Electrochimica Acta 45 (2000) 2623 – 2645

www.elsevier.nl/locate/electacta

Recent developments in faradaic bioelectrochemistry

Fraser A. Armstrong a,*, George S. Wilson b
a
Inorganic Chemistry Laboratory, Uni6ersity of Oxford, South Parks Road, Oxford OX1 3QR, UK
b
Department of Chemistry, Uni6ersity of Kansas, Lawrence, KS 66045, USA

Papers received in Newcastle, 20 December 1999

Abstract

Progress in the area of faradaic bioelectrochemistry over the last decade has been reviewed. Electrochemical and
spectroscopic studies of the protein – electrode interface are discussed along with the use of electron transfer
promoters, self-assembled monolayers (SAMs), and surfactant films. Voltammetric techniques are described that
permit rapid, in-situ measurement of formal potentials while also providing kinetic resolution on the sub-millisecond
time scale. Some enzymes, especially those of analytical interest, must be coupled or ‘wired’ to the electrode using
mediators in order to achieve rapid electron transfer. Supramolecular structures designed to facilitate electron transfer
and to immobilize co-factors are described. The use of electrochemically-based biosensors for in-vivo measurements
is documented. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Protein voltammetry; Wired enzymes; In vivo electrochemistry; Chemically modified electrodes; Electron transfer kinetics

1. Introduction trons and finally, we document the development of

More than 200 years have passed since the death of
Luigi Galvani, who could certainly be considered a
father of bioelectrochemistry, so the field is hardly a
new development. Because of the broad range of activ-
ities we have decided to focus only on faradaic bioelec-
trochemistry, meaning that important biological
phenomena such as membrane potentials and currents
resulting from ion transport across membranes will not
be discussed. Instead, we focus on three areas that have
been especially prominent in the 1990s. First we note
the extensive use of voltammetric techniques to extract
essential physicochemical data concerning the kinetics
and energetics of protein redox reactions. Second, we
consider fundamental and practical considerations in
linking or ‘wiring’ otherwise electroinactive enzymes to
electrodes so that they can efficiently transport elec-
* Corresponding author. Tel.: + 44-1865-270841; fax: + 44-
1865-272690.
E-mail address: fraser.armstrong@chem.ox.ac.uk (F.A.
Armstrong)
biosensors to be used especially in monitoring events
occurring about single cells, in tissue slices or in intact
brain.
The use of voltammetric methods to study redox
proteins and their active sites has gained increasing
attention. Following the early pioneering studies of the
1970s and 1980s, voltammetric methods have become
routine tools for determining formal potentials of redox
centers in small proteins. It is now well established that,
with suitable electrodes, small electron carriers such as
cytochromes, ferredoxins and blue Cu proteins give
reversible, electrochemistry without the need for small
mediators. Progress is being made with increasingly
complex systems, including multi-centered enzymes,
and it is becoming acknowledged that the electrochemi-
cal response of active sites provides a useful diagnostic
signal, in many ways analogous to that provided by
various spectroscopic methods. The electrode replaces
physiological redox partners, providing instead a driv-
ing force that is variable in the potential and time
domains to energize reactions and a sensor to measure
the response.

0013-4686/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 1 3 - 4 6 8 6 ( 0 0 ) 0 0 3 4 2 - X
2624 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645

2. The protein– electrode interface
Little more than 20 years ago, the idea that dynamic
electrochemical methods could be applied to protein
molecules as large as cytochrome c (let alone much
larger enzymes) was regarded with scepticism. The no-
table pioneers of this era were the groups led by Niki
(voltammetry of cytochrome c3 on mercury) [1], Hill
(Au modified with a monolayer of organic ‘promoter’)
[2] and Kuwana (cytochrome c at a metal oxide elec-
trode) [3]. In each case it was established that important
information on a protein’s redox properties could be
obtained from experiments in which direct electron
exchange took place between the electrode and the
active site. Since that time, many important develop-
ments have taken place in the design and structural
characterization of electrode surfaces that are active
with proteins [4]. The misleading dogma that protein
adsorption always presents an undesirable problem has
given way to more constructive ideas in which the
adsorbed state actually provides the key to studying
and understanding these complex systems, many of
which are intimately associated with interfaces (e.g.
membranes) in their natural environment. Accordingly,
efforts have been made to achieve strong adsorption of
proteins on electrodes modified in such a way as to
preserve native properties or provide a well-defined
environment in which electron-transfer or spectroscopic
features can be probed. The various types of protein–
electrode interface currently under study are depicted in
Fig. 1.
Interactions between protein and electrode may be
tailored to be weak or strong. Weak interactions might
ideally give rise to diffusion controlled voltammetry of
solution species, whereas with strong interactions the
experiment may address just a stable protein film, ide-
ally a monolayer. Following the pioneering work by the
groups of Hill and Taniguchi on the modification of
gold electrodes with organic adsorbates that yield diffu-
sion-controlled electrochemistry of cytochrome c [5,6],
further efforts have been made to understand how these
‘promoters’ work. In particular, do they provide tran-
sient interaction sites for the protein or do they prevent
blocking by contaminants? It appears that both these
factors are important. Lateral interactions among the
adsorbate molecules are important and lead to self
assembled monolayers (SAMs), the X-functionality
(nowadays usually a thiol) being bound to the metal (as
thiolate) and the Y-functionality interacting with the
solution and protein molecule. Selectivity of modified
electrodes for particular proteins can be tuned by
changing the functionality Y; thus whereas cytochrome
c prefers acidic groups such as COO(H) which can
interact more favorably with the lysine-dominated re-
gion around the exposed heme edge, acidic proteins
such as plastocyanin or ferredoxins respond best if Y is

