Observation of direct electron transfer from the active center of glucose oxidase to a graphite electrode achieved through the use of mild immobilization

Bioelectrochembtry and Bioenergetics, 33 (1994) 191-199

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Observation of direct electron transfer from the active center of glucose oxidase to a graphite electrode achieved through the use of mild immobilization Manuel Alvarez-Icaza

and Rolf D. Schmid

GBF-Gesellschaft fiir Biotechnologische Forschung mbH, Department of Enzyme Technology, Mascheroder Weg 1, 38124 Braunschweig (Germany)

(Received 27 August 1993; in revised form 25 October 1993)

Abstract An immobilization method based on the use of aminophenylboronic

acid enhanced the rate of electron transfer between glucose oxidase and a graphite electrode. Under anaerobic conditions, a group of 21 electrodes responded to a 10 mM change in glucose concentration with an average change of current of 0.5 f 0.1 PA. Experimental evidence ruled out the possibility of (natural or artificial) mediated electron transfer or a mechanism based on orientation of the enzyme. A mechanism for electron transfer promotion based on the prevention by aminophenylboronic acid of the removal of the FAD from enzymes well situated and/or oriented to transfer electrons directly is postulated. To support this mechanism, experimental evidence was found confuming the removal of FAD from the enzyme by the graphite electrode and a protective role against it of aminophenylboronic acid.

1. Introduction

Traditionally, electron transfer in biosensors occurs via natural mediators (oxygen) or artificial mediators such as ferrocene or its derivatives [ll. For example, the reaction involving the enzyme glucose oxidase (GOx) can be described by the following set of equations: GOx,+S+GOx,,+P GOx,, + M, + GOx, M,, + M, + 2e-

(1) + M,,

(2) (3)

where S is the substrate (glucose), P is the product CD-gluconolactone) and M is the mediator. Reactions (1) and (2) take place at the enzyme active site, while reaction ,(3) takes place at electrode surface. Apparently, for an enzyme the size of glucose oxidase (radius 4nm) [21 the distance between the electrode and the active site may be too large for electron transfer to occur directly when standard immobilization methods are used. In general, the presence of a mediator is required to produce the overall enzyme reaction: S-,P+2e0302-4598/94/$7.00 SSDI 0302-4598(93)01681-C

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In some analytical situations, such as bioprocess monitoring or in-vivo measurements, the natural mediator (oxygen) is not available or is only present in limited amounts. In the latter case the availability of the mediator for the enzymatic reaction can be increased by using membranes which favour the diffusion of oxygen while limiting the diffusion of glucose [3], or using special sensors with cylindrical geometry which allow the diffusion of oxygen through a substantially larger surface than that available for glucose [4]. This approach has permitted in-vivo monitoring of glucose for extended periods of time (several weeks) [5], but the special geometry required limits the possibilities of fabrication. The addition of artificial mediators allows the enzymatic reaction to be completed in all analytical situations and, because it does not require of any particular geometry, is ideal for planar fabrication technologies. Mass production and reduction to microscopic scale is possible for these sensors. However, because a mediator needs to be free to diffuse to shuttle electrons between the enzyme and the electrode, it eventually affects the stability of the sensor and releases potentially toxic substances. Therefore, alternative methods to complete reaction (4) are required. 0 1994 - Elsevier Sequoia. All rights reserved

