1 Introduction A CURSORY consideration of any general undergraduate biochemistry text book reveals that electron transfer reactions and electron transport play a key role in the processes of life. In living cells electrons are transferred between redox partners in an orderly and ordered fashion. At each step in these chains of electron transport specific interactions between the redox partners ensure molecular recognition. The disruption of the sequential transfer of electrons along such chains, for example by small, indiscriminate electron transfer catalysts such as methyl viologen (paraquat), is frequently fatal. Amperometric biosensors seek to exploit the high specificity of the components of the electron transport chain to make sensors which can be used to detect particular components of complex mixtures. The very features which confer this advantage of specificity also present a problem for the electrochemist: frequently the biological molecules we wish to use show very poor electron transfer kinetics at metallic electrodes. With hindsight this is not surprising, as a simple metallic electrode rarely presents a surface attuned to the particular requirements of the biomolecule. A way around this dilemma is to try to design, or engineer, the surface of the electrode at a molecular level so that it will be recognised by the biological redox species and rapidly exchange electrons with it. Although this sounds fraught with difficulty significant advances have been achieved in this field over the past few years by building on fundamental work in the area of the electrochemistry of modified electrodes. In this paper we will briefly review the various types of electrode modification which have been used and the types of electrochemical behaviour characteristic of such modified electrode surfaces. We will then consider the application of modified electrodes in three areas of

bioelectrochemistry and their applications in amperometric biosensors.

Received 12th September 1989

surface Fig. 1 Redox catalysis at a modified electrode surface

~) IFMBE: 1990 BIO

2 Chemical modification of electrode surfaces The first studies of modified electrodes were carried out in the 1970s and concentrated on the adsorption of organic monomers at clean metal surfaces (LANE and HUBBARD, 1973a; b). Since then a variety of different modification strategies have been developed and a number of excellent reviews of the field have appeared (e.g. ALBERY and HILLMAN, 1981; FAULKNER, 1984; MURRAY, 1984; MURRAY et al., 1987; BARTLETT,1987; HILLMAN, 1987a, b). For our present purposes, the basic reason for modification of the electrode surface is to catalyse the reaction of the solution species with the electrode. This may be achieved through mediation of electron transfer by bound redox centres (Fig. 1) or, in the case of large biomolecules, by the transient binding of the solution redox species at the electrode surface in the correct orientation for electron transfer (Fig. 2). To achieve our goal of a suitable electrode surface for our target electrochemical reaction, we must first identify the functional group or groups we wish to deploy at the surface, and then select a suitable immobilisation or modification method. The various possible strategies can be represented on a dendrogram (Fig. 3). Starting from the top, modified electrodes can be divided into monolayer or multilayer struc-

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ox ~

product

red

substrate

to bulk ~, from bulk

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tures, depending on the amount of material present on the electrode surface. A multilayer structure has the advantage that many more catalytic (or binding) sites are present, and so the stability may be improved; it has the concomitant disadvantage that electrons must propagate through the multilayer film if the sites at the outside are to be of any practical use in catalysis. Moving downwards, monolayer modified electrodes are subdivided into those prepared by the covalent attachment of species to the electrode surface

is to create hydroxide groups on the metal surface, this is frequently achieved by electrochemical oxidation in acidic solution, and then to react these surface hydroxides with reactive silanes such as chlorosilane derivatives. Elimination of HX, where X is the leaving group, for example C1 of OMe, produces a silicon oxygen linkage, binding the silane to the electrode surface (MosEs et al., 1975). This approach has been used with electrodes made from platinum, tungsten, silicon (p- and n-type), indium tin oxide,

0\ adsorption

R2SiX2 -HX

0

/

SiR 2

0\

I electron transfer

/

SiR 2

0

~

~

Fig. 4 Silanisation of an electrode surface

desorption=

I

surface

bulk

Fig. 2 Cartoon of a large biomolecule binding to an electrode surface and being reduced

and those in which the molecules are absorbed at the surface. Multilayer modified electrodes, on the other hand, are frequently based on polymeric films either produced at the electrode surface by in situ polymerisation or polymerised ex situ and subsequently applied to the electrode. In the former case the method is simple and convenient, although the characterisation of the exact structure of the film is often problematical. In the latter case the polymer can be characterised, and purified, prior to deposition and this can be advantageous. Let us consider some of the options in a little more detail, beginning with the covalent attachment of species to electrode surfaces. The most popular methods are to use reactive silanes, to use cyanuric chloride, or to use the functional groups present on the surface of a carbon electrode. The use of silane coupling is illustrated in Fig. 4. The basic principle

modified electrodes

covalent adsorption polymers crystalline attachment ~ I films reversible

~

cyanuric carbon si[anisation chloride functionalisotion S ~ ' ] ~ electrochemical dip or covalent polymerisation dropcoat cross linking conducting redox polymers polymers Fig. 3 The different types of modified electrodes MBEC

