Electrical contact of redox enzymes with electrodes: novel approaches for amperometric biosensors

ELSEVIER

Bioelectruchemistry and Bioencrgetics42 (1997) 95-104

Electrical c mtact of redox enzymes with electrodes: novel approaches for amperometric biosensors Eugenii Katz a, Vered Heleg-Shabtai a, Bilha Willner a Itamar Willner ~'*, Andreas F. Biickmann b Instituw nf Chemistm'. The Hebrew University of Jerusalem. Jerusalem 91904. Israel b Gesellschaft fiir Biotechnologische Forschung. Department of En~'mology. Mtt~cheroder Weg I, D-38124 Brunswick. Gemmny

Received 3 May 1996; accepted 9 May 1996

Abstract Electrical communication of the redox-active center of enzymes with an electrode surface is a fuadamemal elemem fc, the development of amperometric biosensor devices. Different methods to assemble enzyme-electrodes exhibiting electrical collect between the redox protein and electrode surface are discussed with specific examples for tailoring glucose sensing electrodes. By one apimmch, a multilayer ~rray of glucose oxidase is assembled on a Au-eleetrode. ~ number of enzyme laye~.s is contr~led by the syi~hetic methodology to assemble the electrode. Electrical contact between the enzyme array and the electrode is esta~ished by cherak:al modification of the protein layer with N-(2-methyl-ferrocene)-capt'oic acid. acting as an electrical wiring redox mediator. The number of enzyme layers associated with the electrode allows one to control the sensitivity of the resulting enzyme electrode. Afm,ther ~ of enhancing the sensitivities of enzyme electrtales involves the application of rough Au-elactredes as base-support to assemble the enzyme network. The high surface area of these electrodes (roughness factor ~- 20) allows the increase of the Ifiucatalyst comem in a sin#¢ monolayer, and the resulting ampemmetric responses of the electrodes are ca. g-fold enhanced compared to enzyme [aye~ a s s e ~ aa smooth electrodes of identical geometrical areas. A novel method to electrically wire flavoenzymes with electrode .retraces was d e v e h ~ by reconstitutiot,, of the apo-flavnenzyme with a ferrocene-tethered FAD diad. Raconstimtion of apo-glucose o x ~ with the ferrucene-FAD diad yields an active binelectrucatalyst of direct electrical communication with the electrode. "eh:ctroenzyme'. The reconstitution methodology was further applied to tailor enzyme-electrodes of superior prolxrdns for electrical contact with the electrodes. A pynoloquinoline quinone-FAD diad monolayer was assembled on a Au-electrode. A~,m-glucose oxidase was reconstitmed on the surface with the FAD-cofactor site to yield the aligned biucatalyst on the electrode. The pylxok~]uir~line q~inone, PQQ. redox m ~ ~:ts as an electron relay that electrically contacts the FAD redox-site of the enzyme with the e,'eetrode. The surface reconsdt~ed enzynm exhibits direct electrical communication with the electrode and acts as bioeleetrucatalyst for the ox~mion of glucose. The e~,-'uical communication of the reconstituted glucose oxidase on the PQQ-FAD monolayer is extremely efficiem. The experimental cm~ent density at a glucose concentration of 80 mM is 300 5:100 # A . cm -2. This value overlaps the theo~tical current density of glucose oxMnse electrode (290 + 60 p,A. cm -2) taking into account the limiting turnover-tree of the enzyme, 9L~O+ 150 s - t (a~ 35°C). The ex~emcly efficient electrical contact of the reconstituted enzyme and the electrode yields an enzyme-electro~.~e that is insensitive to oxygen and is not affected by glacosc-sensing interferants such as ascorbic acid. The application of the differem enzyme-elec.mxic configmmkms as bioalectronic devices for the determination of glucose is addressed. © 1997 Elsevier Science S.A. Keywords: Enzyme monolayer; Enzyme electrode; Glucose oxida.se; Glucose sensor, Reconstituted enzyme: Pyn'otcqui~mlinequimme; Fla,~oer,zyrae

1. Introduction Electrical communication between redox proteins ,and electrode surface is a general means to electrically activate