M. Alvarez-Icaza, R.D. Schmid / Direct electron transfer from glucose oxidase

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Several approaches have been used to facilitate the direct transfer of electrons. Degani and Heller 161and Bartlett et al. [7] covalently immobilized electron relays to each GGx molecule. However, they found that most of the electron relays were lost during the initial stabilization period [8]. Higher stability has been achieved by retaining the electron relays using a polymer instead of attaching them directly to the enzyme structure. Redox polymers have been used since 1983 [9]. Degani and Heller [lo] used polyionic redox polymers fixed to the enzyme structure, electrostatically and also covalently [ll]. More recently, they cross-linked the polymer with a diepoxide to produce a redox-conducting epoxy cement [12] which was used for a reagent-free glucose sensor [13]. In other approaches, a flexible ferrocene-modified siloxane polymer has been used [14,15] to produce reagentless glucose electrodes [16,17], or the enzyme has been modified with polyethylene glycol [18] or hydroquinone sulphonate [19]. Although initial observations of possible direct electron transfer used enzymes in solution and differential pulse voltammetry on gold electrodes 1201, it has recently been reported using GOx adsorbed in polypyrrole microtubules [21]. The achievement of this phenomenon through an immobilization technique has generally been based on the use of modified carbon electrodes. Direct electron transfer using differential pulse voltammetry from GOx immobilized onto a modified graphite electrode via an intermediate group (cyanuric chloride) between the enzyme and the electrode was reported in 1982 [22]. A previous attempt, using an immobilization technique without an intermediate group, was unsuccessful [23]. More recently, Szucs et al. 1241 studied the reactions of GOx at graphite electrodes. They observed reduction peaks that can be associated with direct electron transfer to GOx. However, they also measured a strong adsorption of FAD by graphite. Narasimhan and Wingard [25] reported an immobilization method on glassy carbon based on another intermediate group; aminophenylboronic acid (APBA). Because this work may have important future applications in the promotion of electron transfer from new enzymes produced by protein engineering, we have adapted it using a highly porous graphite pellet to increase the enzyme loading to amplify the effect of the enzyme in the observed signal. 2. Experimental .,

2.1. Materials. Spectrographic grade graphite discs (4 mm in diameter by 2 mm thick) were obtained from RingsdorffWerke GmbH (Bonn-Bad Godesger, Germany). l-

Cyclohexyl-3-(2-morpholinoethyl)~carbodiimide methop-toluenesulphonate and m-aminophenylboronic acid hemisulphide salt (APBA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The hemisulphide salt was converted into free base using the method described by Weith et al. 1261. Native GOx grade II from Aspergiflus niger was purchased from Boehringer (Mannheim, Germany). Deglycosylated 1271and highly purified GOx from As. niger were obtained from Dr. H. Kalisz (GBF, Braunschweig, Germany). Enzyme (GOx) loaded with electron relays was modified using the method reported by Bartlett et al. [7]. Catalase was obtained from Sigma. N-[2_hydroxyethyllpiperazineN’-[Zethanesulphonic acid] (HEPES), magnesium chloride hexahydrate, urea and anhydrous D-glucose were purchased from Merck (Darmstadt, Germany). Analytical grade (99.999%) nitrogen was obtained from Linde (Hanover, Germany). Glucose solutions were prepared in 0.04 M phosphate buffer (pH 7). They were left to mutarotate overnight, and oxygen was removed before the anaerobic test by passing a nitrogen stream for 30 minutes. . 2.2. Electrode preparation Blue plastic pipette tips (1000 ~1) were cut to a length of 51 f 0.5 mm to leave a front opening of approximately 3.5 mm. A graphite disc was then pushed from the back of the pipette until it was aligned with the rim of the cut. Spring-loaded electrical contact was made through the internal (back) surface of the graphite disc.

2.3. Enzyme immobilization Initially and after each of the immobilization steps (except the final one) the graphite discs, mounted in the plastic tips, were washed with distilled water, extracted overnight in methanol and vacuum dried the next morning. The immobilization was performed in four steps. (1) Surface oxidation (to increase porosity and density of active groups) by passing a 100 mA current for 20 s in a solution of 10% nitric acid and 2.5% potassium dichromate. (2) carbodiimide activation by immersing the graphite discs in 5 mg ml-’ carbodiimide solution in pure water for 90 min. (3) APBA immobilization by immersing the discs in an aqueous solution of APBA free base (10 mg ml- ‘) for 90 min. (4) enzyme attachment by one of the following two methods. For high cost enzyme, a 5 ~1 drop of the deglycosylated, highly pure or relay loaded GOx solution (2 mg ml-i) in 50 mM HEPES buffer (pH 7.0) was deposited on the electrode surface which was then