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gold, nickel, germanium, gallium arsenide and glassy carbon. Many examples can be found in the literature (ARMSTRONG and SrmPARD, 1980; BURT et al., 1976; WILLMANet al., 1980). The cyanuric chloride method is very similar, again surface hydroxide groups are used, but in this case the cyanuric chloride is employed to activate the surface. This is followed by a second step in which an alcohol or amine is attached to the activated surface (Fig. 5, L ~ et al., 1977). In the case of carbon electrode surfaces many functional groups are already present prior to modification. The exact nature and distribution of these groups depends both on the type of carbon (pyrolytic graphite, glassy carbon etc.) and its detailed pretreatment. However, these surface functional groups can be used to anchor molecules to the electrode surface by covalent modification (KoVAL and ANSON, 1978). Fig. 6 shows some examples. One of the great advantages of electrochemistry is that it is a very sensitive technique, and this is particularly true for the detection of surface-bound electroactive species. Thus, once the electrode surface has been modified, it is very easy to detect the presence of the bound redox groups. Even a monolayer gives large, easily measured signals, and integration of the current passed gives a direct measure of the number of electroactive molecules present at the surface. Fig. 7 shows a nice example, from the work of WRIGHTON et al. (1978), of the cyclic voltammetry of a gold electrode modified by the attachment of 6.9nmolcm -2 of a ferrocene silane derivative. It is noticeable that the two peaks in the voltammogram are almost directly above each other. This is the behaviour predicted in the absence of diffusional or other kinetic limitations, and provided that there are no strong interactions between redox species at the surface (LAY[RaN et al., 1980; ANDRIEUX et al., 1986). Frequently these conditions are not met and some peak separation is observed. For further details of techniques for the characterisation of modified electrodes see MLrRRAV (1984), BARTLETT (1987) and H I L L M A N (1987a). The desire to increase the number of functional sites immobilised at the electrode surface, and thus to possibly increase the stability or activity of the electrode, led to an interest in the modification of electrode surfaces with polymeric films~ In redox polymers such as poly(vinylferrocene) charge propagation through the film occurs by successive self-exchange reactions between the immobilised redox sites (Fig. 8, PEERCE and BARD, 1980). It should be Bll

~ --.~_ N

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~

N

o.~_/~-~

/>--CI

o~-- N R0H

-HCI

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0 "-~_ N

0

o ~_Nb-cl Fig. 5 The cyanuric chloride method for the attachment of species to an electrode surface

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Different ways to functionalise a carbon electrode surface

borne in mind that these polymers are typically swollen by solvent and contain mobile counter ions so that a certain degree of flexibility of the polymeric chains is clearly possible. Many examples of polymers of this type have been studied. An alternative approach is to deposit a film of polymeric electrolyte at the electrode surface and to entrap redox species of opposite charge within this film. In essence the redox species partition into the film and are strongly held by electrostatic interactions with the fixed charged sites.

i

i

--0 " ~

~

012

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016/

Again, these polymers are often extensively swollen by solvent, and charge propagation occurs by self-exchange between the redox ions (Fig. 9). Examples of polyelectrolytes which have been used in this context include Nation (SzENXaRMAYand MARTrN, 1984; ESPENSCrmtD et al., 1986), poly(vinylpyridine) (OYAU_Aand ANsor~, 1980), and protonated poly(lysine) (ANsoN et al., 1983a; b). A common feature of the multilayer, or polymer, modified electrodes is the fact that charge must be able to move through the film if all the redox, or catalytic, sites are to play a role. In principle, a number of processes controlling the overall behaviour can be identified. These include the propagation of charge through the film, the diffusion of reactants from bulk solution into the films, the rate of

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o Fig. 7 Cyclic voltammetry of a surface-bound ferrocene. Adapted from WRI6HTON et al., 1978 B12

Fig. 8

Cartoon representing the propagation of charge through a redox polymer film, for example poly(vinylferrocene). Note that the oxidation of a bound ferrocene site is accompanied by the movement of a counter ion, X - , into the film to compensate for the charge within the film

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reaction within the film between the bound redox groups and reactants from the solution, and the kinetics of the interfacial reactions. The interplay between these different, potentially rate-limiting, processes produces considerable complexity. Nevertheless, these processes have been analysed and approximate analytical treatments of the problem presented (ALBERYand HILLMAN, 1981; 1984; ANDRIEUX et al., 1982). Based on the results of this type of analysis it is possible to identify a number of different cases and hence to undertake the rational design of polymer modified electrodes for particular catalytic applications. One method which can be used to overcome the charge transport problem is to use an electronically conducting polymer. In these materials the polymer is itself a good electronic conductor so that the electrical resistivity of the