Fig. 1. Cartoon depicting the various types of electrode surfaces for which protein voltammetry is commonly observed. Shown are
a metal electrode modified with an ‘XY’ SAM, a metal oxide electrode, a pyrolytic graphite ‘edge’ electrode often used in
conjunction with mobile co-adsorbates such as aminocyclitols, and an electrode modified with a surfactant layer in which protein
molecules are embedded.
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
a basic group such as NH2 (H+) [7,8]. Although it has
often been assumed that the adsorbate itself is stable,
recent work has shown that 4-mercaptopyridine (or
bis(4-pyridyl) disulfide, which adsorbs as 4-mercaptopy-
ridine decomposes slowly at gold to give adsorbed
elemental sulfur, thereby inactivating the surface for
protein interaction [9]. The loss of activity gives
changes in waveform similar to those expected on the
basis of the ‘microscopic model’, as active areas of the
electrode become progressively blocked [10].
The microscopic model accounts for the rounded and
drawn-out shapes of voltammograms attributed to ‘ir-
reversible’ electrochemistry, in terms of electron trans-
fer that is in fact reversible but occurs only at isolated
active zones on the electrode, i.e. those sites that are
suitably functionalized to interact with the protein
molecule. It has found fairly wide acceptance, with
support stemming from studies on electrodes coated
with lipids and presenting only ‘pinholes’ to allow
interaction with proteins such as cytochrome c [11]. The
effect of blocking protein interaction sites has also been
examined using mixed SAMs comprising active ‘X– Y’
adsorbates diluted with non-active co-adsorbates that
lack the appropriate ‘Y’-functionality [12]. With solu-
tions of cytochrome c, the classical, peak shaped
voltammograms which are exhibited at gold electrodes
modified with just 3-mercaptopropionic acid or bis(4-
pyridyl) disulfide (which is bound as 4-mercaptopy-
ridine) become increasingly sigmoidal as n-alkylthiols
are added to the monolayer. Interestingly, with 3-mer-
captoethanoic acid, the effect is much more marked as
n is increased from 3 to 18, whereas with bis(4-pyridyl)
disulfide it is the shorter chain n-alkylthiols that are
more effective in blocking the surface. It is proposed
that this is due to the way the co-adsorbates interdis-
perse on the surface, with interaction between bis(4-
pyridyl) disulfide and long chain n-alkylthiols being
sufficiently weak so as to leave open large areas of
adsorbed bis(4-pyridyl) disulfide suitable for cy-
tochrome c interaction. Relevant to this are recent
studies on how stabilities of n-alkyl SAMs depend on
chain length; notably shorter chains produce a less
stable layer because the hydrophobic effect is smaller
[13].
Further refinements in the analysis and interpretation
of diffusion-controlled protein voltammograms have
been reported. Honeychurch and Rechnitz have consid-
ered the influence on the appearance of cyclic voltam-
mograms arising from the requirement that for
SAM-modified electrodes, electron exchange must oc-
cur at the outer extremity of the SAM and not the
electrode surface itself [14]. For cytochrome c solution
voltammetry at a bis(4-pyridyl) disulfide-modified gold
electrode, the peak separation is always greater than 57
mV; further, the diffusion coefficient is smaller than
that obtained from studies at simple electrodes such as
2625
bare gold or indium oxide, or at a rotating modified
gold electrode. Assuming that the surface is not blocked
so as to generate inactive sites, these observations are
predicted by a model that takes into account the effect
of the potential drop that occurs across the adsorbed
layer.
Important advantages are gained if proteins can be
immobilized at the electrode surface, giving an elec-
troactive film that is ideally of monolayer coverage.
Armstrong and co-workers have termed this approach
‘protein film voltammetry’ to encompass a variety of
different configurations [15]. Without the need to have
protein molecules free in bulk solution, minuscule sam-
ple quantities can be used, and there is greatly increased
resolution of thermodynamic and kinetic information.
Referring back to Fig. 1, immobilization can be
achieved in several ways. Most simply, the electrostatics
can be optimised. For example, at low ionic strength,
cytochrome c adsorbs strongly at gold electrodes
modified with v-carboxylate alkanethiol SAMs, as ex-
pected if this is governed by electrostatic interactions
involving the lysine residues surrounding the exposed
heme edge [16]. Stable hydrophilic electrode materials
such as metal oxides or polished carbon (glassy carbon
or pyrolytic graphite edge-PGE, which has acidic CO
functionalities [17] also have a good capability for
electrostatically controlled adsorption of proteins with-
out denaturation or loss of biochemical activity
[15,18,19]. Proteins have even been shown to be elec-
troactive when immobilized in carbon nanotubes [20].
For the more acidic proteins, adsorption may require or
be enhanced by the inclusion of co-adsorbates such as
neomycin, polymyxin or other polyamines. Although
the structures of the resulting electrode surfaces are not
well understood, carbon and metal oxides offer certain
advantages over SAM-modified metal electrodes: for
example, pyrolytic graphite provides a very wide poten-
tial window, while metal oxides are optically transpar-
ent and permit UV-visible spectral changes to be
observed as the surface-bound protein is redox-cycled
[21]. Protein molecules may also be attached to elec-
trodes by covalent bonding. Dong and co-workers have
reported the use of carbodiimide coupling to link the
normally membrane-bound enzyme cytochrome c oxi-
dase to a Au/3-mercaptopropionic acid SAM [22]. The
‘chemisorbed’ enzyme shows reversible voltammetry
due to one or more active sites, and can transfer
electrons on to cytochrome c in solution. Hill and
co-workers achieved adsorption of Azurin on gold by
engineering a cysteine residue into the structure, The
resulting AuS bond orients the enzyme for electron
exchange and restricts its lateral movement on the
electrode [23].
Various spectroscopic studies have been undertaken
to examine the structural integrity of proteins adsorbed
at electrodes. For heme proteins, the Raman spectrum
2626 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
is a sensitive probe of spin state and hence of any
disruption to the active site. Surface-enhanced reso-
nance Raman spectroscopy (SERRS) studies show
that oxidized and reduced cytochrome c remain in
their native conformations when adsorbed at a silver
electrode coated with a monolayer of bis(4-pyridyl)
disulfide or 4-mercaptopyridine, the depth of which is
short enough to allow resonance transfer to the
protein [24]. By contrast, at a bare silver electrode,
the spin-state marker bands of adsorbed cytochromes
are shifted, signalling conformational changes that
may be relevant to function. Exploiting this further,
Hildebrandt and co-workers have reported an applica-
tion of time-resolved SERRS to measure the kinetics
of electron transfer and coupled conformational
changes in a bacterial counterpart of cytochrome c,
cytochrome c552, adsorbed at a silver surface [25].
Techniques such as ellipsometry and quartz crystal
microbalance (QCM) measurements are widely used
to quantitate the progress of adsorption of proteins
and supporting nanostructure on electrode surfaces
[26]. The question of how protein molecules are ori-
ented at the electrode has provoked some interesting
experiments, such as that reported by Wilson and co-
workers who used antibodies to study the potential-
dependence of orientation of adsorbed cytochrome c3
[27]. Orientation distributions of molecules adsorbed
on various surfaces have been studied by a combina-
tion of absorption linear dichroism and emission an-
isotropy [28]. The results indicate that for protein
adsorption at SAMs there is a quite a broad distribu-
tion of orientations, so that the protein molecules are
rather disordered. This is relevant to the problem of
peak broadening due to dispersion, noting that, ide-
ally, all the molecules in a monolayer should behave
identically. This aspect has been identified in different
kinds of experiment and modeled [29,30]. In cyclic
voltammetry, reversible electrochemistry for an immo-
bilized sample is characterized by a peak half-height
width of 90.6/n mV at 25°C. This value varies with
temperature (width 8T/K), or interactions between
sites (cooperative or anticooperative). However it can
also be increased by dispersion, i.e. inhomogeneity in
thermodynamics or kinetics.
Another way to prepare electroactive films of
protein molecules is to immobilize them in surfactant
films, which resemble lipid bilayers and multilayers.
Rusling and co-workers have developed procedures
for preparing surfactant films into which proteins are
embedded. These may be multi-layer assemblies within
which protein molecules diffuse, but do not escape to
the bulk aqueous phase [31]. The surfactants are wa-
ter-insoluble, with two long-chain alkyl chains; exam-
ples being didodecyldimethylammonium bromide
(DDAB), dimyristoyl-phosphatidyl-cholate (DMPC)
and dihexadecylphosphate (DHP). Films containing
protein molecules are obtained by several methods.
The simplest way is to evaporate a chloroform solu-
tion of surfactant onto the electrode, which is typi-
cally pyrolytic graphite, gold or platinum. The
electrode is then placed in a solution of the protein
which then diffuses into the film. Another method is
to mix an aqueous vesicle dispersion of surfactant
with protein solution then evaporate this onto the
electrode. These procedures give multiple layers, and
for gold or platinum deposited on a quartz crystal,
the depth of the layer can gauged by QCM measure-
ments. Initial studies were carried out with myo-
globin, a heme-containing oxygen binding protein that
displays only slow electron transfer at bare electrode
surfaces [32]. Rusling’s group found that myoglobin
(Mb) gives reversible voltammetry when contained in
a DDAB film at basal plane graphite, a discovery that
has been elegantly exploited (see below) by Farmer
and co-workers [33]. The approach was subsequently
extended to cytochrome P450, enabling direct detec-
tion of redox transitions of the heme group [34]. Re-
duction potentials are sensitive to the nature of the
surfactant, i.e. whether DDAB or DMPC are used,
and also (as expected) to the presence of CO. The
enzyme film also catalyzes reduction of O2 and reduc-
tive dechlorination of trichloroacetic acid. These films
behave electrochemically as thin layers within which
the protein molecules diffuse freely, although the rea-
son for this high mobility is not clear. As liquid crys-
tals, it is easy to envisage a very dynamic medium
with alkyl chains moving around like a solvent.
There is substantial spectroscopic evidence to sug-
gest that these proteins retain most if not all of their
native structure when immobilized in these surfactant
films. Rusling and co-workers have employed UV-visi-