M. Alvarez-Icaza, R.D. Schmid / Direct electron transfer from glucose oxidase

covered overnight with a cap to avoid evaporation. For low cost enzymes, the discs were immersed in a 2 mg ml-i native enzyme solution in 50 mM HEPES buffer (pH 7.0 or pH 8.0) containing 0.1 M magnesium chloride hexahydrate and various concentrations of urea (0, 0.5, 1 and 3 M). The electrodes were thoroughly washed (30 min with stirring) in distilled water before use. After immobilization, the amount of enzyme (0.39 mg) was estimated from the change in absorption (at 287 nm) of the enzyme solution used for the immobilization. Similarly, 0.18 mg of APBA was estimated to be immobilized as determined from the change in absorption at 300 nm. 2.4. Apparatus A potentiostat equipped with a current-voltage converter (VA-detector E611) and a voltage scanner (VA-Scanner E612) from Metrohm (Herisau, Switzerland) were used for the electrochemical measurements. The graphite disc was connected as the working electrode, a Ag/AgCl electrode (363-S7) in a 3 M KC1 solution from Ingold (Urdorf, Switzerland) was used as the reference and a platinum wire (0.5 mm in diameter and 50 mm long) was used as the auxiliary electrode. The three electrodes were held together either in a 35 mm thick silicone bung, which sealed the test cell containing 30 ml of 0.04 M phosphate buffer (pH 7), or using a polyphenylene sulphide titration vessel lid (Metrohm 6.144.010) which sealed a test cell containing 40 ml of the same buffer. The lids also provided connections for a moisture-saturated nitrogen stream, for gas ventilation and for changing the glucose concentration of the solution in the cell. To ensure anaerobic conditions during the experiments, the nitrogen stream was transported from the source using thick rubber-walled tubes (vacuum tubes). The stream was passed through the measuring cell for 30 minutes before the experiments. During the experiments the tube was lifted over the surface of the liquid so that a nitrogen blanket was formed. To avoid oxygen diffusion from the back of the graphite disc, a continuous stream was also passed through the back of the graphite disc. When more rigorous anaerobic conditions were required, the experiment was performed inside an anaerobic chamber obtained from COY Laboratory Products (Grass Lake, MI, USA) where the oxygen concentration was guaranteed to be below 5 ppm. 2.5. General measuring procedure The presence and activity of enzyme in the electrodes was determined by testing them under aerobic conditions. For this test, oxygen was not removed from the buffer. The electrode was polarized to 800 mV.

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After the current reached a steady value, the glucose concentration was changed from zero to 10 mM and the difference in current was recorded. The test cell was washed and the same experiment was repeated at a polarization of 400 mV under anaerobic conditions. 3. Results and discussion A set of 21 electrodes was prepared using APBA and different forms of GOx (14 with native GGx, one with purified GGx, two with deglycosylated GOx and four which had been electron relay modified). All of them responded to the 10 mM change in glucose concentration under aerobic conditions (average 19 rf: 2 PA), and all of them produced a measurable response (average 0.5 f 0.1 PA) to the change in glucose concentration under anaerobic conditions. In contrast, control electrodes for APBA, i.e. prepared with enzyme but without APBA, responded during the aerobic test (12 f 3 PA) but no detectable change (within a noise band of kO.01 PA) was observed under anaerobic conditions. Control electrodes for direct electrocatalytic oxidation of glucose, i.e prepared with APBA but without enzyme, also produced no detectable change in current as a result of a change in glucose concentration when polarized to 400 mV. A large dispersion was observed for aerobic and anaerobic responses (standard deviations of 9.2 /.LA and 0.4 PA respectively), probably because of variations in the enzyme used and because the electrodes were prepared in three different batches. The average anaerobic response was 38 times lower than the aerobic response. Nevertheless, the anaerobic response was noticeably slower than the response under aerobic conditions. Figure 1 shows an example of the different responses of the same electrode under the two conditions. The slower anaerobic reaction is probably due to diffusion-reaction phenomena occurring near the surface of the electrode. With lower enzymatic activity due to the low density of enzyme molecules capable of transferring electrons directly, the substrate can diffuse into deeper layers of the porous graphite. This process requires a longer time and involves a larger area of enzyme. Therefore it implies an even lower activity under anaerobic conditions. To a first approximation, and to give an idea of the difference in response under the two conditions, we can consider the situation as pure diffusion, i.e. the depth of the diffusion is proportional to t’/’ where t represents time [28]. Then if the response time (O-90%) is 40 times longer, as in the example shown in Fig. 1, the volume of electrode material participating in the reaction is 6.3 times greater. Therefore the anaerobic response is about 240 times lower than the aerobic response.

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thlin Fig. 1. Time response of an AF’BA+ GOx electrode to a 10 mM change in glucose concentration: (A) under aerobic conditions and at 800 mV polarization potential; (B) under anaerobic conditions and at 400 mV polarization potential.