X-

(CN)6)3-

N

Fig. 9 Cartoon representing the propagation of charge through a polymer film consisting of fixed cationic sites and chargecompensating redox anions (ferri/ferrocyanide). Note that reduction of ferricyanide within the film is accompanied by expulsion of a anion X to maintain charge neutrality within the film

after deposition, or by first preparing a suitable derivative and then forming the polymer. Finally, a wide range of types of monomer can be employed and copolymers can be formed. This great flexibility offers many exciting possibilities for the design of electronically conducting polymer electrodes for biosensor applications. Some examples of conducting polymers are shown in Fig. 10. Recent reviews of the properties of these materials include those of CHANDLERand PLETCHER(1985), DIAZ and BARGON(1986) and STREET(1986). In the following sections we will consider some examples of the applications of modified electrodes in bioelectrochemistry and biosensors. We will begin by considering applications in redox protein electrochemistry and then move on to look at applications in the electrochemistry of redox coenzymes and enzymes.

3 R e d o x protein e l e c t r o c h e m i s t r y The electrochemistry of the redox proteins has been an active area of scientific investigation for a number of years. Work in this area has been important in shaping many of our ideas about the role of interactions between electrodes and biological macromolecules and their importance in electron transfer. Excellent reviews of this work can be found in the literature (ARMSTRONGet al., 1986; 1988). We will not to go into great detail here but rather simply illustrate the role of modified electrodes in successful redox protein electrochemistry. In this context it is important to realise the constraints of distance on electron transfer. The experimental evidence indicates that the probability of electron transfer falls exponentially as the distance increases (see for example reviews by MARCUS and SLrnN, 1985; and McLENDON, 1988). Thus lgel =

l~el.0 exp (--fl[r -- to] )

(1)

where re~ is the electronic transmission coefficient, xet' o its value when the separation is r o , r is the distance between

film is low. One of the paradigm examples of a polymer of this type is poly(pyrrole) but many other examples are now known. Conducting polymers combine a number of features which make them exciting potential electrode materials for bioelectrochemistry. First, they can be deposited electrochemically so that their thickness and location are controlled. Secondly, they can be derivatised either

/ / I 0.5nm

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Fi~ 11 Schematic representation of the best and worst orientaFig. 10 Some fragments of conducting polymers: poly(pyrrole), poly(thiophene), poly(aniline) MBEC

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tions of cytochrome c at an electrode surface for electron transfer to occur. Approximate values of the distance are shown

B13

centres, and fl is a constant. Values of fl estimated from experimental measurements of long-range electron transfer rates suggest that fl is about 12 nm- 1. This implies that the probability of electron transfer decrease by an order of magnitude for every 0.2nm increase in distance between the two centres. This is a significant point when we come to consider electron transfer between redox centres in macromolecules, such as cytochrome c, and electrodes. Fig. 11 shows a schematic representation of cytochrome c. The molecule is approximately 3.4 nm in diameter with the Fe redox centre located near the periphery. If the cytochrome c presents itself to the electrode in the worst possible orientation, with the Fe centre away from the electrode surface, the distance is large and the probability of electron transfer is vanishingly small. If the molecule presents itself to the electrode in the best possible orientation, with the redox centre towards the electrode surface, the distance is much smaller and electron transfer is facile. This situation mirrors the situation in the living cell. The natural redox partner for cytochrome c is cytochrome c peroxidase, another larger macromolecule. For electron transfer to occur between the two they need to approach each other in the correct way; molecular recognition and orientation are all-important. This recognition and orientation is believed to be achieved by complementary interactions between positively charged lysine residues on the cytochrome c and negatively charged regions on the surface of the cytochrome c peroxidase. Hill and coworkers have shown (ALLENet al., 1984) that cytochrome c electrochemistry is possible at electrode surfaces.which can mimic these interactions. For example, EDDOWESand HILL (1979) have shown that gold electrodes modified by adsorption of a monolayer of 4,4'-bipyridyl exhibit reversible electrochemistry for cytochrome c. In this case the 4,4'-bipyridyl modified gold electrode presents a negatively charged surface to which the cytochrome c can bind (ALBERY et al., 1981). Similar effects can be achieved for other redox proteins such as plastocyanin and azurin where reversible electrochemistry can be achieved if the electrode surface is engineered to interact in the correct manner with the redox protein (HILL et al., 1985; ARMSTRONGand BROWN, 1987). These ideas can be extended to the design of conducting polymers for cytochrome c electrochemistry. Using electrodes coated with electrochemically polymerised films of poly(5-carboxyindole) we are able to observe electron

a

b

ii

5 A

I

O'IV I

Fi~ 12 The electrochemistry of cytochrome c at a poly(5carboxyindole) electrode surface ,recorded at (a) 100 mV s- 1 and (b) 50 mV s- 1. (i) background current, (ii) in the presence of 2Oltm cytochrome c

B14

transfer to and from the redox centres in cytochrome c adsorbed at the electrode surface (Fig. 12, BARXLEXT and FARINGTON, 1989). In this case we believe that the carboxylate groups on the polymer (Fig. 13) interact with the lysine residues on the protein surface and ensure adsorption in the correct orientation for facile electron transfer. When poly(indole) is used in place of the carboxylate derivative no corresponding cytochrome c electrochemistry is observed. It should be possible to extend this approach to other redox proteins and other conducting polymers.