ble, EPR and reflectance FT-IR to establish the in-
tegrity of myoglobin and cytochrome P450 in films
deposited on suitable substrates [35]. Amide IR bands,
which reflect backbone CO stretching and are sensi-
tive to protein conformation, do not alter significantly
when Mb is transferred from aqueous solution to the
DDAB film.
Boussaad and Tao have used atomic force mi-
croscopy (AFM) to examine the interactions of
proteins (myoglobin or cytochrome c) and DDAB
with a pristine, highly-ordered pyrolytic graphite
(HOPG) surface [36]. Cytochrome c adsorbs ran-
domly, rapidly giving a complete layer. By contrast,
adsorption of Mb from solution proceeds slowly, but
eventually results in chain-like structures about 60 nm
long consisting of blobs about 6 nm wide. At this
point in time, voltammetry reveals a reversible redox
couple with a potential close to that expected for
myoglobin. If the electrode is coated with DDAB,
adsorption of Mb is much more random, suggesting
that strong DDAB – protein interactions break up the
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
inter-protein interactions responsible for aggregation at
the bare electrode. The corresponding voltammetry is
less reversible than at the bare electrode, most likely
reflecting the increased distance over which electron
transfer must occur. The structure of the DDAB layer
was also found to be potential dependent, with the
liquid crystal phase only being favored above 0 V.
These results are somewhat at variance with previous
conclusions that Mb is inactive at a bare graphite
electrode, but nevertheless provide an alternative and
revealing perspective.
Rusling, Lvov and co-workers have also examined
the properties of films prepared using an alternating
layer-by-layer adsorption strategy [37]. Stacked bilayers
can be formed starting from a layer of mercapto-
propanesulfonic acid on gold or quartz, followed by
successive layers of protein (Mb, cytochrome P450),
polyanion/DNA, protein, etc. The progress of assembly
can be monitored using a QCM. These films do not
support protein mobility and most of the resulting
electrochemical activity stems from proteins closest to
the electrode surface. Using this method, it has recently
proved possible to detect active sites in a photosyn-
thetic reaction center [38].
The scope for developing membrane-mimetic surfaces
appears very promising. Salamon and co-workers re-
ported studies on two membrane bound proteins,
spinach chloroplast cytochrome f and beef heart mito-
chondrial cytochrome c oxidase integrated into a phos-
phatidylcholine bilayer coated on a tin-doped indium
oxide electrode [39]. Voltammetric signals due to re-
versible redox couples were obtained in each case, one
from cytochrome f at 365 mV and two from cy-
tochrome c oxidase, at 250 and 380 mV. These values
are quite close to reported values, the latter correspond-
ing to the CuA and cytochrome a electron-transfer
centers of the terminal oxidase, although no catalytic
activity was described. The shapes of the voltam-
mograms suggest that these proteins are immobilized in
the bilayer.
Hawkridge and co-workers have developed an inter-
esting way to immobilize cytochrome c oxidase, based
on the formation of a submonolayer of octadecylmer-
captan (OM) on a metal surface (Ag deposited on Au)
[40]. This serves as the template/anchor for assembling
a bilayer that is completed by amphiphiles (L-phos-
phatidylethanolamine or L-phosphatidylcholine). The
cytochrome c oxidase/membrane mimetic electrode is
prepared by entrapping an aliquot of solution contain-
ing detergent (deoxycholate)-solubilised enzyme and
amphiphile against the OM-modified electrode surface
with dialysis membrane, the principle being that as the
detergent dialyzes out, the enzyme becomes incorpo-
rated into the OM/amphiphile bilayer. The immobilized
enzyme exhibits voltammetry in which a broad oxida-
tive wave observed at low scan rates is joined by a
2627
reductive wave as the scan rate is increased. The pres-
ence of a bilayer is supported by QCM measurements,
while the catalytic enhancement that is observed with
cytochrome c in solution suggests that the enzyme is
oriented with its binding site for this natural partner
directed away from the electrode. However, the charge
that is passed during potential sweeps is far greater
than expected for a monolayer, prompting the sugges-
tion that the excess charge is capacitative and arises
from a redox-coupled conformational change that in-
creases ion transport in the lipid bilayer. This is an
interesting idea, since redox-driven conformational
changes are believed to be functionally important in
this enzyme’s role as a proton pump.
3. What factors determine ET rates at
protein– electrode interfaces?
The most revealing experiments in this area have
been carried out with SAM-modified electrodes since
these provide the best defined surfaces. As with non-
protein systems, it is expected that ET rates for protein-
SAM-electrode systems depend on a donor-acceptor
distance, and indeed electrochemical studies with func-
tionalized (X– Y) n-alkanethiol SAMs have confirmed
that rates depend on the length of the spacer. Bowden
and co-workers used cyclic voltammetry to determine
electron exchange rate constants (k0) between cy-
tochrome c and gold across medium-length
HS(CH2)n COOH SAMs [16], whereas Niki and co-
workers studied chain lengths as short as n =2 using ac
potential-modulated UV-visible electroreflectance [41].
With this method, ET rates are obtained from the
frequency dependence of the reflected light, and it is
worth noting that it can be applied to systems such as
‘blue’ Cu proteins which do not have such intense
optical transitions as hemes [42]. With the longer spac-
ers 9 B nB 11, rate constants depend exponentially on
chain length, consistent with a through-bond tunneling
mechanism and a decay factor of 1.1 per CH2− group.
This result is in excellent agreement with results ob-
tained for simple redox species attached to alkanethiol
SAMs [43,44]. As the chain length is shortened, rate
constants do not increase as expected but instead ap-
proach a limiting value; accordingly it is proposed that
electron exchange becomes gated by changes in confor-
mation/orientation of the protein. The implication is
that the most stable orientation does not correspond
with the most favorable orientation for electron trans-
fer, so that some rate-limiting adjustment is required.
However, studies carried out on n-alkyl SAMs termi-
nated with a covalently attached small-molecule redox
couple have indicated that complications arise here also
as the chain length is shortened [45]. Whatever the
reason for kinetic limitations, it is clear that rate con-
2628 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
stants well in excess of 1000 s − 1 are readily attainable
with proteins whose active sites lie quite close to the
surface that contacts the electrode. Rate constants of
this magnitude are observed also in studies of proteins
adsorbed at graphite electrodes [46].
Evidence that the rates of electron transfer depend in
a very subtle way on how the protein interacts with the
electrode is provided by Bowden and co-workers, who
have reported an intriguing result obtained for the
voltammetry of horse heart and yeast cytochrome c
adsorbed at mixed SAMs [47]. With horse heart cy-
tochrome c, the ET rate constant obtained using a
mixed SAM derived from equimolar amounts of
HS(CH2)10COOH and HS(CH2)8OH is about 5-fold
faster than one comprised only of HS(CH2)10COOH.
By contrast, with yeast cytochrome c, the pure
HS(CH2)10COOH SAM gives only a very low rate, but
there is a 2500-fold rate enhancement when switching
to the mixed HS(CH2)10COOH/HS(CH2)7OH mono-
layer. The two proteins have similar ET reorganisation
energies and surface charge distribution, but there are
large differences in amino acid composition that could
affect the ET coupling to the electrode.
Continuing this theme, the question of why the elec-
trochemical rates observed for myoglobin depend so
much on the electrode environment is unclear. Here we
are comparing heterogeneous rate constants (cm s − 1)
among different studies in which myoglobin is diffusing
in the aqueous or surfactant film phase. Rusling and
co-workers have suggested that the high activity ob-
served in surfactant films is due, at least in part, to
inhibition of adsorption of deactivating contaminants
[48]. Another possibility concerns the nature of the
ligation to iron and how this affects the ET reorganisa-
tion energy. Hawkridge and co-workers showed that
the CN−-ligated form displays much more reversible
voltammetry than the native aquo-Fe(III) form at an
indium tin oxide electrode [49]. Variations also exist
between myoglobins from different species; the protein
from horse heart shows a higher rate constant than the
recombinant protein from sperm whale [50]. Arm-
strong’s group later showed that a significant increase
in the rate constant for myoglobin electrochemistry at a
pyrolytic graphite edge electrode could be achieved
simply by replacing the distal histidine (H64) by amino
acids such as glycine or leucine [51]. The structures of
many of these H64 mutants have been crystallographi-
cally characterized. Histidine-64 stabilizes the H2O lig-
and that is bound to Fe(III) but not Fe(II), and also
links this coordinated H2O to bulk solvent water via a
chain of H-bonds; the mutants break this connection.
Consequently, placing myoglobin in the more hydro-
phobic environment of a surfactant film can disrupt
these interactions and lower the reorganization energy
for electron transfer.
4. Studies of protein redox reactions using
voltammetric techniques
An obvious advantage of voltammetric methods over
the more conventional and widespread use of mediated
potentiometric titrations is that a large amount of data
can be accumulated in a short time-an obvious asset in
such laborious tasks as measuring the variation of
reduction potentials with pH. Freedom from the depen-
dence on chemical titrants and mediators or the need
for a characteristic spectral feature further facilitates
experiments that are not readily undertaken with tradi-
tional methods. Some of these aspects will now be
described before dealing with some of the more recent
developments in which the protein is interrogated as a
film on the electrode.
First, unlike potentiometric studies, there is no re-
quirement for a distinctive and unambiguous change in
some spectroscopic parameter-for example, an EPR
signal that may be observable only at cryoscopic tem-
peratures and thus require rapid freezing. A problem
with freeze quenching is that re-equilibration of elec-
trons can occur during the freezing process, particularly
if quenching from elevated temperatures [52]. By con-
trast, direct electrochemical methods allow in situ mea-
surements of reduction potentials. This aspect is
exemplified in measurements of the temperature depen-
dence of the reduction potential of the ferredoxin from
Pyrococcus furiosus [52– 54]. This organism is a hyper-
thermophile found naturally near deep sea hydrother-