• Corresponding author. Tel: +972 2 658 5272; fax: +972 2 652 7715.

redox-acdve biecatalysts [l]. The electrical contact between the protein redox-center and electrode interface is, however, usually l~'.hibited due to the insulation of active site by the pretein matrix. The electron transfer theory [2,3] implies that t.~ electron transfer rate ~etween a donor and an acceptor site is given by Eq. 1. where AG ° and A are the free energy c h ~ g e and the reorganization energy associated with the electron transfer, respectively,

03024598/97/$17.00 Copyright © 1997 Elsevier Science S.A. All rights reserved. PII S0302-4598(96)05142-2

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and d is the distance separating the donor--acceptor units. The protein assembly spatially separates the redox-site from the electrode surface and practically insulates the active center [tom electrical contact with electrode interfaces. Ko, e~p[--/31( d - d,. )1. exp[ - ( Z C

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(i) Various methodologies were employed to impose electrical contact between redox prc,teins and electrodes. Diffusional electron mediators such as ferrocenes [4-6], ferricyanide [7-9], N,N'-bipyridinium ,.Ats [10,1 I] and quinone derivatives [12,13] were applied as charge transporters that connect the active redox center and electrode interface. These electron mediators operate by a diffusional route where penetration into the protein matrix permits short electron transfer distances with respect to the active site and simultaneous diffusion of the electron carrier from the protein facilitates electrical contact with the electrode surface [I]. A further means to establish electriczl communication of redox proteins with electrodes includes the surface modification of the electrode with promoter molecules that bind and align the protein to a configuration that facilitates electron transfer [14.15]. This method is usually applicable to relatively low molecular weight proteins, i.e. cytochrome c [16-18]. but sometimes this approach was successfully applied even for large enzyme molecules [19]. Chemical modific,ation of redox proteins by redox-tethered components was rep.)rted as a method to yield electrical contact between the redox center of enzymes and electrode interlaces [20,21]. By this method, electron transfer distances are shortened and the redo~ groaps associated with the protein act as electron relays for transporting the charge to or from the protein active site. For example, glucose oxidase was "electrically wired" with electrode surfaces by the covalent linkage of ferrocene units to the protein [20.21]. Immobilization of redox-enzymes in redox polymer matrices was used as an additional means to electrically communicate the redox center with electrode surfaces [22-24]. For example, immobilization of glucose oxidase in fcrrocene-modified polymers or in Os(ll)-polypyridine-substituted polymers, yielded bioelectrocatalytic as~mblies for electrooxidation of glucose [25]. Similarly, immobilization of nitrate reductase in N,N'-bipyridiniummodified polythiophene yield an enzyme exhibiting electrical contact with electrodes [26,27]. Electrochemical activation of redox-enzymes is of specific importance in the development of amperometric biosensor devices [28-30]. Electrobiocatalytic oxidation (or reduction) of the enzyme substrate allows the quantitative determination of the substrate/analyte by the transduced amperometrie output of the electrode. Tailoring amperometric biosensor devices requires the integration of redox-enzymes with electrode surfaces in an organized configuration exhibiting electrical contact. We have developed a method to assemble enzyme electrodes by covalent

attachment of protein monolayers and multilayers to Au electrodes [31-37]. Covalent modification of the immobilized protein molecules by electron relay groups enabled the establishment of electrical contact between the biocatalyst and the conductive substrate. For example, glutathione reductase was assembled as a monolayer on a At, electrode, and chemical modification of the protein layer yielded a bioelectrocatalytic system for the reduction of oxidized glutathione [31,32]. The effectiveness of electrical contact between the redox protein and the electrode is a fundamental feature of the resulting biosensor device. Effective electrical communication will yield sensitive interfaces. High sensitivities of enTvme electrodes would then contribute to two important parameters: (i) High current densities would allow the miniaturization of the electrode dimensions: (ii) Effective electrical activation of the biocatalyst would eliminate non-specific electrochemical oxidation (or reduction) of the interfering components to the enzyme substrate/analyte. That is. the electrobiocatalytic amperometric response would prohibit non-specific electrochemical processes of the interfering reagent, leading to selective sensing surfaces. In the present account we address different methods to tailor enzyme-electrodes exhibiting electrical communicatl0n and discuss various means to enhance the specificity of the electrodes.