H02C~

N~~/C02H H0~2CN Fig. 1:3 Supposed structure of poly(5-carboxyindole) 4 M o d i f i e d electrodes f o r N A D H electrochemistry NADH (fl-nicotinamide adenine dinucleotide) is an important coenzyme, with over 250 NADH-dependent dehyrogenases known. Consequently there is much interest in NADH electrochemistry and its application in biosensors. At clean metallic electrodes the oxidation of NADH only occurs at large overpotentials and proceeds through radical intermediates. This can cause problems with electrode fouling or interferences when applied in electrochemical sensors. Hence there have been a number of attempts to produce a modified electrode surface at which the oxidation of NADH to enzymatically active NAD + can be achieved at more modest potentials. Much of this work has been reviewed elsewhere by GORa'ON (1986), and so we will only consider selected examples here. The general approach adopted towards this problem has been to try to identify species which react rapidly in homogeneous solution with NADH and then to immobilise these at the electrode surface using one or other of the techniques already discussed. For example Miller's group (DEGRAND and MILLER, 1980; FUKUI et al., 1982; LAU and MmLm~, 1983; CARLSON and MILLER, 1985) have investigated the use of polymers containing ortho-quinone groups (Fig. 14). They found that the electrodes initially catalysed the oxidation of NADH to NAD § but that the effect rapidly decayed with use. They also found that there were problems with the propagation of charge through the films and that as a result only those quinone groups close to the electrode were in electrical communication with the electrode. A more successful approach is to use redox dyes adsorbed on graphite (Fig. 15). Using this approach GORTON et al. (1984; 1985) were able to obtain good stability and to achieve the oxidation of NADH at low potentials. They have subsequently applied this approach in flow systems using sensors based on NADH-dependent MBEC

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dehydrogenases such as glucose dehydrogenase (APPELQVISTet al., 1985). Conducting organic salts can also be used as electrode materials for these types of application. For example, N M P .TCNQ (where N M P is N-methyl phenazine and TCNQ is tetracyanoquinodimethane) is a good electrode material for the oxidation of NADH (ALBERY and BARTLETT, 1984) and can be used with alcohol dehydrogenase or steroid dehydrogenases to make alcohol (ALBERY et al., 1987b) or bile acid sensors (ALBERY et al., 1987e). In this case the reaction is thought to proceed by

Et2N

0

0

-

~

I Me

Me

I

I C ~ CH2

C M CH2

I

co

0"41

I

C02H

0.59

I NH(CH2 )2 OH

--fHCH21H--CH2 r tLeO

0.37

LCO,H

NH(CH2)2

OH

Fi~ 14 Structures of two ortho-quinone polymers used to prepare modified electrodes for N ADH oxidation

adsorption of the NADH on the surface of the conducting salt followed by hydride transfer to the Nmethylphenazinum present in the crystal lattice at the electrode surface. An advantage of this approach is that the material is sufficiently conducting that it can be formed into pressed pellets and used to make electrodes. In this form a fresh electrode surface is then prepared simply by polishing and stability is not a problem (BARTLETT, 1990). 5 Flavoprotein electrochemistry Organic conducting salts can also be used as electrodes for flavoprotein electrochemistry (KuLYS et al., 1980; ALBERY and CRASTON, 1987). For this application TTF. TCNQ, where TTF is tetrathiafulvalene, appears to be the best choice (ALBERYet al., 1985). Electrodes of this type have been used with a range of flavoproteins including glucose oxidase, amino-acid oxidases, xanthine oxidase and choline oxidase (ALBERYe t aL, 1987a; M c K E ~ A and BRAJTER-ToTH, 1987). At present the precise mechanism of the oxidation of the reduced flavoprotein is the subject of some debate (CENAS and KOLYS, 1981; KULYS, 1986; ALBERY et al., 1987c; HILL et al., 1988). Nevertheless, electrodes of this type obviously work very well and have been developed for in vivo applications (BouVELLE et al., 1986; HALE and WIGrrrMAN, 1988). Another recent approach has been to immobilise glucose oxidase within conducting polymer films at electrode surfaces. This is attractive for several reasons; the process is Biosensors special feature

for N A D H oxidation

0-63

OH

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Fig. 15 Redox dyes used to prepare modified carbon electrodes