mal vents at temperatures exceeding 100°C; an
understanding of its electron-transport chains thus re-
quires knowledge of the thermodynamic properties of
individual components measured under these extreme
conditions. Earlier potentiometric titrations, monitored
by EPR spectroscopy after freezing equilibrated sam-
ples, had shown a non-linear variation of reduction
potential at high temperature, with a discontinuity oc-
curring at 80°C [53]. A change in conformation was
proposed, and this was discussed in terms of its rele-
vance for an organism that grows at these elevated
temperatures. However, recent in situ voltammetric
studies (at a glassy carbon electrode) have revealed that
no such discontinuity occurs [52,54].
A second example is the ability to measure redox
thermodynamics at high pressures. The variation of
reduction potentials with pressure yields V 0, the change
in molar volume between oxidized and reduced forms,
and hence the difference in compressibility ( 8 #V 0/
·
#P). Sligar and co-workers used a high-pressure
voltammetric cell to study cytochrome c up to pressures
of 5 kbar [55]. Over the range from ambient to 5 kbar,
the potential increased by 76 mV, enabling the authors
to quantify independent observations that cytochrome
c(II) has a more compact structure than cytochrome
c(III). Smith and coworkers have applied voltammetric
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
Fig. 2. Voltammograms of films of 7Fe ferredoxins on a PGE
electrode at 0°C. Azotobacter 6inelandii (scan rate 20 mV s − 1,
pH 7.0). Desulfo6ibrio africanus (scan rate 191 mV s − 1, pH
7.0) and Sulfolobus acidocaldarius (scan rate 10 mV s − 1, pH
7.4). In each case an electroactive coverage of approximately
one monolayer is obtained in the presence of polymyxin as
co-adsorbate. Signals A%, B% and C% refer to the redox couples
[3FE – 4S] + /0, [4FE – 4S]2 + / + and [3FE – 4S]0/2, respectively.
methods to study both high-temperature and high-pres-
sure effects on rubredoxins (Rd, active site = Fe(SR)4)
from a mesophile (Clostridium pasteurianum) and hy-
perthermophile (P. furiosus) [56]. The effects are appre-
ciable, for example the reduction potential of P.
furiosus Rd decreases from 31 mV at 25°C to −93 mV
at 95°C, while reduction potentials increase slightly
with pressure.
Highly reducing species with reduction potentials
that are too negative to access with conventional chem-
ical titrants such as dithionite can be studied by voltam-
metric methods. The result of this is that there are now
many examples of proteins containing a [4Fe – 4S]2 + / +
cluster with reduction potentials more negative than
− 600 mV. In particular, voltammetry has made possi-
ble the extensive characterisation of crystallographi-
cally-defined mutants of the 7Fe ferredoxin from
Azotobacter 6inelandii in which the environments of the
[3Fe – 4S] and [4Fe – 4S] cluster are altered systemati-
cally [57,58]. The [4Fe – 4S] cluster in this protein has a
2629
very negative reduction potential and was once thought
to be of the ‘HiPIP’ type ([4Fe – 4S]3 + /2 + ). Until re-
cently, the so-called ‘clostridial 8Fe’ ferredoxins were
the only well studied class of small proteins containing
two [4Fe – 4S] clusters, and invariably these have very
similar reduction potentials, at around − 400 mV.
Through voltammetric methods, and again through the
ability to probe active sites with very negative reduction
potentials, it has recently become clear that there is a
distinct class of naturally occurring 8Fe ferredoxins in
which one of the [4Fe – 4S] clusters has a much lower
potential which prevents its reduction by dithionite
[59,60].
Even when very negative potentials do not pose a
problem, iron-sulfur clusters are often difficult to study
by conventional methods, their broad and uncharacter-
istic absorption spectra providing only poor signatures
for real-time measurements at ambient temperature.
The situation is particularly unsatisfactory if dealing
with labile, air-sensitive species. Voltammetry provides
an attractive solution to these problems, a good exam-
ple being the ability to engage and monitor reactions
occurring at [3Fe – 4S] clusters. As shown in Fig. 2,
[3Fe – 4S] clusters display two signals, one due to the
well-studied [3Fe – 4S] + /0 couple and one at very nega-
tive potential which integrates to double intensity and is
assigned to the [3Fe – 4S]0/2 − couple [61,62]. The latter
signal has the narrow width expected for cooperative
two-electron transfers and the variation of reduction
potentials with pH shows that 2 – 3 H+ are also
transferred.
Proton transfer is not a widely acknowledged prop-
erty of Fe– S clusters, but [3Fe – 4S] clusters appear to
be an interesting exception. The two signals serve as a
diagnostic feature of the presence of [3Fe – 4S] clusters
in proteins, and this has been exploited in studies of the
potential-dependent transformations to cubane adducts
[4Fe – 4S] or [M3Fe– 4S] that are observed for the ferre-
doxins from Desulfo6ibrio africanus and P. furiosus
[62– 65]. These redox-coupled reactions (see below) are
conveniently induced and monitored in proteins ad-
sorbed at a pyrolytic graphite edge electrode. Several
novel clusters have been ‘synthesised’ in this way, in-
cluding those incorporating Tl, Cu and Pb. The result-
ing [M3Fe– 4S] clusters undergo reversible ligand
exchange, which presumably occurs at the new subsite
M [66].
Coupling between electron transfer and chemical/
conformational changes is conveniently depicted in
terms of a square-scheme such as that shown (Scheme
1), in which the species are interconnected by electro-
chemical (E) and chemical (C) transformations [66].
The latter include protein/prosthetic group conforma-
tional changes, and transfer of ions (including protons).
Whereas equilibrium methods like potentiometry re-
veal only the thermodynamics of these systems, dy-
2630 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
namic methods provide kinetic information. In biology,
electron transfer drives chemical reactions; equally im-
portant, perhaps, are ‘gated’ electron-transfer reactions,
in which electron-transfer rates are determined (gated)
by the preceding chemical process. Voltammetry is a
powerful tool for resolving and quantifying these com-
plex reactions, and several examples involving cy-
tochromes and Fe– S clusters have recently been
reported.
The redox properties of cytochromes depend greatly
on the nature of the axial ligation to iron. In particular,
since Fe(II) may prefer different ligands to Fe(III),
electron transfer can drive changes in axial coordina-
tion and vice versa, and the resulting kinetics can be
observed and deconvoluted by electrochemical meth-
ods. Studies by Haladjian and co-workers [67], then
Barker and Mauk [68] revealed the ligand interchange
kinetics accompanying redox cycling of mitochondrial
cytochrome c. Under mild alkaline conditions, the axial
methionine ligand to Fe(III) is easily replaced by com-
peting ligands, and cyclic voltammograms recorded on
appropriate timescales (i.e. scan rates of the order of a
few volts per second) reveal the interconversion kinet-
ics. Interestingly, this type of problem can be tackled by
other electrochemically-based methods, notably
Hawkridge and co-workers used double potential step
chronoellipsometry, in which circular dichroism is mon-
itored through an optically transparent indium oxide
electrode [69]. Feinberg and co-workers have recently
studied a mutant form of yeast cytochrome c, in which
phenylalanine-82, situated close to Met-80, is replaced
by histidine [70]. This directs the formation of a well-
defined Fe(III) state in which His-82 replaces the origi-
nal Met-80 ligand present in Fe(II). The results are
depicted in Fig. 3. Recently Mauk and co-workers have
been able to detect states formed in mutants in which
one or more of the lysine residues that normally replace
Met-80 in the Fe(III) form are replaced by alanine [71].
Even more complex transformations occur in the
di-heme enzyme cytochrome c peroxidase from P. deni-
trificans. Here, cyclic voltammetry displays how reduc-
tion of one heme induces a change at the other, and
Scheme 1.
activates the enzyme to catalyze peroxide reduction
[72].
The redox-coupling experiments outlined above in-
volved free protein molecules undergoing reversible dif-
fusion-controlled electrode reactions. Alternatively,
coupled chemistry can be studied in adsorbed proteins.
Immobilising the protein sample creates important ad-
vantages in terms of sample size and response time, and
even cyclic voltammetry can provide kinetic resolution
on the millisecond timescale or lower, and allow the
detection and measurement of electron-transfer gating
[73].
Many redox reactions in proteins are coupled to
proton transfer, and indeed this forms the basis of
energy conservation whereby transfer of electrons down
a thermodynamic gradient is used to pump protons
uphill so that energy is stored as a proton gradient.
Some of the details of how this can occur at an active
site have been revealed by a study of the [3Fe – 4S]
cluster in A. 6inelandii 7Fe ferredoxin [73]. One-electron
reduction (to give [3Fe – 4S]0) is coupled to the uptake
of a single proton: this further example of proton
transfer at Fe– S clusters is significant because high-res-
olution crystal structure studies on the different oxida-
tion and protonation states reveal (a) that the cluster is
buried with no access for water molecules, and (b) that
a carboxylate group from an aspartate (D15) is located
close to the cluster on the protein surface. The mecha-
nism of proton transfer between the cluster and solvent
has been determined using protein film voltammetry in
conjunction with site-directed mutagenesis. As indi-
cated in Fig. 4, data analysis procedures (‘trumpet
plots’) compare the scan rate dependence of voltammet-
ric peak positions to those expected for suitable kinetic
models. With the D15N mutant, proton transfer is very
slow, and oxidation of [3Fe – 4S]0 is gated by release of
H+. The native protein exhibits much faster proton
transfer, and rate constants vary with pH in accordance
with the proton being transferred by the D15
carboxylate.
As mentioned above, [3Fe – 4S] clusters are unique in
exhibiting further redox chemistry in the form of a
two-electron/two-proton reduction of [3Fe – 4S]0 to
yield a novel ‘hyper-reduced’ state in which all three
iron atoms are formally 2+ [61]. The physiological
significance of this reaction is unclear. However the
achievement of formal oxidation levels ranging from
all-Fe(III) to all-Fe(II) is unprecedented for Fe– S clus-
ters, as is the coupling to multiple proton transfer. The
process occurs at potentials of around − 700 mV at pH
and may involve some rearrangement of the cluster
environment. Recent experiments using fast scan cyclic
voltammetry reveal that the hyper-reduced 2-state can
undergo a very fast and reversible two-electron/two-
proton oxidation, yielding a state that differs from the
normal ‘0’ level into which it converts within a second
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645 2631

