ferritin (figure 3.3) looked like the scans of the 61

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-0.6 v I= 1.25 JJA Figure 3.2 Cyclic voltammogram of background with 2-mercaptoethylamine modified gold, after scans in ferritin 62 +0.20V

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-0.50 v Figure 3.3 Cyclic voltammogram of ferritin at ammonia plasma modified edge-plane pyrolytic graphite electrode 63 +0.80V

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background (figure 3.4), indicating no activity at this electrode. It may have been possible that the HOPG edge plane was not modified with amine groups by the ammonia plasma apparatus used. As we did not perform an analysis of the electrode surface with a surface-sensitive technique, such as x-ray photoelectron spectroscopy before and after treatment in the plasma tube, the nature of the surface was not characterized. 3.3 Polypyrrole The majority of the work to modify the gold electrodes was performed using polypyrrole. This polymer was chosen, as it is conductive in the potential range being used to study ferritin. Additionally, in its conductive form, there is a positive charge on every three to four repeating amine units. Dilute solutions of pyrrole were electrochemically oxidized to form a polypyrrole layer on a gold substrate. The counter ion is incorporated into the polypyrrole layer and contributes to the film's properties. The characteristics of the polymer film may also be controlled with careful selection of the counter ion. The polymerization of pyrrole starts at a potential that is high enough to oxidize pyrrole at an anode, to form a radical cation. The concentration of radical cations is high at the electrode surface and dimerization 64

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-0.50 v +0.80 v Figure 3.4 Cyclic voltammogram of background at ammonia plasma modified edge-plane pyrolytic graphite electrode 65

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of the radicals occurs. The dication formed deprotonates to form a pyrrole dimer. Oxidation of dimers occurs at a lower potential than monomers, oxidation of trimers at a potential lower than dimers, and so forth. The dimer is oxidized to form a radical cation and reacts with a pyrrole molecule forming a radical trication, which in turn, is deprotonated to form a trimer. The trimer is oxidized and so forth until a polymer is formed. Oxidation of the polymer and incorporation of counter ion lead to the formation of a conductive polymer (Figure 3.5).21 3.3.1 Observation Of Charging Current With Polypyrrole On A Gold Electrode After the gold/polypyrrole electrode was cycled three times in 1 M sulfuric acid and then rinsed with water. Two scans were performed in 1 M potassium chloride, and a charging current that had nearly parallel traces between the scan directions was obtained. A few crystals of potassium ferrocyanide were added to the cell and the solution stirred by bubbling nitrogen from the bottom. After the solution had been mixed, a scan was obtained that showed the anodic and cathodic waves of ferrocyanide superimposed on the charging current previously observed (figure 3.6). 66

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Figure 3.5 Polypyrrole and counter ion 67

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tJA -0.15 v +0.80 v I =soo J.LA -0.20 v +0.60 v Figure 3.6 Cyclic voltammogram of background and ferrocyanide at polypyrrole-perchlorate modified gold foil electrode. 68

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Based upon these observations it is believed that this electrode is conductive and its use to achieve direct electron transfer of fenitin is worth pursuing. 3.3.2 Diffusion-Controlled Direct Electron Transfer Of Electron Ferritin On Polypyrrole A polypyrrole layer was formed on a gold electrode as described above, with the exception of omitting the treatment in sulfuric acid, as subsequent attempts to treat with sulfuric acid did not produce the charging current observed in the first experiment. After rinsing to remove excess pyrrole, counter ion, and acetonitrile, the electrode was placed in a solution offenitin (5 mg per mL 0.1 M KCl, phosphate pH 7 buffer) and the cyclic voltammogram shown in figure 3.7 was obtained. The voltammogram was obtained using a scan rate of 10 mV/s due to the slow theoretical diffusion of fenitin, based upon calculations using the Einstein-Stokes equation. This voltammogram possesses features suggesting diffusion-controlled electron transfer of fenitin. The voltammogram is symmetrical and the peak separation is 480 millivolts at 10 millivolts per second. The E0 estimated by the potential midway between the cathodic peak potential and anodic peak potential was -0.06 volts, which matches well with the E0 of -0.07 volts of fenitin adsorbed at a bare gold electrode. The 69