May 1990

simple, it is readily controlled, it is well suited to the immobilisation of enzymes on microelectronic substrates and microelectrodes, and multilayer structures can be developed. The immobilisation is achieved by adding the enzyme to the monomer solution before polymerisation. The enzyme appears to be entrapped because of its high negative charge ( - 9 at neutral pH); in effect it is incorporated as a counter ion into the polycationic conducting polymer film. To date the immobilisation of glucose oxidase in poly(pyrrole) (FOULDS and LowE, 1986; UMANA and WALLER,1986) poly(aniline) (BARTLETT and W~TAKER, 1987/1988; SHINOHARA et al., 1988), poly(indole) (PANDEY, 1988), poly(N=methylpyrrole) (BARTLETT and WHITAKER, 1987) and ferrocene substituted poly(pyrrole) (FOULDS and LowE, 1988) films has been reported. In nearly all cases there is little evidence of direct electron transfer between the flavin prosthetic group of the enzyme and the polymer although FOULDS and LowE (1988) suggest that this may happen in the ferrocene derivatised polymer. Direct, unmediated, electron transfer between redox proteins and conducting polymers remains a tantalising goal. Finally, an alternative approach which looks very promising is to modify the enzyme by the covalent attachment of redox centres to the protein (HILL, 1984). In this case the suggestion is that the covalently attached redox centres act as 'stepping stones', allowing the electrons to get from the active site out to the electrode by a number of sequential, short steps (Fig. 16). Note that this is very similar to the mechanisms of charge propagation through a redox polymer film, discussed above. Thus when glucose oxidase is modified by attachment of ferrocene carboxylic acid (DEGANI and HELLER, 1987) or ferrocene acetic acid (BARTLETT et al., 1987) direct electrochemical oxidation of the reduced flavoprotein at a clean metal electrode becomes possible. This approach can be extended to other flavoproteins (DEGANI and HELLER, 1988) and may prove to be a general one. B15

Fig. 16 Cartoon of a redox mediator modified enzyme. Electron transfer from the active site to the electrode is proposed to occur by a number of shorter steps involving the bound mediators, in this case ferrocene groups

6 Conclusions In this paper I have tried to provide an overview of the field of chemically modified electrodes and the way that it impinges upon bioelectrochemistry. Chemically modified electrodes and conducting polymer electrochemistry are extensive and vibrant fields of research in their own right and it is not possible to do them justice in such a small space. Nevertheless, I hope that this brief introduction will stimulate the reader's interest and provide an entry into the literature in this fascinating area. References ALBERY, W. J. and HILLMAN,A. R. (1981) Modified electrodes. Ann. Rep. Prog. Chem., Sect. C, 377-437. ALBERY, W. J., EDDOWES,M. J., HILL, H. A. O. and HILLMAN,A. R. (1981) Mechanism of the reduction and oxidation reaction of cytochrome c at a modified gold electrode. J. Am. Chem. Soc., 103, 3904-3910. ALBERY,W. J. and HILLMAN,A. R. (1984) Transport and kinetics in modified electrodes. J. Electroanal. Chem., 170, 27-49. ALBERY,W. J. and BARTLETT,P. N. (1984) An organic conductor electrode for the oxidation of NADH. J. Chem. Soc., Chem. Commun., 234-236. ALBERY, W. J., BARTLETT, P. N. and CRASTON, D. H. (1985) Amperometric enzyme electrodes, part II. Conducting organic salts as electrode materials for the oxidation of glucose oxidase. J. Electroanal. Chem., 194, 223-235. ALBERY,W. J. and CRASTON,D. H. (1987) Amperometric enzyme electrodes: theory and experiment. In Biosensors: fundamentals and applications. TURNER, A. P. F., KARUBE,I. and WILSON,G. S. (Eds.), Oxford University Press, 180-210. ALBERY, W. J., BARTLETT,P. N., BYCROFT, M., CRASTON,D. H. and DRISCOLL,B. J. (1987a) Amperometric enzyme electrodes, part III. A conducting organic salt electrode for the oxidation of four different flavoenzymes. J. Electroanal. Chem., 218, 119~ 126. ALBERY, W. J., BARTLETT,P. N., CASS, A. E. G. and SIN, K. W. (1987b) Amperometric enzyme electrodes. Part IV. An enzyme electrode for ethanol. Ibid., 218, 127-134. ALBERY, W. J., BARTLETT, P. N. and CASS, A. E. G. (1987c) Amperometric enzyme electrodes. Phil. Trans. R. Soc. Lond., B316, 107-119. ALLEN, P. M., HILL, H. A. O. and WALTON,N. J. (1984) Surface modifiers for the promotion of direct electrochemistry of cytochrome c. J. Electroanal. Chem., 178, 69-86. ANDRIEUX, C. P., DUMAS-BOoCmAT, J. M. and SAVEANT,J. M. (1982) Catalysis of electrochemical reactions at redox polymer B16