2. Results and d~scussion

One method to enhance the electrical response of an enzyme electrode involves the organization of an enzyme network on the electrode surface and tailoring an electrical contact between the enzyme matrix and electrode surface to yield a bioelectrocatalytic electrode assembly [33]. Fig. 1 shows the method we employed to assemble a glucose oxidase network on a Au electrode. A primary cystamine monolayer was assembled on the gold surface. This monolayer was reacted with trans-stilbene-4,4'-dfisothiocyanate3,3'-disulfonate, (DIDS), (1), to yield an isothiocyanatefunctionalized monolayer. The latter monolayer was reacted with glucose oxidase, GOx, to generate the thiourealinked protein monolayer. By a two-step procedure involving the chemical modification of the base protein monolayer with trans-stilbene-4,4'-diisothiocyanate-3,3'-disulfonate. (1). and GOx, the enzyme network is assembled onto the electrode surface. The number of enzyme layers associated with the electrode is controlled by the number of treatment cycles of the electrode with the bifunctional coupling reagent, (1), and GOx. The enzyme network assembled on the electrode surface is then modified with N-(2-methyl-ferrocene)-caproic acid. (2), in the presence of urea. Addition of urea is essential as it partially unfolds the respective protein units and permits modification of inner-protein sites by the redox, electrical-contacting unit. Physical cha;acterization of the enzyme network revealed

E. Kat: et al. / Bioelectrochemist~" and Bioenerfetics 42 (1997) 9~-104

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•,b::t the average surface coverage of the enzyme per layer c, rresponds to ca. 7 x 10 -t2 mole - cm -2 and ti~ average loading of each enzyme unit by the ferroeene redox unit is 8. The electron-relay-modified GOx network on the Au electrode exhibits electrical communication and reveals bioelectrocatalytic activity. Fig. 2 shows the cyclic voltammograms of one, four and eight enzyme layers network electrodes in the presence of glucose. A single-layer electrode does not yield any delectable amperometric response but the four and eight enzyme-layer electrodes generate an electrncatalytic anodic current indicating that the enzyme assembly is electrocatalytically active, and bioelectrocatalytic oxidation proceeds. That is, the ferrocene electronrelay units are oxidized by the electrode and mediate the oxidation of the active site, where glucose is oxidized to gluconic acid (Fig. 3). The amperometric response of the enzyme network is controlled by the number of layers assembled in the protein network and, as the number of enzyme layers increases, the amperometric response of the electrode is enhanced. This allows us to tune the electrode sensitivity by the number of layers immobilized onto the electrode. This latter statement should, however, be considered with some reservation. We find that the sensitivity

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(amperometric signal) of the GOx network electrode is indeed increased, up to 1 I - 12 protein layers, and then the transduced current signal levels-off and even drops. This is attribtJted to the perturbation of the electrical contact between enzyme units and the electrode surface in thick protein networks, or to diffusion bmriers of the substrate to inner biocatalyst components in the thick protein assemblies. Fig. 4 shows the amperometric responses of a fourlayer GOx electrode at different glucose concentrations. This curve represents a calibration curve for the use of the electrode as a glucose biosensor. Control of the number of enzyme layers associated with the electrode provides one approach to regulate the electrnde sensitivity. Note that these experiments were performed with regular smooth Au surfaces (roughness factor ca. 1. I - 1.2). As the transduced amperometric signal of the electrode relates to the surface density of the protein on the elee~ode, roughe.ni~g of the electrode surface could provide an alternative method to increase the protein content in a single layer. Au electrodes were roughened by amalgamation to yield rough Au surfaces (roughness factor ca. 15-25) [38]. The rough electrodes were modified by GOx multilayer [36] by a method similar to that outlined in Fig. 1. The amperometric responses of a two-layer GOx assembly on a rough and smooth Au surface of identical geomet-