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-1.00 v I / l ii ;I I / 41 / I=}Oj..LA Figure 3.7 Cyclic voltammogram of ferritin at polypyrrole-perchlorate modified gold foil electrode, electrode area approximately 1 cm2 scan rate 10 m V /s, Ag/ AgCl reference electrode 70 +0.40 v

PAGE 84

similarity of the potentials suggests that ferritin does not unfold appreciably when it adsorbs. This result suggests that absorbed ferritin probably retains the redox activity of native ferritin. A systematic approach was begun to determine the conditions needed to reproduce the original voltammogram. New cleaning solutions were prepared and all glassware was cleaned. Acetonitrile was purified to remove any water and new reagent solutions were made. Careful monitoring was made of the amount of charge passed per unit area to form the polypyrrole layer and the amount of water in the monomer solution was controlled. The polypyrrole layer was formed at several potentials. Attempts to repeat this experiment were initially unsuccessful. However, after continued trials, the voltammogram was reproduced. A polypyrrole film was formed and transferred to a blank buffer solution and a background obtained after three scans from +0.60 V to -0.75 Vat 5 mV per second (figure 3.8). After the background scan, the electrode was transferred to a solution of ferritin (34 mg/mL) diffusion-controlled direct electron transfer of ferritin was observed (figure 3.9). A plot of the peak cathodic current versus the square root of the scan rate produced a linear fit, providing evidence of a diffusion controlled process (figure 3.1 0). After rinsing the electrode, the background buffer was scanned and the blank was again observed (figure 3.11), again indicating the voltammetric response in the ferritin solution was indeed due to ferritin. Repeated attempts to reproduce the above 71

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-0.75 v I =so J1A Figure 3.8 Cyclic voltarnmogram ofbackground polypyrrole-perchlorate modified gold foil electrode 72 +0.60V

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-0.75 v +0.60 v Figure 3.9 Cyclic voltammogram of ferritin at polypyrrole-perchlorate modified gold foil electrode 73

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peak cathodic current vs square root of scan rate08/23/01 <( 50.0 ::::l. "tf 40.0 0 0 30.0 0 0 20.0 8. 10.0 y = 28.5036x18.5453 R2 = 0.9687 0.750 0.950 1.150 1.350 1.550 1.750 1.950 2.150 square root scan rate, m V /s I sqrt rate vs Ipc --Linear (sqrt nrte vs lpc) I Figure 3.10 Peak cathodic current versus square root scan rate, 34 mg ferritin/mL, 1 M KCI, pH 7 buffer, 1 cm2 polypyrrole electrode 74 2.350

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-0.75 v I= 10 jJA Figure 3.11 Cyclic voltammogram of background at polypyrrole-perchlorate modified gold foil electrode, after scans in ferritin 75 +0.60V

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experiment again met with failure. The flattened gold rod was used instead of the gold wire/gold foil flag assembly to determine if the problem was due to material trapped between the gold foil and wire. No differences were observed. The lack of differences between the flattened gold rod and the gold foil/wire assembly support the hypothesis that the inability to reproduce the direct electron transfer of ferritin is not due to contamination at the gold foil/wire interface. Additionally, as the flags were surrounded by a glass sheath and the flattened rod was not, the implication was that the presence of the glass sheath is not a factor in reproducing the voltammogram of ferritin at gold/polypyrrole. 3.3.3 Treatment With Hydrogen Peroxide An experiment was performed to verify whether non-uniformity of the polypyrrole film was responsible for the non-reproducibility of the results. Polypyrrole perchlorate films are known to be porous and it may be possible for a species to traverse the pores in the polypyrrole film and react at the gold surface. A background (figure 3.12) was obtained in 1M potassium chloride, pH 7, 0.1 M phosphate buffer for a gold/polypyrrole-perchlorate film. A voltammogram of ferritin (51 mg/mL) in the same buffer, is same as the background (figure 3.13) indicating the layer was not 76

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I =5o JJA -0.60 v +0.55 v Figure 3.12 Cyclic voltammogram of background at polypyrrole-perchlorate modified gold foil electrode 77

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-0.60 v +0.55 v Figure 3.13 Cyclic voltammogram offerritin at polypyrrole-perchlorate modified gold foil electrode 78