electrodes. Kinetics for stationary voltammetric techniques. Ibid., 131, 1-35. ANDRIEUX,C. P., HAAS,O. and SAVEANT,J.-M. (1986) Catalysis of electrochemical reactions at redox polymer coated electrodes. Mediation of the Fe(III)/Fe(II) oxido-reduction by a polyvinylpyridine polymer containing coordinatively attached bisbipyridine chlororuthenium redox centers. J. Am. Chem. Soc., 108, 8175-8182. ANSON, F. C., OSHAKA,T. and SAVEANT,J,-M. (1983a) Diffusional pathways for multiply-charged ions incorporated in polyelectrolyte coatings on graphite electrodes. Cobalt oxalate in coatings of protonated polylysine. J. Phys. Chem., 87, 640-647. ANSON, F. C., OSHAKA,T. and SAVEANT,J.-M. (1983b) Kinetics of electron transfer cross-reactions within redox polymers. Coatings of a protonated polylysine copolymer with incorporated electroactive anions. J. Am. Chem. Soc., 105, 4883-4890. APPELQVIST,R., MARKO-VARGA,G., GORTON,L., TORSTENSSON,A. and JOHANSSON,G. (1985) Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode. Anal. Chim. Acta, 169, 237247. ARMSTRONG, N. R. and SI-mPARD, V. R. (1980) Voltammetric studies of silane-modified SnO 2 surfaces in selected aqueous and non-aqueous media. J. Electroanal. Chem., 115, 253-265. ARMSTRONG, F. A., HILL, H. A. O. and WALTON,N. J. (1986) Electron-transfer reactions of redox proteins at electrodes. Quart. Rev. Biophys., 18, 261-322. ARMSTRONG, F. A. and BROWN, K. J. (1987) Studies of proteinelectrode interactions by organosilyl derivatisation of pyrolytic graphite electrodes. Ibid., 219, 319-325. ARMSTRONG, F. A., HILL, H. A. O. and WALTON, N. J. (1988) Direct electrochemistry of redox proteins. Acc. Chem. Res., 21, 407-413. BARTLETT,P. N. (1987) The use of electrochemical methods in the study of modified electrodes. In Biosensors: fundamentals and applications. TURNER, A. P. F., KARUBE,I. and WILSON,G. S. (Eds.), Oxford University Press, 211-246. BARTLETT, P. N. and WmTAKER, R. G. (1987) Electrochemical immobilisation of enzymes. Part II. Glucose oxidase immobilised in poly-N-methylpyrrole. J. Electroanal. Chem., 224, 37-48. BARTLETT, P. N., WmTAKER, R. G., GREEN, M. J. and FREw, J. (1987) Covalent binding of electron relays to glucose oxidase. J. Chem. Soc., Chem. Commun., 1603-1604. BARTLETT,P. N. and WHITAKER,R. G. (1987/1988) Strategies for the development of amperometric enzyme electrodes. Biosensors, 3, 359-379. BARTLETT,P. N. and FARINGTON,J. (1989) The electrochemistry of cytochrome c at a conducting polymer electrode. J. Electroanal. Chem., 261,471-475. BARTLETT, P. N. (1990) Conducting organic salt electrodes. In Biosensors: a practical approach. CASS, A. E. G. (Ed), IRL Press, (in press). BOUTELLE,M. G., STANFORD,C., FILENZ, M., ALBERY,W. J. and BARTLETT,P. N. (1986) An amperometric enzyme electrode for monitoring brain glucose in the freely moving rat. Neurosci. Lett., 72, 283-288. BURT, R. J., LEIGH,G. J. and PICKETr, C. J. (1976) Modification of a tin oxide electrode by attachment of iron-sulphur clusters. J. Chem. Soc., Chem. Commun., 940-941. CARLSON, B. W. and MILLER, L. L. (1985) Mechanism of the oxidation of NADH by quinones. Energetics of one-electron and hydride routes. J. Am. Chem. Soc., 107, 479-485. CENAS, N. K. and KULYS,J. J. (1981) Biolytic oxidation of glucose on conductive charge transfer complexes. Bioelectrochem. Bioenerg., 8, 103-113. CHANDLER,G. K. and PLETCHER,D. (1985) The electrochemistry of conducting polymers. Electrochem., Specialist periodical report, 10, 117-150. DEGANI, Y. and HELLER, A. (1987) Direct electrical communication between chemically modified enzymes and metal electrodes. 1. Electron transfer from glucose oxidase to metal electrodes via electron relays, bound covalently to the enzyme. J. Phys. Chem., 91, 1285-1289. DEGANI, Y. and HELLER, A. (1988) Direct electrical communication between chemically modified enzymes and electrodes. 2.