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rical area in the presence of glucose, are shown in Fig. 5. The electrocatalytic anodic current in the presence of the rough electrode is ca. 8-fold higher (Fig. 5A) than with the smooth electrode (Fig. 5B). Assuming that the protein layers on the rough and smooth Au surface exhibit similar activities, the substantially higher amperometric response with the rough surface is attributed to an increase in the biocatalyst content in a single monolayer on the roughened surface. Fig. 6 shows the transduced amperometric signals of the rough and smooth two-layer electrodes at different glucose concentrations. It is obvious that the sensitivity of a layered-enzyme electrode is substantially improved by the application of rough electrode surfaces (curve (a)). The approaches discussed up to now to enhance the enzyme-electrode sensitivities, basically involved the de-

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concentrations. Results are recorded by maintaining the enzyme electrode (geometrical area 0.4 cm z. roughness coefficient ca. I.I) at a fixed potential ( E = + 0.4 V vs. SCE) and sequential injection of glucose into the electrochemical cell. Electrolyte consists of phosphate buffer. 0. I M. pH = 7.3. Amperometrie responses were recorded at 3 5 + 0 . 5 ° C under Ar.

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E. Katz et al. / Bioelectrochemist~" and Bioenergetics 42 ( 1997~ 95-104

velopment of means to increase the content of biocatalysts in electrical contact with the electrode surface. A different approach to face the sensitivity issue of the enzyme-electrode could involve the improvement of the electrical communication between the biocatalyst and the electrode surface. Chemical mc;dification of the protein by redox relay units generates a random, non-specific, attachment of the charge transporting ccmponents on the protein. The electron transfer distances in the redox-modified biocatalyst are ce~:'.Jnly not optimized and cons:~quently, the electrical communication is limited. Maximal loading of the protein with the electron-relay units could p:ovide a general method to shorten electron transfer distances and

improve the electrical contact of the redox-center with the electrode. This approach is limited, however, since

introduction of non-native chemical componenls ~ the protein partially denamrates the bioca~ysL Thus. ~ appropriate balance between the loaded relay urfits and the resulting biocamlyst activity must be m~mained. An ~tractive method to improve the elecuical c ~ i o n ber,Jeen the redox center of the biocam!yst and the e~ctrode, could be the site-specific modification of the pin.in with a single redox relay unit at optimized distaaces for electron transfer. Site-specific modification of flavoenzymes by a redoxtethered relay unit was achieved by the reconstim~ion

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methodology that is schematically shown in Fig. 7 [39]. N6-aminoethyl-FAD, (3), was coupled to N-(2-methyl-ferrocene).caprnic acid (2), to yield the ferrocene-FAD diad (4) (Fig. 7A). The native FAD-cofactor of the flavnenzyme is excluded from the protein to yield the apo-protein. Reconstitution of the apo-protein with the ferrocene-FAD diad~ (4), implants the redox-tethered diad into the protein (Fig. 7B). This method was applied to reconstitute various flaveenzymes and the features of (4).reconstituted glucose oxidase, (4)-GOx, will be discussed here. The reconstituted (4).GOx retained ca. 60% of the native biocatalyst activity. Fig. 8 shows the cyclic voltammograms of the ferroceneFAD diad, (4), prior to reconstitution (curve a) and after reconstitution with apo-GOx, (curve b). The diad itself reveals two reversible redox waves at E ° = - 0 . 5 V and E ° = 0.35 V (vs. SCE, pH = 7.0), characteristic of the FAD and ferrocene units, respectively. After recoh:*_!lution, only the cyclic voltammogram of the ferrocene unit is :,~ ~erved. Tim FAD-component is embedded in the protein and electrically shielded, whereas the ferrocene component is sufficiently exposed to the protein periphery and exhibits effective electrical communication with the electrode surface. The resulting reconstituted glucose oxidase, (4)GOx, exhibits binelectrocatalytic activity and its redox active-site electrically communicates with the electrode. Fig. 9(A) shows the cyclic voltammograms of (4).GOx in the presence of different concentrations of glucose. An electrocatalyfic anodic current is observed indicating that bioelectrocatalyzed oxidation of glucose proceeds. The electrocatalytic anodic current increases as the concentration of glucose is elevated (Fig. 9(B)). Fig. 10 outlines schematically the pathway of electrical communication between the reconstituted biecatalyst, (4).GOx, and the electrode surface. The reconstitution methodology yields a site-specific modified bioelectrocatalyst, "electroenzyme'. To apply such a biocatalyst as active biological material in a sensing