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promoted direct electron transfer of ferritin. A few crystals of potassium ferro cyanide were added to the background buffer, and it was verified that a voltammogram could be obtained (figure 3.14). The film was air-dried overnight and a voltammogram of 8.9 mM ferrocyanide in the above electrolyte verified that the electrode film was still conductive. Exposure to hydrogen peroxide is known to destroy the conductivity of polypyrrole films. The electrode was soaked in 30 % hydrogen peroxide for fifteen minutes, rinsed with water and allowed to air dry. After the conductivity of the polypyrrole layer was destroyed, a scan in 8.9 mM ferrocyanide showed an attenuated charging current superimposed by two new peaks, an anodic peak at +0.28V and a cathodic peak at+ 0.23 V (figure 3.15). These two new peaks appear to be a redox couple. Scans in blank buffer and ferritin solutions revealed similar voltammograms. From these observations, it is concluded that the observed voltammetric responses are due to activity at the polypyrrole surface and not to diffusion into pores on the surface and reaction at the gold surface. 79

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-0.20 v +0.60 v Figure 3.14 Cyclic voltammogram offerrocyanide at polypyrrole-perchlorate modified gold foil electrode 80

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-0.20 v J =so J.LA Figure 3.15 Cyclic voltammogram offerrocyanide at polypyrrole-perchlorate modified gold flag electrode, after destruction of polypyrrole conductivity 81 +0.60 v

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3.3.4 Troubleshooting Whether A Polypyrrole Film Will Give Cyclic Voltammograms Of Ferrocyanide Unless otherwise noted, the scans were performed in an H-cell with a luer-lock combination auxiliary and reference electrode. A gold disk electrode was immersed in a 4.1 mM solution offerrocyanide in 1M potassium chloride and a voltammogram obtained (figure 3.16). From the anodic peak current, the electrode area was calculated and the value was in agreement with that calculated from the disk radius. The electrode was then coated with a polypyrrole film and a background obtained in I M potassium chloride. The background was clean with parallel currents on the anodic and cathodic sweeps (figure 3 .17). A voltammogram was obtained in the 4.1 mM ferrocyanide solution and the voltammetric waves were observed to be superimposed on the background charging current (figure 3.18). The disk was next placed in a I M potassium chloride, 0.2 M phosphate, pH 7 buffer and a new background was obtained. This new background was very similar to the background in 1 M potassium chloride, with the exception of increasing currents as the sweeps approached the switching potentials (figure 3.19). A voltammogram obtained in 4.4 mM ferrocyanide in 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer showed the expected waves superimposed on the new charging current (figure 3.20). To 82

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-0.20 v +0.60 v Figure 3.16 Cyclic voltammogram on 4.1 mM ferrocyanide in 1 M potassium chloride, at gold disk electrode 83

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I-s !1A -0.20 v +0.60 v Figure 3.17 Background of I M potassium chloride, at polypyrrole-perchlorate on gold disk electrode 84

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-0.20 v +0.60 v Figure 3.18 Cyclic voltammogram on 4.1 mM ferrocyanide in 1 M potassium chloride, at polypyrrole-perchlorate on gold disk electrode 85

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-0.20 v +0.60 v Figure 3.19 Background 1M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole perchlorate on gold disk electrode 86

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-0.20 v +0.60 v Figure 3.20 Cyclic voltarnmogram of 4.4 mM ferrocyanide in 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole perchlorate on gold disk electrode 87

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check for possible effects caused by the reference and auxiliary electrodes, the 4.4 mM ferrocyanide solution was scanned using Vycor auxiliary and Vycor reference electrodes. The scan was identical and could be superimposed on the scan using the luer-lock electrodes. A scan was obtained in a solution of ferritin (24 mg/mL 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer) in a cutoff vial cell with Vycor auxiliary and reference. (figure 3.21) The scan appears to be the same as those in blank buffer. The electrode was rinsed with water and the 4.4 mM ferrocyanide solution rescanned, using a Vycor reference and Vycor auxiliary electrodes. The ferrocyanide waves are still visible, but are not as large as those seen in figure 3.22. This result indicates that the electron transfer is occurring between the polypyrrole and ferrocyanide. The effects of the phosphate buffer, the reference electrodes, and the auxiliary electrodes can be ruled out as causes for the inability to consistently reproduce the voltammograms of ferritin. On this electrode, ferritin is inactive and appears to have been adsorbed, perhaps forming a partially insulating layer. 3.3.5 Peak Current Dependence On Ferritin Concentration Plots were constructed of data obtained from four ferritin concentrations, scanned at four scan rates, using a 0.08 cm2 polypyrrole disk electrode. The ferritin 88