Fig. 3. Top. Experimental cyclic voltammogram (circles) and simulation (solid line) for Phe82His yeast Iso-1-cytochrome c (in
solution) obtained using a gold electrode modified with a monolayer of bis(4-pyridyl) disulfide. Scan rate 50 mV s − 1. Bottom.
Square scheme showing redox-coupled interchange of Met-80 and the residue His-82 that is introduced. The voltammogram is
simulated using an equilibrium constant KR/R% = 3.6 × 105 (i.e. R favored) and k (R%“ R) =10 s − 1. Adapted from original figure in
reference [70], with permission.
2632 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
Fig. 4. Plots of peak position as a function of scan rate
(logarithmic scale) for proton-coupled reduction and reoxida-
tion of the buried [3FE – 4S] + /0 cluster of the D15N mutant of
Azotobacter 6inelandii ferredoxin, in which proton transfer is
retarded. The detailed kinetics and energetics can be analyzed
by simulation of the shapes of the ‘trumpet plots’. In this case
the black diamonds correspond to pH 8.34 (pH pK, thus no
protonation occurs) while the gray diamonds correspond to
pH 5.50 (pH  pK). The solid line is a simulation with pK=
6.9, and the proton release rate koff is 2.5 s − 1. The shapes are
explained as follows using the scheme shown above. At pH\
pK, no proton transfer occurs and the peaks separate in an
approximately symmetric fashion. By contrast, at pH  pK,
the reduced cluster becomes protonated and three regimes are
observed: (a) at low scan rate, the peaks lie close together at
the formal reduction potential which is at a more positive
value because protonation is coupled to electron transfer; (b)
at intermediate scan rates, electron transfer to the cluster is
followed by proton transfer (E1CR), but re-oxidation is pre-
vented because the proton ‘off’ rate is too slow and gates the
reaction, so that no return peak is observed; (c) at fast scan
rates the electron is ‘re-called’ from the reduced cluster before
protonation can occur, thus giving peaks that coincide in
position with the high pH experiment, i.e. protonation is
decoupled (E1 only). Reference [46].
[73]. These results are supported by recent experiments
carried out at − 85°C in which the protein coated
electrode is transferred to a cryosolvent (70%
methanol), enabling this chemistry to be observed at
slow scan rates [74]. The results suggest that the cluster
is performing disulfide chemistry, although its physio-
logical relevance is unclear.
Interesting voltammetry has been observed for the
iron storage protein ferritin. This protein has a giant
cavity which can accommodate over 4000 Fe(III) min-
eralized as a hydrated oxide. Although it is widely
accepted that uptake and release of iron involves reduc-
tion to Fe(II), details of the processes involved are yet
to be established. Zapien and coworkers observed that
ferritin adsorbs at a tin-doped indium oxide electrode
to give films of submonolayer coverage, thus providing
the opportunity to control and observe iron mobiliza-
tion [75]. The voltammetry is not straightforward; in
the first reductive scan, two large reduction peaks ap-
pear at potentials of approximately − 100 and − 300
mV versus SHE, and correspond to two different forms
of the protein. Subsequent cycles show just single oxi-
dation and reduction peaks at an average potential of 0
mV. However, in the presence of EDTA, the voltam-
meric peaks vanish after only one cycle, showing that
iron is released and then complexed. This establishes
the capability of voltammetry to probe this complex
chemistry and further studies should provide some in-
teresting alternative perspectives on this important
problem.
For electron-transport enzymes, an important crite-
rion for voltammetric experiments should be that elec-
trons exchanged with the electrode should be translated
into catalytic action. Without this, there must be doubt
about whether or not the enzyme is catalytically active
or able to function with the electrode as a redox
partner. A theoretical analysis of electrocatalytic wave-
forms due to adsorbed enzymes has been described, the
aim being to recognize different modes of kinetic con-
trol and extract mechanistic information [76]. The inter-
face consisting of electrode, enzyme and electrolyte is
considered as a three-resistor series through which cata-
lytic current flows. Three limiting cases have been con-
sidered, corresponding to control by interfacial electron
transfer (least useful), substrate mass transport, and
enzyme catalysis (most useful).
Farmer and co-workers have discovered that myo-
globin entrapped in surfactant films catalyzes interest-
ing transformations of NO and NO2− [33]. In this
confined and controllable state, myoglobin provides an
excellent small-protein model system for examining the
NO metabolizing activities of certain heme enzymes.
Some results are shown in Fig. 5.
Under N2, Mb confined within a DDAB film at
basal-plane graphite displays two reversible redox cou-
ples, assigned to Fe(III)/(II) and Fe(II)/(I) transforma-
tions of the active site. The protein appears to diffuse
within this film, as noted also by the Rusling group.
When N2 is replaced by NO, the voltammetry changes
dramatically, with the appearance of a catalytic reduc-
tion peak at − 0.75 V versus SCE which is dependent
on the partial pressure of NO but independent of pH
over the range 5.5– 10. Controlled potential electrolysis
with mass spectrometric analysis of the head gas reveals
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
that the major product is N2O. A film of pre-formed
MbNO cycled under a N2 atmosphere shows a re-
versible couple at − 0.87 V, which persists indefinitely
at high pH provided the potential is cycled sufficiently
fast to outrun an irreversible chemical reaction which
results in the reappearance of Fe(III)/(II) and Fe(II)/(I)
signals. Piecing these observations together and draw-
ing on known reactions of NO species in solution, the
authors have modeled the MbNO electrochemistry in
terms of the following hypothesis and scheme (Scheme
2) the result of which is the reduction of NO to give
N2O.
Production of NO2 is accounted for by side reactions
including reduction of the Fe(II) nitrosyl Mb which is
followed by irreversible dissociation of reactive NO−.
Mb-DDAB films catalyze reduction of NO2− in reac-
tions that produce NH3, NH2OH, N2 and N2O [33].
Fig. 5. Catalytic reduction of NO by myoglobin incorporated
in a surfactant film. Cyclic voltammograms in deaerated pH
7.4 phosphate buffer, 0.1 M NaBr, scan rate 500 mV s − 1: (a)
graphite electrode cast with a DDAB film; (b) myoglobin
incorporated into DDAB film; (c) as for (b) but solution
saturated with NO. Modified from original figure in reference
[33]b, with permission.
2633
Mb confined to these surfactant films is also active in
oxygenation reactions. Under aerobic conditions, large
catalytic reduction currents are observed, which are
ascribed to the reduction of coordinated O2 to produce
H2O2 [77]. However, the oxy-intermediate that is pro-
duced in this reaction is active also in oxygenating
substrates such as styrene. Styrene oxidation is not
rate-determining in the electrochemical mechanism but
the transformations are clearly evident from glc analy-
sis of bulk electrolysis products.
For adsorbed redox couples, half-height widths vary
as 1/n, while peak heights depend on n 2, where n is the
number of electrons transferred in the process. The
significance of this relationship is that cooperative two-
electron active sites appear much more prominent in
voltammograms and can quite easily be diagnosed. This
is illustrated in the voltammetry of yeast cytochrome c
peroxidase, an enzyme that catalyses reduction of hy-
drogen peroxide by cytochrome c and which has long
been a valuable system for studying oxygen activation
by hemes. Adsorption of cytochrome c peroxidase
(Fe(III) resting state) at a pyrolytic graphite edge elec-
trode from dilute ice-cold 20 mM phosphate buffer
gives rise to a prominent signal at + 750 mV versus
SHE (pH 5.4) [78]. The signal consists of oxidation and
reduction peaks that each have half widths below 80
mV, and it is therefore assigned to a cooperative two-
electron couple, i.e. oxidation of Fe(III) to a state that
is equivalent to ‘Fe(V)’. The peaks transform to a
sigmoidal catalytic reduction wave when H2O2 is
added. Studies of the rotation rate and peroxide con-
centration dependence yielded a turnover number com-
parable to that reported from solution studies, and it
was concluded that the oxidized form of the redox
couple is either ‘compound I’ (Fe(IV) and radical) or a
closely related species. The electron-exchange kinetics
of adsorbed cytochrome c peroxidase were also mea-
sured using staircase cyclic voltammetry, although in-
terpretation of the value of approximately 6 s − 1 is
complicated by the fact that two electrons are trans-
ferred [79]. Comparisons have been made with a mu-
tant W51F in which the distal tryptophan (not the same
tryptophan that houses the radical) changed to pheny-
lalanine. The mutant has a more positive reduction
potential ( +883 mV at pH 5.4) and higher catalytic
activity, but is less stable at the electrode. The result
showed that this tryptophan plays a significant role in
stabilizing the Fe(IV) heme, possibly by creating favor-
able interactions with the bound oxide ion.
The flavin group provides a further illustration of
how cooperative two-electron centers may be particu-
larly ‘visible’ to voltammetric methods. Normally, FAD
or FMN cofactors in enzymes are detected and studied
via their visible absorption spectrum (400– 500 nm) or
occasionally through EPR if a one-electron radical is
stabilized. Flavocytochrome c3, a periplasmic fumarate
2634 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645