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c O,eose I mM Fig. 9. (A) Cyclic voltammograms of systems that contain (4}-reconstituted GOx, 1.75 rag. ml- :. and different concentrations of glucose: (a) 0 mM, (b) l nfiVl. (c) 3 raM. (d) 20.5 raM. All experiments were performed in O.I M phosphate buffer, pH 7.3, at 35:t:0.5°C, using a cystamine-modified Au electrode; scan rate. 2 mV.s -t, Potentials are given vs. SCE. (B) Calibration curve of electrocatalyticanodic currents at different glucose concentrations in the presence of (4)-reconstitutedGOx. Currents determined by chronoamperometryat final potential +0,2 V vs. SCE. device, its integration with an electrode surface is required. Proper alignment of the bioelectrocatalyst on the electrode surface is a crucial element [40]. The electron-mediator unit must be positioned at the electrode surface in an optimized configuration to allow electron transport between the biocatalyst active-site and the electrode, With these limiting constrains, we decided to assemble the relay-FAD diad on the electrode surface and reconstitute the apo-protein on the surface itself [41]. Fig. i I(A) shows the method to assemble the reconstituted GOx on a Au electrode surface. A primary cystamine monolayer is linked to a roughened Au electrode (roughness factor ca. 20) and pyrroloquinolino quinone, PQQ, (5), is covalently associated to the base monolayer. N6-aminoethyI-FAD, (3), is

Fig. 10. S c h e m e for electrical c o m m u n i c a t i o n b e t w e e n the reconstituted biocatalyst, ( 4 ) - G O x , and the electrode surface.

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8ioelectrochemistry and Bioenergetics 42 f 1997) 95-104

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coupled to the carboxylic functions of PQQ to yield the redox-active FAD diad on the monolayer. The functionalized monolayer electrode is then reconstituted with apoGOx to form an active biocatalyst monolayer on the electrode surface. Fig. 12 shows the cyclic voltammogram of the PQQ-FAD diad monolayer prior to the tz~onstitution (curve a) and after reconstitution with apo-GOx (curve b). Prior to the reconstitution process, the functionalized monolayer shows two reversible redox waves at E~ = -0.125 V and E ~ z - 0 . 5 V vs. SCE, pH =7.0, corresponding to the two-electron redox proce~nes of PQQ and FAD, respectively. By integration of the charge associag, d with the PQQ and FAD redox units, the surface density of these components is estimated to he ca. 1 × !0-ti mole. cm-z. After reconstitution of the apo-GOx with the functionalized monolayer, the redox wave of the PQQ is almost unaltered, but the FAD redox wave is substantially decreased in its intensity. This is consistent with the fact that

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E. Katz et al. / Bioeleetrochemisto" and Bioenergetics 42 (1997) 95-104

I02

reconstitution of apo-GOx with the monolayer insulates the FAD-sites towards electrical communication with the electrode interface. The PQQ redox unit is sufficiently exposed to the protein periphery and hence reveals nonperturbed electrical contact with the electrode. The residual redox-wave of the FAD units is attributed to the free diad that was not reconstituted into apo-GOx. By integration of tl~ deple~.ed charge of FAD units, and assuming that two FAD-cofactor components participate in the anchoring o f apo-GOx onto the surface in the reconstitution process, the surface density o f the protein on the electrode surface is ca. 1.7 × !0 -12 m o l e - c m -2. Using the footprint dimension of GOx (58 nm 2) [42], the surface coverage of the electrode by the enzyme corresponds to a densely packed protein monolayer assembly. Fig. 13(A) shows the cyclic voltammograms o f the reconstituted GOx monolayer electrode in the absence o f g!ucose (curve a) and in the presence of glucose (carve b). With glucose, a high-magnitude electrocatalytic anodic current is observed, indicating that the reconstituted GOx on the monolayer yields a bioactive monolayer interface exhibiting direct electrical contact with the monolayer. A control system that includes the reconstituted GOx on a FAD monolayer without the PQQ redox mediating unit, was also assembled on a Au electrode, Fig. I l(B). In this system, no direct electrical communication between the embedded FAD-cofactor and