For several APBA electrodes, we tested the response for different concentrations of glucose under anaerobic conditions (Fig. 2). Although the response is still too low to be used in practical applications where oxygen is present and shows large variations among the different electrodes, the figure shows that reagent-free anaerobic glucose determination is a possibility. 3.1. Evidence that APBA does not act as a mediator After completing the immobilization, becausecarbodiimide is only an agent [29] to produce the imide bond between the carboxyhc groups on the surface of the graphite and the amino groups in the APBA, the only substance not present in an oxidized graphite

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Fig. 2. Response of six different APBA + GOx electrodes to various glucose concentrations under anaerobic conditions and at a polarization potential of 400 mV. Electrodes represented with circles (open and closed) were prepared with enzyme modified with electron relays. The remainder were prepared with native enzyme.

electrode modified with enzyme is APBA. From the results obtained with control (electrode prepared with APBA only) described above, direct electrocatalytical participation of this compound is excluded. Therefore the only other active role for this compound in the oxidation of glucose is as a mediator, moving electrons (by diffusion) between the active site and the electrode. Other forms of mediation, not connected with APBA, were excluded because of the lack of anaerobic response of electrodes prepared without AF’BA. Mediation due to APBA is, in principle, not possible for these electrodes because this compound is covalently bound to the electrode surface. In addition, the electrodes were extracted in ethanol overnight. Hence the only APBA remaining on the electrode surface should be covalently attached and cannot diffuse to act as a mediator. However, it is known 1301 that the oxidation of aminophenol (a compound very similar to APBA) produces a quinone-imine that is subsequently hydrolysed to benzoquinone, a compound which is a good mediator for GOx [311. Therefore it is necessary to determine whether APBA or its derivatives (compounds which are probably similar to benzoquinone) can act as mediators for GOx. For this purpose, a 33 mgml-’ solution of APBA in phosphate buffer (pH 7) was tested using cyclic voltammetry with a glassy carbon electrode. No electrochemical activity was found in the range between 0 and 500 mV. However, at higher potentials (above 550 mV) strong oxidation of APBA occurred (initial cycle in the cyclic voltammogram in Fig. 3A). Furthermore, this oxidation produced a redox couple (second cycle> with E” = 100 mV, a region of electric potential where it could act as a mediator in our experiments. Further investigation of this redox couple showed that when the potential was scanned between -400 and 500 mV at rates from 10 to 600 mVs_’ the height of the oxidation peak increased proportionally to the square root of the potential scan rate. However, the position of the oxidation peak, showed the shift to more positive potentials proportional to the rate of scan characteristic of irreversible electrochemical reactions, which is not a favourable characteristic for a substance acting as a mediator. In this case the measured shift was 1.9 mV for each increase of 10 mVs_’ in the scanning rate. In addition, the initial height of the peak depended on the initial concentration of APBA. However, continuous scanning to potentials higher than 550 mV produced a reduction in the size of the peaks (those around 100 mV and that starting at 550 mV) on every cycle. Further addition of APBA produced no increase in any of the peaks. This last observation probably implies passivation of the electrode surface due to the electropolymerization of

M. Alvarez-Icaza, R.D. Schmiu’ / Direct electron transfer from glucose oxidase

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Fig. 3. Cyclic voltammogram of (A) a solution of APBA (0.33 mg ml-‘) in 0.04 M phosphate buffer (pH 7) using a glassy carbon electrode and (B) the buffer alone. The electrode potential was scanned at 10 mV s-l.

APBA, similar to the passivation due to the electropolymerization of phenol [32,33]. This. polymerization process reduces even further the possibility of using APBA as a mediator. In a further test, a graphite electrode with adsorbed GOx was immersed in a solution containing APBA (0.5 mgml-‘1 and polarized to 200 mV (a potential high enough to oxidize the new compound). No response to a change in glucose concentration (100 mM) was observed after increasing the polarization to 800 mV (to generate the new compound on the surface of the electrode) and then returning it to the measuring potential. Finally, the possibility that this electrochemitally generated compound could act as a mediator was completely excluded because no change in the peaks was observed when either GOx or glucose was added to the solution under anaerobic conditions. If the compound under test acted as a mediator, the oxidation peak should have increased and the reduction peak should have decreased to produce a catalytic wave. Therefore neither APBA nor its derivatives obtained by electrochemical oxidation act as mediators for GGx. Hence its participation in the promotion of electron transfer from the enzyme to the electrode must be passive and connected with the immobilization process. 3.2. Evidence that the modified electrodes did not detect hydrogen peroxide at low potential A series of test were performed to demonstrate that the signal observed under anaerobic conditions was not