Scheme 2.

reductase from the marine bacterium Shewanella turnover’ signals consist of (a) oxidation and reduction
frigidimarina, provides an interesting example of an envelopes covering the range 0.1 to −0.2 V, each
enzyme in which the FAD is very difficult to observe dominated by a prominent peak (the covalently-bound
because its spectrum is obscured by the intense Soret FAD) which overlays the weaker features due to two
bands from four heme groups; however, it is rendered Fe– S clusters, and (b) a weaker feature at − 0.3 V
clearly visible by voltammetry. The enzyme adsorbs which is assigned to a [4Fe – 4S] cluster having a singu-
from dilute solution at a pyrolytic graphite edge elec- larly negative reduction potential. Addition of fumarate
trode to give a dense coverage equivalent to an elec- gives rise to an intense sigmoidal catalytic reduction
troactive monolayer [80]. The resulting voltammetric wave whose amplitude is dependent on rotation rate.
signal consists of complex reduction and oxidation As mass-transport control is relaxed, i.e. at high fu-
peaks enveloping the contributions of all redox centers. marate concentrations and high rotation rates, the cata-
As shown in Fig. 6, the FAD cofactor is clearly visible
as a sharp and prominent component of this envelope.
Despite not being covalently bound, the FAD does not
dissociate from the adsorbed enzyme. The adsorbed
enzyme is very active: when fumarate is added, the
complex envelope collapses to a sigmoidal wave the
magnitude of which is very dependent on electrode
rotation rate.
The ability to probe activity as a function of poten-
tial makes it possible to detect even the subtlest of
changes in rate that may accompany redox transforma-
tions of centers in an enzyme. This may indicate a
regulatory activity for certain centers or help confirm
their involvement in catalytic electron relays. An inter-
esting example is the fumarate reductase from E. coli.
This is a membrane-bound enzyme consisting of four
subunits, two of which are catalytic and membrane-ex-
trinsic (i.e. protrude from the membrane surface) while Fig. 6. Baseline corrected voltammogram for a film of flavocy-
the other two are membrane-intrinsic anchors and tochrome c3 (Shewanella frigidimarina) adsorbed at a PGE
provide a binding site for the natural electron donor electrode in the presence of polymyxin as co-adsorbate. Tem-
menaquinol. A soluble form consisting only of the perature 0°C, pH 6.0. The FAD cofactor is clearly observed
above the redox transitions due to the four heme groups. The
membrane-extrinsic subcomplex can be obtained, either
different components have been resolved (gray lines within
by chemical resolution or genetic engineering. This envelope) and the resulting overall simulation is overlaid on
adsorbs strongly at a rotating disk pyrolytic graphite the experimental result with excellent agreement. When fu-
edge electrode and yields informative voltammetry marate is added to the solution and the electrode is rotated,
[81,82]. In the absence of fumarate, the observed ‘non- the signals transform into a strong catalytic wave.
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
lytic wave exhibits a second boost in current close to
the potential of the [4Fe – 4S] cluster. This shows that
despite its low potential, this center is somehow in-
volved in catalytic electron transfer.
Mitochondrial complex II (succinate – ubiquinone ox-
idoreductase) is structurally similar to fumarate reduc-
tase (two membrane anchors and two
membrane-extrinsic subunits containing a covalently-
bound FAD and three Fe– S centers) and can also be
prepared as a membrane-extrinsic soluble form (succi-
nate dehydrogenase) which adsorbs on a pyrolytic
graphite edge electrode. In this state it is catalytically
active, but the response is not stable and the coverage is
too low to observe signals from the active sites. How-
ever, the short-lived activity is informative [83]. Experi-
ments conducted with a 1:1 mixture of fumarate and
succinate produce a characteristic current response: suc-
cessive cycles show succinate oxidation at high poten-
tial and fumarate reduction at low potential, each
decreasing in amplitude but crossing at isosbestic po-
tentials in each direction. The average values of these
potentials (at which there is no net reaction) corre-
sponds to the formal reduction potential of fumarate.
Catalytic waveforms at different pH values are deter-
mined by computing difference voltammograms, i.e.
subtracting an early cycle from a later one. The result-
ing profiles show: (a) that at pH values above 7.4 the
enzyme is actually biased to operate in the direction of
fumarate reduction, and (b) that although fumarate
reductase activity is high, it is limited to just a narrow
region of potential. As the potential is made more
negative, intending to drive fumarate reduction faster,
the activity shuts down. Because this resembles negative
resistance, the behavior was termed the ‘tunnel diode’
effect [81]. The decrease in activity can be fitted to a
two-electron reaction that occurs close to the potential
region expected for the FAD. This suggests that in its
FAD-reduced form, the enzyme favours a less-active
conformation which is avoided when the steady-state
potential is maintained at a higher value. In support of
this, non-electrochemical assays showed a negative or-
der in viologen concentration, i.e. the rate increases as
the reductant is consumed [84]. Succinate dehydroge-
nase from E. coli gives the same result as the beef heart
enzyme despite there being a significant difference in
the isoelectric point and only 50% amino acid similarity
(mostly confined to those residues believed to lie in the
active site) [85]. These results argue against, but do not
rule out the possibility, that the ‘tunnel diode’ effect is
due instead to a potential dependent re-orientation.
Armstrong and co-workers used this system to explore
how voltammetry can be used to observe and resolve
H/D isotope effects in electron-transport catalysis [86].
Hydrogenases have featured in several reports, and
useful information has been obtained. An early exam-
ple of faradaically monitored catalytic activity was re-
2635
ported by Bianco and Haladjian, but details on the
enzyme properties were not revealed [87]. More re-
cently, the all-Fe enzyme from Megasphaera elsdenii
has been studied [88]. The enzyme adsorbs at a glassy
carbon electrode, and from the relative catalytic cur-
rents obtained in either direction it was possible to
determine electrochemically the degree to which it is
biased to operate in the direction of H2 production. The
NiFe enzyme from Chromatium 6inosum has recently
been studied; in this case ‘non-turnover’ signals are
revealed quite clearly in the CO-inhibited enzyme,
which are tentatively assigned to the [3Fe – 4S] cluster
and the two [4Fe – 4S] inhibited enzyme, that are tenta-
tively assigned to the two [4Fe – 4S] clusters (each at
approximately − 0.3 V) and the [3Fe – 4S] cluster at
− 0.03 V [89]. Catalytically, the enzyme shows a clear
preference for H2 oxidation over proton reduction.
Oxidation of H2 (10% atmosphere) is mass-transport
controlled, as revealed by strong sigmoidal currents
which are very dependent on electrode rotation rate,
and the estimated turnover number is in excess of 1500
s − 1, considerably higher than values obtained by con-
ventional dye reduction assays in solution. Significantly,
the H2 oxidation activity is very high at potentials
below that of the [3Fe – 4S] cluster, so that this site must
remain reduced throughout turnover.
5. Mediated protein electron transfer
Most of the proteins discussed above have as their
biological function the transfer of electrons to other
proteins. They typically function in ordered structures
such as mitochondria and the redox active centers are
generally accessible to the outer surface of the protein.
In contrast, the redox reactions catalyzed by a class of
enzymes known as oxidoreductases involve small
molecules. Thus one of the two required substrates
(redox couples) for the enzyme will be designated the
substrate and other the co-factor or co-substrate. Be-
cause the catalytic center of the enzyme is buried well
within the protein, communication between this center
and an electrode serving as a source or sink for elec-
trons is frequently poor or non-existent. Because oxi-
doreductases selectively catalyze reactions of analytical
importance, it is of interest to efficiently couple the
enzyme electron transfer to the electrode, which then
effectively serves as one of the substrates. With a few
exceptions such as horseradish peroxidase (HRP)
[90,91], heterogeneous electron transfer rates for en-
zymes are vanishingly low so that the enzyme redox
activity must be coupled to the electrode using media-
tors whose redox states are alternatively recycled at the
electrode, at adjacent mediators, and the enzyme active
site. If successfully accomplished, the rate of recycling
of the redox state of the enzyme then becomes propor-
2636 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
tional to the rate of the enzymatic reaction and there-
fore to the concentration of the substrate of interest.
Such a device is known as an enzyme electrode and has
been widely employed as an analytical tool, especially
for the determination of glucose and lactate. There are
two broad classes of oxidoreductases: oxidases that use
oxygen as a co-substrate and dehydrogenases that typi-
cally use NAD or NADP as a co-factor. It should be
said at the outset that critical properties of the enzyme
electrode as a practical device (sensitivity, stability, size,
selectivity and the medium in which measurements will
be made, etc.) will determine whether the device is
useful or not. One important goal is to make the sensor
‘reagentless’, meaning that nothing has to be added to
the medium for proper functioning. This requirement is
a major reason for the overwhelming popularity of
oxidases, because many media have sufficient oxygen
present that none need be added. The reaction sequence
is:
Sred + Eox ? Sox + Ered (1)
S%ox + Ered ? S%red + Eox (2)
If, for example, glucose oxidase (GOx) is the enzyme
employed, then Sred, Sox, S%ox and S%red correspond to
glucose, gluconic acid, oxygen, and hydrogen peroxide,
respectively. The goal is to make reaction 1 rate limit-
ing, and this necessarily requires a large excess of
oxygen (S%ox). The overall rate of the reaction is moni-