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with the FAD-FQQ monolayer bound to the gold electrode: (b) alter addin£ glvcose *~ 80 mM concentration.Ar atmosphere,0.01 M phosphate buffer and 0.1 M sodium sulfate, pH = 7.0, 35±0,5°C; scan rate. 5 mV-s-I; electrode gomefricalarea, O~ cm-'; roughness factor, ca. 20. P.otcntials arc given vs. SCE. (B) Amperometricresponsesof the FADPQQ reconstitutedGOx monolayerelectrodeat differentglucoseconcentrations. C,~rten~ dete~.-miaedb) chronoamperometryat final potential +0.2 V vs. SCE.

the electrode exists, but the reconstituted GOx exhibits biocatalytic activity, and bioelectrocatalyzed oxidation of glucose proceeds in the presence of a diffusional electron mediator, ferrocene carboxylic acid. These results clearly demonstrate that the reconstitution of apo-GOx on a FADmonolayer represents a novel means to link and align flavoenzymes onto surfaces. Reconstitution of apo-GOx onto the PQQ-FAD diad yields an electroactive biocatalyst interface, where direct electrical contact between the enzyme-active site and the electrode is achieved. The intermediate PQQ component that acts as a redox-relay unit, transports the electrons from the active site of the enzyme to the electrode. Fig. 13(B) shows the transduced currents by the PQQFAD-reconstituted GOx monolayer electrode at different glucose concentrations. The current responses are almost linear in a concertration range of 1-80 mM of glucose. The upper limit of the turnover-rate of glucose oxidase at 25°(2. is 6 0 0 + 100 s -~ [43] and the activation energy is 7.2 kcal - mole- t [44]. At the temperature employed in our measurements (35 °C), this translates to a limiting turnover rate of 9 0 0 + 150 s -~. Realizing that the recnnstituted GOx surface coverage is 1.7 × 10 -12, the maximum current density that can be observed for the theoretical turnover of the enzyme is 290 + 60 I~A. cm -2. Fig. 13(B) shows that at a glucose concentration of 80 mM, the observed current is ca. 1.9 mA (for an electrode with a surface area of 0.4 cm 2 and roughness factor of ca. 20). Thus, the experimental current density, 3 0 0 + 100 ~.A. cm -2. is within ",.;,,e range of the limiting turnover rate o f the enzyme. "1his suggests that at the potential employed in the chronoamperometric experiments to analyze glucose ( E = 0.2 Volt, Fig. 13(B)), all FAD sites are electrically connected with, the electrode and exist ie the oxidized form. The resulting current is then controlled by the diffusion of glucose to the active site. Our discussion indicates that the reconstituted apo-GOx on the PQQ-FAD- monolayer electrode exhibits unique electrical communication features and the theoretical turnover rate of the active center with molecular oxygen is achieved for the electron exchange between the active site and the electrode. Also, the electrobiocatalyzed oxidation of glucose proceeds at relatively low positive potentials, E > - 0 . I V vs. SCE, characteristic to the PQQ electron mediator component. These two features have important implications for the use of the monolayer-modified electrode as a glucose sensing interface. The high electrical turnover rate of the reconstituted biocatalyst suggests that the enzyme-electrode would not be influenced by oxygen. The presently available amperometric glucose enzyme electrodes are sensitive to the oxygen content in the environment, and most analyses are performed in an oxygenfree medium. Fig. 14 shows the amperometric response of the reconstituted enzyme electrode in the presence of glucose in an oxygen-free environment (curve b) and in the presence of air (cu.we c). The transduced amperometric