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-0.20 v +0.60 v Figure 3.21 Cyclic voltammogram of 24 mg ferritin/mL 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole perchlorate on gold disk electrode 89

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-0.20 v +0.60 v Figure 3.22 Cyclic voltammogram of 4.4 mM ferrocyanide in 1 M potassium chloride, 0.2 M phosphate, pH 7 buffer at polypyrrole perchlorate on gold disk electrode, after scans in ferritin 90

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concentrations were 4.5 mg/mL. 9.0 mg/mL, 18 mg/mL and 36 mg/mL. The scan rates were 5 mV/s, 10 mV/s, 20 mV/s and 40 mV/s. The results show a linear relationship between the peak cathodic currents and concentration (figure 3.23), as predicted for a diffusion-controlled process by the Randles-Sevcik equation (equation 3.1). n = number of electrons transferred A = electrode area D = diffusion coefficient of the electroactive species Cb = bulk concentration of the electroactive species v112 =square root of the scan rate (3.1) The four plots show good linear fits for both 5 m V /s and 10 m V /s. The plots at 20 m V /s and 40 m V /s do not show linear relationships. The obsetvations from the later two scan rates support our earlier hypothesis that fast scan rates cannot be used due to the slow diffusion of ferritin to the electrode surface. 91

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peak cumDI YS COIIceD!nDOD, 02/16/02 ..... --, ............. -.. .. .... . . . . . . .... . .. .. ... ... . .... .. ---------r------lr -_,.-0 10 IS 20 mgFt/mL 2S 30 3S 40 Figure 3.23 Peak current dependence on bulk concentration at four scan rates, 0.08 cm2 polypyrrole electrode, Ag/ AgCl reference, Pt auxiliary, 1M KCl, 0.2 M phosphate pH 7 buffer 92 --.-smvls IOmv/s mvls ...,._40mvls

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3.4 Conclusions We can, at unsubstituted polypyrrole on gold electrodes, effect the diffusion controlled electron transfer of ferritin, as evidenced by background and ferritin scans. This evidence is reinforced by the peak current's linear dependence upon the square root of the scan rate and upon the bulk concentration. Analysis of the morphology of the ferritin voltammograms and the P' imply that we have effected a diffusion controlled direct electron transfer response and that n, the number of electrons being transferred, is not 1500. The E0 of diffusion-controlled ferritin was within 25 millivolts of the adsorbed ferritin, indicating that adsorbed ferritin retains most of its native structure. The small values for the peak currents observed in the diffusion controlled voltammograms indicate that the diffusion coefficient for ferritin, in the solutions used, is smaller than that calculated by empirical methods. The effects of a protective sheath around the polypyrrole working electrode, the construction of the reference electrode and the auxiliary electrode seem not to effect the electrochemical activity at the working electrode. The reasons for ferritin exhibiting electrochemical activity at some polypyrrole films and not at others have not been discovered. The experiments suggest, based upon the variables that were carefully controlled, that more work is needed to gain tighter control over the variables studied, and possibly others not yet studied, to consistently produce diffusion-controlled direct electron 93

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transfer of ferritin at conductive polymer-modified gold electrodes. Possible variables that may impact the performance of polypyrrole as an electrode at which the diffusion-controlled direct electron transfer of ferritin may be observed are 1) variations in the structural conformation of the polypyrrole layer, 2) variations in the conductivity of the polypyrrole layer, and 3) variation in the surface charge of the polypyrrole. It is possible that another counter ion or aprotic solvent combination may produce a polypyrrole film that is less sensitive to the variables controlled, and thus produce a film that has more reproducible behavior. 94

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