tored either by following the rate of consumption of
oxygen (requires two measurements: one in the presence
of enzyme, one in its absence) or the formation of
hydrogen peroxide. The advantage of the former
method is the selectivity that can be achieved using a
gas permeable membrane, the disadvantage being the
added complexity of the device. In the latter case an
applied potential of around 600 mV versus AgCl/Ag
reference electrode is needed, and in this region numer-
ous endogenous species such as ascorbic acid, uric acid,
and a variety of biogenic amines are electroactive. A
major and largely successful solution to this problem is
the use of permselective membranes to exclude interfer-
ing species. The electrochemistry of hydrogen peroxide
at physiological pH at a platinum electrode is actually
quite complicated and has been the subject of recent
study [92,93]. The rate of electron transfer depends on
the potential-dependent formation of Pt(II) sites on the
electrode surface.
Mediated electron transfer has therefore attracted
considerable attention not only for reasons of funda-
mental interest but also to make a more practical
enzyme electrode. Mediators should be distinguished
from electrode modifiers which are not themselves elec-
troactive at the potential of the protein electron transfer
reaction. If reaction 2 above could be replaced by one
involving coupling or ‘wiring’ to the electrode, then, in
principle, the sensor response would no longer be sensi-
tive to fluctuations in S%ox. This condition can only be
met if the rate of electron transfer via the ‘wired’ route
is high compared to the rate of reaction of the enzyme
with endogenous oxygen. Most mediator-based sensors
involving oxidases do not meet this requirement and
exhibit parasitic effects of oxygen on their response
which in some cases are as large as 70% [94]. Willner’s
group has elegantly addressed the question of whether
it is possible to ‘wire’ an enzyme with sufficently high
efficiency to prevent competition with oxygen [95]. This
objective was apparently met by removing the non-co-
valently bound FAD redox center from the enzyme
(GOx) and reconstituting the enzyme on a tether con-
sisting of cystamine chemisorbed on a gold surface, a
pyrroloquinoline quinone (PQQ) link, and FAD. The
transfer of electrons from the FAD to the PQQ and
thence to the electrode was sufficiently rapid that a high
turnover number (maximum rate for the enzymatic
reaction) was obtained (900 9 150 s − 1), which com-
pares favorably with a value of about 1000 s − 1 for the
enzyme in solution. There is only a monolayer of
enzyme on the electrode surface, thus aiding efficient
communication. Such a device might have, however,
only limited stability because of the small amount of
enzyme present. Heller’s group has examined a variety
of approaches to ‘wiring’ of enzymes with the goal of
finding a mediator that reacts rapidly with the enzyme,
but has also a low potential. In the case of oxidases, the
formal potential of the flavin redox center is low
enough that a relatively weak oxidant with a potential
in the range of + 30 to −100 mV versus AgCl/Ag
reference would be sufficient to drive reaction 2 to
completion without at the same time effecting the elec-
trocatalytic oxidation of species such as ascorbate. The
approach has involved the covalent attachment of re-
dox centers such as [Os(bpy)2Cl] + /2 + [96] or [Os(4,4%-
dimethoxy-2,2% bipyridine)2Cl] + /2 + (E o% = 0.035 V
versus SCE [96] to polymeric hydrogels. The low poten-
tial of the mediator reduces but does not eliminate the
electrocatalytic oxidation of ascorbate. Although the
hydrogels are somewhat fluidic, the redox centers are
still not mobile enough to compete favorably with
freely diffusing oxygen. Some success in reducing oxy-
gen dependence has been achieved by employing mem-
branes that will ‘salt out’ oxygen so that it does not
reach the enzyme layer [97].
A number of other mediators have been tried includ-
ing ferrocene derivatives [98], phenoxazines, phenothi-
azines, and Wurster’s salts [99] and the conclusions are
consistent: mediated systems frequently show poor sta-
bility when operated continuously for extended (hours –
days) periods of time. In general, it appears that the
demise of the mediator itself, through leaching or chem-
ical degradation, is the primary problem [100]. A solu-
tion has been the dissolution of the mediator in carbon
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
paste to provide a largely renewable supply. Wang has
proposed this strategy for ‘storing’ oxygen [101]. This
approach is acceptable provided that there is no prob-
lem with soluble mediator leaking into the sample. On
the other hand, electrochemically-based ‘fingerstick’
strips which aid diabetic patients in monitoring blood
glucose use non-covalently immobilized mediators such
as ferrocene and ferricyanide, but such devices need
only to give a reliable response for perhaps 30 s [102].
Enzymes have also been adsorbed on or trapped within
electrodeposited conducting polymers [103,104].
One mediator system that yields surprisingly stable
sensors is the so-called organic salt electrode. This is
based on the use of an electrode coated with the
organic salt tetrahydrofulvalene (TTF) tetra-
cyanoquinodimethane (TCNQ). First reported by
Kulys [105] in the 1980s it became the focus of signifi-
cant study. It is thought to mediate electron transfer
with GOx, resulting from the corrosion of the electrode
to produce a layer of soluble cationic TTF+ close to
the electrode surface that can reoxidize the enzyme
according to reaction 2 [106]. It has been suggested that
TTF+ is also not stable [99], thus the apparent sensor
stability is probably due to the replenishment by corro-
sion of the TTF+ available for electrocatalysis. Khan
[107] has investigated in some detail the importance of
the morphology of the organic electrode surface in
defining the sensitivity and stability of a glucose sensor
constructed from this material. A high rate of mediator
efficiency was achieved due in part to the nature of the
TTF+TCNQ− salt deposit that was dendritic and
shaped like a tree. This could have caused efficient
corrosion with the result that the sensor showed very
little dependence on oxygen. Ascorbate is a potential
interferent to the operation of sensors using this system,
but in this case the interference was about 1% at 10
mM glucose. The apparent Michaelis constants for
glucose, that are again indicative of efficient mediation,
were in the range of 20 – 60 mM, depending on the
applied potential.
Another approach to the facilitation of electron
transfer is the use of colloidal gold particles [91]. HRP
is immobilized on gold particles (30 nm diameter)
which are then deposited on an electrode surface. By
comparison, efficient coupling of the enzyme to a
smooth gold electrode is not observed, due no doubt to
the ability of the colloidal gold particles to interact
intimately with the protein. The authors postulate that
only the monolayer of gold particles in direct contact
with the electrode is really effective and that the addi-
tion of mediators to ‘connect’ subsequent layers does
not appear to have any effect. Hydrogen peroxide
produced by reaction 2 above oxidizes the HRP and a
potential of 0 V versus AgCl/Ag is required to reduce
the HRP to its native state. The advantage of this
arrangement is that the applied potential is in a range
2637
where few endogenous species in biological fluids are
electroactive, the disadvantage is that HRP can non-
specifically catalyze the oxidation of various organic
molecules present in the medium. Direct electron trans-
fer of HRP has also been demonstrated at suitably
prepared carbon electrodes [90].
Several fundamental studies have enhanced under-
standing of electron transfer between mediators and
enzymes: investigation of single electron donors and
acceptors (largely ferrocene derivatives) shows that
their reactivities do not follow simple outer sphere
electron transfer [108]. Mikkelsen and co-workers [109]
have developed the theory for intramolecular electron
transfer involving the multiple attachment of ferrocene
derivatives to GOx. The highest rate of intramolecular
electron transfer was observed for a ferrocene car-
boxylic acid (0.9 s − 1). If the rate of reaction with
oxygen with the enzyme is assumed to be 2 × 106 M − 1
s − 1, then the ‘relay rate’ would have to be ] 5 ×103
s − 1 if the sensor is placed in an air saturated solution
([O2] =240 mM). The observed rates are far short of
this limit, again emphasizing the influence of oxygen on
the relay process. The theory for electron transfer to
enzymes through mediator layers has also been devel-
oped [110,111].
There are over 300 enzymes that employ either
NAD(P) or NAD(P)H as a co-factor, and for this
reason the issues surrounding both the immobilization
of such enzymes (dehydrogenases) and the recycling of
the NAD/NADH couple become important. Many
electrochemical studies of NADH oxidation have been
carried out following the classical work of Elving and
co-workers in the 1970s [112]. The objective has been to
find a modified electrode that will reduce the overpo-
tential for NADH oxidation and prevent or minimize
adsorption of oxidation products on the electrode sur-
face. A number of mediators including phenoxazine
derivatives [113], catechols [114] bipyridines, metal
complexes [115], and hydroxybenzaldehydes [116] have
been used for this purpose. A significant advantage of
the latter materials, especially 3,4 dihydroxybenzalde-
hyde, is the property of improved stability and the
possibility of electrodeposition to form a chemically
modified electrode. The pseudo first order rate con-
stants for reaction with NADH are in the range of
2.6× 103 M − 1 s − 1. The above functionalities have
been attached to polymers [117] that are then deposited
on the electrode surface. There has been considerable
discussion over the years as to whether the oxidation of
NADH involves hydride transfer (one step) [118] or
whether instead the process involves three steps (elec-
tron/proton/electron) but within one complex [119].
The goal of the chemically-modified electrode is to
lower the barrier to electron transfer and prevent the
dimerization of the one-electron oxidation product. The
quinone-like structures offer the possibility of meeting
2638 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645