E. Katz et aL / Bioeleetroehemistry and Bioenergetics 42 f 19971 95-104 0.2

'< ~1. ILl

e

0.05

3

3.5

4 t

4.5

5

5.5

I S

Fig. 14. Amperometric responses produced by the GOx reconstituted with the FAD-PQQ monolayer (a) in the presence of (b) gluon~. 50 raM. in the ab~nce of O,, (c) glucose, 50 mM, in a ~lution saturated with air, (d) glucose, 50 raM. and ascorbic acid. 0.1 raM. in the absence of O z. Currents determined by chronoamperometry at final potential 0.0 V vs, SCE. Electrolyte composed of 0.01 M phosphate buffer and 0.1 M sodium sulfate, p H = 7 . 0 , 35_+0.5°C; electrode geometrical area. 0.4 cm", roughness factor, ca. 20.

signal is only slightly affected (less than 2%) by oxygen. Similarly, the effective electrical contact between the biocatalyst and the electrode suggests that the non-specific oxidation of interfering components will he screened by the highly efficient bioelectrucatalyzed oxidation of glucose. Ascorbic acid is a common interferant in glucose analysis. Fig. 14 shows the transduced currents by' the reconstituted enzyme electrode in the presence of glucose without added ascorbic acid (curve b) and in the presence of ascorbic acid (curve d). The transduced current in the presence of ascorbic acid is only slightly increased (less than 5%) implying that the observed current originates essentially from the bioelectrocatulyzed oxidation of glucose.

3. Conclusions and perspectives The present account has discussed novel approaches to tailoring enzyme electrodes for biosensor devices by the assembly of enzyme monolayer and multilayer films on Au surfaces. An important aspect for consideration in designing enzyme-electrodes is the sensitivity of the resulting sensing interface. This issue was addressed by different approaches: increasing the biucatalyst content at the electrode surface provides one method to enhance the sensitivity of the resulting electrode sensitivity. This was achieved by two alternative methods: (i) Organization of a multilayer enzyme network on the electrode support, and (ii) the application of rough, high surface area electrodes. The method to construct the multilayer enzyme network on the electrode surface involved the step~,t~e assembly of a controlled number of layers in the protein network. This procedure allows the tuning of the electrode sensitNity to the desired value. The stepwise chemical methodology to assemble the raultilayer enzyme network has further important implications in biosensor technology. It permits the

103

organization of ordered layered arrays of two or mote enzymes. Thus, one of the enzymes coukt generate from the analyte-substrate, a product acting as the subswate for the secondary electroactive biocatulyst. For exa,q~e, organization of an ordered choline oxidase/acetyk:hohne esterase enzyme network on a Au electrode, enabled the electrochemical detection of acetylcholine [36]. A further method to enhance the sensitivity of 1mmolayer-enzyme-electrodes involves the improvement of the electrical communication between the redox active-cemer of the biocatulyst and the electrode. Reconsti~zlkm of the apo-flavoprotein, apo-GOx, on a redox relay-modified monolayer electrode consisting of a PQQ-FAD ~ offered a novel method to generate highly eff'uzient b~oelectrocatulytic sensing interfaces. Reconstitution of apo-C.Ox onto the PQQ-FAD monolayer electrode, demonslmted a new method to link and align flavoenzymes on the conducting support. The reconstituted biucamlyst revealed extremely efficient electrical communication with the electrode surfaces which resulted in unique features of the enzyme electrode: (i) High current densities were transduced by the electrode. (ii) The enzyme electrode was essentially insensitive to oxygen or common glucose-sensing interferants such as ascorbic acid. The high cmTentdensities produced by the electrode, its specificity, and its composition of ine~'edients of natural origin (PQQ and FAD) should enable the application of miniaturized enzyme-electrodes for invasive, in situ, analyses of glucose.

Acknowledgements The support of The Israel Ministry of Science and Arts and the Gesellschaft fiJr Biotechnologische Forschung, Braunschweig, Germany, is gratefully acknowledged. A.F,B. was supported by the European Commission (E.C. contract No. 2904395 and CI !-CT92-0024).

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