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Methods for bonding electron-transfer relays to glucose oxidase and D-amino-acid oxidase. J. Am. Chem. Soc., 110, 2615-2620. DEGRAND, C. and MILLER, L. L. (1980) An electrode modified with polymer-bound dopamine which catalyses NADH oxidation. Ibid., 102, 5728-5732. DIAZ, A. F. and BARGON,J. (1986) Electrochemical synthesis of conducting polymers. In Handbook of conducting polymers. Vol. 1. SKOTnEIM,T. A. (Ed.), Marcel Dekker, 8-115. EDDOWES, M. J. and HILL, H. A. O. (1979) Electrochemistry of horse heart cytochrome c. J. Am. Chem. Soc., 101, 4461--4464. ESPENSCHEID,M. W., GHATAK-RoY,A. R., MOORE,R. B., PENNER, R. M., SZENTIRMAY,M. N. and MARTIN,C. B. (1986) Sensors from polymer modified electrodes. J. Chem. Soc., Faraday Trans. I., 82, 1051-1070. FAULKNER,L. R. (1984) Chemical microstructures on electrodes. Chem. & Eng. News, 27th Feb., 28-45. FOULDS, C. N. and LOWE, C. R. (1986) Enzyme entrapment in electrically conducting polymers. Immobilisation of glucose oxidase in polypyrrole and its application in amperometric glucose sensors. J. Chem. Soc., Faraday Trans. I., 82, 12591264. FOULDS, C. N. and LOWE,C. R. (1988) Immobilization of glucose oxidase in ferrocene-modified pyrrole polymers. Anal. Chem., 60, 2473-2478. FUKUI, M., KITANI, A., DEGRAND,C. and MILLER, L. L. (1982) Propagation of a redox reaction through a quinoid polymer film electrode. J. Am. Chem. Soc., 104, 28-33. GORTON, L., TORSTENSSON,A., JAEGFELDT,H., and JOHANSSON,G. (1984) Electrocatalytic oxidation of reduced nicotinamide coenzymes by graphite electrodes modified with an adsorbed phenoxazinium salt, meldola blue. J. Electroanal. Chem., 161, 103-120. GORTON,L., JOHANSSON,G. and TOgSTENSSON,A. (1985) A kinetic study of the reaction between dihydronicotinamide adenine dinucleotide (NADH) and an electrode modified by adsorption of 1,2-benzophenoxazine-7-one. Ibid., 196, 81-92. GORTON, L. (1986) Chemically modified electrodes for the electrocatalytic oxidation of nicotinamide coeznymes. J. Chem. Soc., Faraday Trans. I, 82, 1245-1258. HALE, P. D. and WIGHTMAN, R. M. (1988) Enzyme-modified tetrathiafulvalene tracyanoquinodimethane microelectrodes: direct amperometric detection of acetylcholine and choline. Mol. Cryst. Liq. Cryst., 160, 269-279. HILL, H. A. O. (1984) Assay techniques utilising specific binding agents. European Patent Application No. 84303090.9. HILL, H. A. O., PAGE, D. J., WALTON,N. J. and WnlTFORD, D. (1985) Direct electrochemistry, at modified gold electrodes, of redox proteins having negatively-charged binding domains: spinach plastocyanin and a multi-substituted carboxydinitrophenyl derivative of horse heart cytoehrome c. J. Electroanal. Chem., 187, 315-324. HILL, B. S., SCOLARI,C. A. and WILSON, G. S. (1988) Enzyme electrocatalysis at organic salt electrodes. Ibid., 252, 125-138. HILLMAN,A. R. (1987a) Polymer modified electrodes: preparation and characterisation. In Electrochemical science and technology of polymers, 1. LINFORD,R. G. (Ed.), Elsevier, 103-239. HILLMAN, A. R. (1987b) Reactions and applications of polymer modified electrodes. In Electrochemical science and technology of polymers, 1. LINFORD,R. G. (Ed.), Elsevier, 241-291. ](OVAL, C. A. and ANSON, F. C. (1978) Electrochemistry of the ruthenium (3+, 2+) couple attached to graphite electrodes. Anal. Chem., 50, 223-228. KULYS, J. J., SAMALIUS,A. S. and SVIRMICKAS,G.-J. S. (1980) Electron exchange between the enzyme active center and organic metal. FEBS Lett., 144, %10. KULYS, J. J. (1986) Enzyme electrodes based on organic metals. Biosensors, 2, 3-13. LANE, R. F. and HUBBARD, A. T. (1973a) Electrochemistry of chemisorbed molecules. I Reactants connected to electrodes through olefinic substituents. J. Phys. Chem., 77, 1401-1410. LANE, R. F. and HUBBARD, A. T. (1973b) Electrochemistry of chemisorbed molecules. II The influence of charged chemisorbed molecules on the electrode reactions of platinum complexes. J. Phys. Chem., 77, 1411-1421.