Fig. 7. Schematic of Integrated lactate dehydrogenase enzyme electrode. Reference [122]a with permission.

such requirements. When NAD is used as a co-factor it
is generally soluble and freely diffusing. Numerous
attempts have been made to immobilize both the en-
zyme and the co-factor without notable success. How-
ever, Willner’s group has developed a scheme whereby
the NAD is first immobilized via a PQQ-containing
tether that serves, in effect, as a template for lactate
dehydrogenase binding. After the enzyme is in place, it
is then cross-linked with glutaraldehyde to stabilize the
layer. The ordered structure is shown schematically in
Fig. 7. This is the first clear example of a fully inte-
grated and ‘reagentless’ system where NAD is a cofac-
tor. The stability is moderate and the apparent KM for
lactate is about 3.6 mM. [120]. The oxidation of
NADH can be carried out bioelectrocatalytically using
an enzyme (diaphorase), and a variety of quinones or
flavins have been employed as mediators [121].
6. Preparation of ordered protein layers
The desire to couple electron transfer from an en-
zyme layer to an electrode has additionally focused
attention on the ordered immobilization of protein. The
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
strategy is to build up successive layers of enzyme so
that the resulting structure can be studied systemati-
cally. Four approaches have been employed: (1) Build-
ing of successive layers of enzyme by reaction of the
enzyme with bifunctional linkers (tethers) [122]; (2) Use
of biotinylated enzyme linked in successive layers using
avidin [122– 124]; (3) Linking of successive layers using
electrostatic immobilization [110]; (4) Linking of succes-
sive layers using antibodies [125]. Fundamental studies
based on these ordered to quasi-ordered structures have
employed freely diffusing mediators as well as media-
tors covalently attached to the enzyme [126]. The an-
choring of the tether to the electrode can block the
surface thus reducing the effective electrode surface
area available for amperometric detection of substrates.
This can be serious problem especially when using
microelectrodes. As a solution to this problem, Kuhr
and co-workers [127] have developed techniques for
preparing nanostructure domains (photopatterned lines
about 5 mm wide) on carbon electrodes in which alter-
nate lines are ‘bare’ electrode and tether anchor
(biotin).
7. In-vivo electrochemistry
The advances in electrochemical detection of species
in biological milieu in the 1990s were based on a desire
to ask more detailed questions about the real-time
fluctuations of biologically relevant analytes. This ne-
cessitated the development of sensors with dimensions
in the 1 – 10 mm range and with response times (90%
maximum response) of a few seconds or less. Wightman
and co-workers [128] observed current transients at
microelectrodes that they attributed to the single vesicu-
lar release of epinephrine and norepinephrine from
isolated adrenal medullary cells. The signal, obtained
under constant potential amperometric conditions, is
manifested as a series of spikes ranging from 0.2 to 2
pcoulombs (1– 10 attomol catecholamine detected). One
micron diameter electrodes were used to monitor exocy-
tosis from a vesicle whose diameter is B200 nm. This
work established that it was possible to directly moni-
tor and time resolve secretion events at the single-cell
level. Although the quantal hypothesis for neurotrans-
mitter release is well accepted, caution must be taken in
interpretation of results. The actual time-dependent
events are convoluted by diffusion from the vesicle to
the point of observation by the sensor. Catecholamine
exocytosis in PC12 cells was monitored by Chen, Luo
and Ewing [129]. Using a carbon electrode modified
with ruthenium oxide/cyanoruthenate developed by
Cox and Gray [130] Kennedy and co-workers [131]
were able to detect exocytosis of insulin from beta cells.
The sensor is not very specific for insulin, which it does,
however, oxidize rapidly, but because there are few if
2639
any other proteins present, selectivity is not a major
problem. In passing from the cell culture to the brain
slice or intact brain as the measurement medium, sev-
eral issues arise that must be considered. First, the
insertion of the sensor into tissue creates tissue damage,
which is minimized as the diameter of the sensor de-
creases. Implantation can result in the creation of a
liquid layer immediately surrounding the sensor that
has diffusional and other properties different from the
tissue itself. Second, species diffuse through tissue nec-
essarily more slowly, with the result that diffusion
coefficients can be a factor of 3 – 4 smaller in tissue than
in aqueous solution. This issue has been dealt with in
the context of transport of choline in rat brain [132].
Finally, the sensor may lose sensitivity compared to its
in-vitro value as a result of its contact with biological
fluid. The observed signal due to neurotransmitter re-
lease may be distorted due to the finite response time of
the sensor. If the response time is known it is possible
to deconvolute the signal, taking the sensor properties
into account [133]. Electrochemical measurements in
the brain have been recently reviewed [134].
Much of the research involving in-vivo electrochem-
istry has exploited the electroactivity of small molecules
such as catecholamines. Many, if not most, relevant
species, are not electroactive, and therefore they must
be determined indirectly. Because of its importance to
the treatment of diabetes, glucose sensors have become
the ‘holy grail’ in the biosensor area. There are many
approaches being taken to the monitoring of blood
glucose, but at present electrochemical detection ap-
pears the most tractable. Although the electrochemi-
cally-based glucose sensor has been known since the
1960s [135], it was not until the mid-1980s that feasibil-
ity of in-vivo monitoring was demonstrated [136]. This
approach [137] has involved a needle sensor (enzyme
electrode of about 250 mm diameter) that can be im-
planted in the subcutaneous tissue of the forearm or
abdomen for periods of 4 – 7 days, after which they are
removed and replaced. Such a sensor is shown in Fig. 8.
The sensing element is built on a Pt– Ir wire substrate,
followed by a permselective membrane to exclude elec-
troactive interferences such as ascorbate, urate, and
acetaminophen. Next the enzyme is immobilized, and
this is followed by an external membrane having the
property of being highly permeable to oxygen while
exhibiting only limited permeability to glucose. These
properties make it possible to prepare a sensor with a
linear response to glucose over the required range (2–
20 mM glucose) and response characteristics largely
independent of oxygen partial pressure. The short-term
implants give useful results but may need to be cali-
brated one to two times per day due to limited stability
while the long-term implants currently suffer from de-
struction of the implanted instrument package by the
remarkably corrosive elements within the tissue. An
2640 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
important alternative approach might involve a chroni-
cally implanted device that will be expected to function
reliably for 6 – 12 months [138].
Other sensors have also been developed for brain
studies including glucose [139], glutamate [140] and
lactate [141]. The sensors implanted in the brain must
be smaller, faster in response than those in the subcuta-
neous tissue, and free of interferences. For example, the
glutamate sensor must detect 1 mM glutamate in the
presence of 300 – 400 mM ascorbate. The latter elec-
troactive interferent can be removed in two ways: with
a permselective membrane or by using ascorbate oxi-
dase, an enzyme that reduces oxygen to water and not
peroxide, thus making it useful for ascorbate elimina-
tion. Other tissues have also been examined as the
monitoring of lactate release in cardiac tissue has been
demonstrated [142]. If oxidases are employed, it is
essential to establish that low oxygen levels do not
affect sensor response. Progress in sensor design and
methodology has emphasized the value of time-depen-
dent data that an electrochemical sensor can provide.
Of perhaps even greater importance is the possibility of
obtaining simultaneously data on different analytes. By
monitoring glucose, lactate and oxygen simultaneously
in the rat brain, it was possible to demonstrate the
elevation of lactate levels that might otherwise have
been confused with ischemia (oxygen deficiency) [141].
Multiple analyte measurements have also been re-
ported, some combining optical and electrochemical
detection [143].
Fig. 8. Schematic of implantable glucose sensor used for continuous monitoring in humans (see reference [137]).
Electrochemical detection has proven extremely valu-
able in monitoring inorganic species that play an im-
portant role in biological systems. These include
oxygen, hydrogen peroxide, superoxide, and nitric ox-
ide. Oxygen is ideally suited to electrochemical detec-
tion and such methodology was reported in the
physiology literature as early as 1942 [144]. There is
currently significant interest in oxygen in studies of
oxidative stress [145]. For such studies it is necessary to
know not simply concentrations, but fluxes in or out of
cells. One such approach is the ‘self-referencing’ elec-
trode [146]. The concept is to make measurements of
the current due to oxygen at points outside a cell in
culture that fall within the cell concentration gradient.
By physically moving the 1 – 3 mm diameter electrode at
about 0.3 Hz over a distance of 5 mm, two values of the
oxygen concentration can be measured. From this in-
formation and an electrode calibration, the concentra-
tion gradient can be calculated. It is presumed that
electrode motion does not disturb the gradient. The
movement of the electrode has another advantage: it
allows electrode noise to be eliminated through phase
sensitive detection. Electrochemistry has provided some
of the greatest excitement of the decade due to the
possibility of measuring nitric oxide, voted the
Molecule of the Year by Science. This very reactive
species cannot be easily monitored by alternative meth-
ods. Malinski [147] reported on an electrode modified
by a Ni(II) porphyrin. Although the oxidative electro-
 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
chemistry is quite complicated, it appears that an an-
odic current proportional to NO can be obtained to
detection limits in the range of about 10 nM. The
biochemistry of this versatile molecule has been dis-
cussed in the context of electrochemical measurements
[148]. Using a 30 mm platinum disk electrode and
double differential pulse amperometry, it was possible
to selectively measure hydrogen peroxide in the rat
striatum in the presence of dopamine, ascorbic acid,
and uric acid with a detection limit of 0.03 mM [149].
A two-enzyme system (superoxide dismutase and cata-
lase) was used as part of a 50 mm disk microelectrode
to measure hyperoxia (superoxide) in the striatum of a
freely moving rat [150].
Thus far relatively little attention has been paid to
acquisition of detailed understanding of how sensor
performance is affected by contact with tissue and how
the tissue is affected by the presence of the sensor. It is
understood that the initial implantation of sensors can
destroy capillaries and surrounding cells and this inva-
sion triggers the wound healing process the first step of
which involves protein adsorption on the sensor. With
this in mind, membrane materials have been developed
with ‘water-like surfaces’, that tend to minimize ad-
sorption [151]. Biocompatibility studies on biosensors
have been limited, and much needs to be learned about
the interfacial reactions which occur.
8. New electrochemical methodology as applied to
bioelectrochemistry
New technology had a significant impact on bioelec-
trochemistry during the 1990s. Bard’s group extended
scanning tunneling microscopy to the use of the probe
as an electrode (scanning electrochemical microscopy
(SECM)) to detect the activity of glucose oxidase on a
non-conducting surface [152]. The technique has subse-
quently been extended to other applications such as
the detection of guard cells on a plant leaf [153] or the
detection of carcinoembryonic antigen (CEA) mi-
crospotted on a plate and reacted with a CEA–
horseradish peroxidase conjugate [154]. The method
can detect as few as  100 molecules in a 20 mm spot.
SECM has also been used to pattern active enzyme on
a surface. This is accomplished by deactivating enzyme
by oxidation of Cl− or Br− at the enzyme surface
[155].
With increased emphasis on measurements involving
single cells, it has proven useful to exploit microfabri-
cation and micromachining techniques to produce
wells containing electrodes that can accommodate sin-
gle cells. Using this format, Bratten and co-workers
[156] developed a 200 mm diameter well with electrodes
in the bottom, with a depth of 20 mm and a total
volume of  600 pL. The objective was to measure
2641
purine (adenosine and inosine) release during cardiac
ischemia. To accomplish this detection it was necessary
to employ a cascade of three enzymes: adenosine
deaminase, nucleotide phosphorylase, and xanthine ox-
idase. By determining the charge generated from the
oxidation of peroxide, the final product of the serial
enzymatic reactions, femtomole levels of adenosine
could be detected. The detection limit is about 120
amol. The technology lends itself easily to rapid
screening techniques as employed within the pharma-
ceutical industry. Its virtue, the small volume of liquid,
may produce concentrations of the analyte significantly
higher than physiological levels, thus leading to experi-
mental artifacts. Similarly, the effect of incubation of a
single cell in elevated enzyme levels may also affect cell
behavior. Presumably many, if not most, of these
difficulties can be resolved by appropriate control ex-
periments.
Although the electrochemistry of nucleic acids has
been studied for many years [157], there has been
increased interest in oligonucleotide detection for diag-
nostic purposes [158]. The technique of oscillographic
polarography has been revisited to permit the selective
detection of pyrimidine-based nucleotide using a cop-
per electrode and decoupling of the charging and
faradaic current in the frequency domain [159].
9. Bioelectrochemistry in the new millennium
The advances of the last 10 years have been signifi-
cant and have focused strongly on the understanding
of the interface between the electrode and the
molecules reacting with it. We can certainly expect
nanotechnology to heavily influence developments. As
the interfacial chemistry is better understood it will be
possible to more effectively promote biocatalysis and
to further develop biochemical fuel cells [160]. Several
groups have reported on the development of microelec-
trochemical enzyme transistors [161,162]. Amplification
is achieved because peroxide generated by an enzy-
matic reaction alters the conductivity of a poly(aniline)
film [162]. Lower detection limits are possible than
with conventional sensors because the products of the
reaction can accumulate. Miniaturized bioelectronic
devices are also expected to proliferate. As more sen-
sors are developed, it will be increasingly possible to
simultaneously monitor several key analytes in real
time. Finally, we can expect to see bioelectrochemical
techniques used by a growing range of scientists.
Acknowledgements
FAA acknowledges financial support from the UK
Research Councils (EPSRC, BBSRC), Wellcome Trust,
2642 F.A. Armstrong, G.S. Wilson / Electrochimica Acta 45 (2000) 2623–2645
Leverhulme Trust, Royal Society and St. John’s Col-
lege, Oxford. The partial support of the National Insti-
tutes of Health, (US) to GSW, grants DK30718 and
DK55297, is gratefully acknowledged.
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