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Biosensors special feature

May 1990

LAU, A. N. K. and MILLER,L. L. (1983) Electrochemical behaviour of a dopamine polymer. Dopamine release as a primary analogue of a synapse. J. Am. Chem. Soc., 105, 5271-5277. LAVIRON,E., ROULLIER,L. and DEGRAND,C. (1980) A multilayer model for the study of space distributed redox modified dectrodes. Part II. Theory and application of linear sweep poten* tial sweep voltammetry for simple reaction. J. Electroanal. Chem., 112, 11-23. Ln~, A. W. C., YEn, P., YACYNYCH,A. M. and KUWANA,T. (1977) Cyanuric chloride as a general linking agent for the attachment of redox groups to pyrolytic graphite and metal oxide dectrodes. Ibid., 84, 411-419. MARCUS, R. A. and SUTtN, N. (1985) Electron transfers in chemistry and biology. Biochim. Biophys. Acta., 811, 265-322. McKENNA, K. and BRAJ~R-TOTH, A. (1987). Tetrathiafulvalene tetracyanoquinodimethane xanthine oxidase amperometric electrode for the determination of biological purines. Anal. Chem., 59, 954-958. MCLENDON,G. (1988) Long-distance electron transfer in proteins and model systems. Acc. Chem. Res., 21, 160-167. Mos~% P. R., WmR, L. and MURRAY, R. W. (1975) Chemically modified tin oxide electrodes. Anal. Chem., 47, 1882-1886. MURRAY, R. W. (1984) Chemically modified electrodes. In Electrotroanalytical chemistry, vol. 13. BARD, A. J. (Ed.), Marcel Dekker, 191-238. MURRAY, R. W., EWING,A. G. and DURST, R. A. (1987) Chemically modified electrodes. Molecular design for electroanalysis. Anal. Chem., 59, 379-390A. OYAMA,N. and ANSON,F. C. (1980) Factors affecting the electrochemical response of metal complexes at pyrolytic graphite electrodes coated with films of poly(4-vinylpyridine). J. Electrochem. Soc., 127, 640-647. PANDEY, P. C. (1988) A new conducting polymer-coated glucose sensor. J. Chem. Soc., Faraday Trans. I, 84, 2259-2265. PEEgCE, P. J. and BARD,A. J. (1980) Polymer films on electrodes. Part II: film structure and mechanism of electron transfer with electrodeposited poly(vinyl-ferroccne). J. Electroanal. Chem., 112, 9%115. SmNOHARA,H., CmBA, T. and AIZAWA,M. (1988) Enzyme microsensor for glucose with an electrochemically synthesised enzyme polyaniline film. Sensors & Actuators, 13, 79-86. STREET, G. B. (1986) Polypyrrole. From powders to plastics. In Handbook of conducting polymers. Vol. 1. SKOTHEIM,T. A. (Ed.), Marcel Dekker, 265-291. SZENTmMAY,M. N. and MARTItq,C. R. (1984) Ion-exchange selectivity of Nation films on electrode surfaces. Anal. Chem., 56, 1898-1902. UraANA, M. and WALLER,J. (1986) Protein-modified electrodes. The glucose oxidase/polypyrrole system. Ibid., 58, 2979-2983. WILLMAN, K. W., ROCKLIN, R. D., NOWAK, R., KUO, K.-N., SCHULTZ, F. A. and MURRAY, R. W. (1980) Electronic and photoelectron spectral studies of electroactive species attached to silanized C and Pt electrodes. J. Am. Chem. Soc., 102, 76297631. WRIGHTON, M. S., PALAZZOTTO,M. C., BOCARSLY,A. B., BOLTS, J. M., FISCHER, A. B. and NADJO, L. (1978) Preparation of chemically derivatized platinum and gold electrode surfaces. Synthesis, characterization, and surface attachment of trichlorosilylferrocene, (1,1'-ferrocenediyl) dichlorosilane, and 1,1'-bis(triethoxysilyl)ferrocene. Ibid., 100, 7264-7271.

Author's biography Dr Philip N. Bartlett studied Chemistry at Oxford and Imperial College, UK, from where obtained a Ph.D., under the supervision of John Albery, in 1981. He was subsequently awarded an 1851 research fellowship and remained at Imperial until 1984, when he was appointed as a lecturer in Physical Chemistry at the University of Warwick. His research interests centre around the study of the kinetics and mechanisms of reactions at electrode surfaces and he has published over 40 papers on various aspects of electrochemistry. B17

Modified electrode surface in amperometric biosensors.

The electron transfer reactions of biological molecules are frequently very slow at ordinary electrodes. To overcome this problem, and thus to facilit...
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