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due to the production of hydrogen peroxide by the enzyme from trace amounts of oxygen. The first-test was based on the fact that catalase reacts with hydrogen peroxide very fast. Thus it would compete with the electrode for this substrate. In addition, the overall reaction of catalase and glucose oxidase consumes half of the oxygen molecule every turnover [34]. Therefore if the observed signal after the addition of glucose was due to oxidation of hydrogen peroxide, a noticeable decrease should be observed in the presence of catalase. Two new electrodes were prepared and tested for response to glucose under anaerobic conditions. After a response (0.28 PA and 0.26 PA respectively) had been obtained to a change of 10 mM, in glucose concentration 1 mg of catalase (equivalent to 19 900 units) was added to the measuring cell containing buffer and glucose. No change in the response after the addition of catalase was observed even after 2 h. However, a further addition of glucose produced an additional increase in the sensor current. To make the test more rigorous, the same experiment was repeated but, in addition to the catalase, 1 mg of GGx (equivalent to 146 units) was added to the solution so that the possibility that the enzyme immobilized on the electrode could have access to any trace of oxygen would be reduced even further. Again, no change in the response was observed. The opposite approach was also tested using a new electrode (polarized to 400 mV). After obtaining a response of 0.74 PA due to the addition of 10 mM glucose in anaerobic conditions, the cell was opened to allow oxygen to enter the system. After 1 h, the signal was reduced by 26%. The test was repeated with the electrode polarized to 200 mV. A signal of 0.34 PA was obtained in anaerobic conditions, and had already reduced by 20 percent after the measuring cell had been open for 20 min. The signal reduction indicates competition between the electrode and oxygen for the re-oxidation of the enzyme. In a final test to demonstrate that there was no possible connection between the observed signal and oxygen, the whole measuring system was placed inside an anaerobic chamber (oxygen below 5 ppm). After leaving it overnight, glucose was added and a response of 0.28 PA was observed. For comparison, the same electrode was tested under the same conditions as in the other measurements reported in this paper. The same change in current for the same change in glucose concentration was observed in both situations. It is also interesting to notice the difference in the response of AF’BA electrodes to glucose and to hydrogen peroxide. An electrode was analysed for electric potentials between 200 and 1000 mV (allowing the current to reach a steady state). The same electrode

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peared. The absence of a significant change in the observed signal (electrodes prepared with deglycosylated enzyme responded in anaerobic conditions with 40% and 41% of the average response) indicates that the electron transfer enhancement does not depend on the carbohydrate shell. In addition, previous observations [37] have shown that better results using the APBA immobilization occurred when the enzyme was immobilized at pH 7. Since APBA probably holds the enzyme very weakly at this pH (the pK, of APBA is 8.861, the immobilization is more likely to be due to adsorption on the graphite.

1000

EJmV Fig. 4. Dependence of steady state current on electrode potential for an APBA+ GOx electrode measuring 10 mM glucose and 0.17 mM H,O, under anaerobic conditions.

was used later, in the same electric potential range, to detect an amount of hydrogen peroxide (0.17 mM) such that, for the initial potential (200 mV), the current was slightly lower than that detected using glucose. A comparison of the sensitivity of the electrode in the two situations is shown in Fig. 4. When the potential was increased, the current due to hydrogen peroxide increased faster than the current due to glucose. Therefore the process limiting the current in the two situations should be different. 3.3. Evidence that the enzyme is not oriented through the carbohydrates In an attempt to augment the enhancement of electron transfer under anaerobic conditions we investigated whether the enhancement was related to enzyme orientation promoted by APBA. The boronic part of APBA is specific for the cis-diol groups [35], for example in the carbohydrates of the enzyme. If the effect is due to the presence of specific sugars in the external shell of the enzyme, a noticeable difference should be detected when deglycosylated enzyme is immobilized to the electrodes. Ninety-five percent of the carbohydrates present in the native enzyme used in this work were removed by deglycosyl action [36]. Hence, if the observed response under anaerobic conditions using APBA was due to binding from any or some of the remaining 5% of carbohydrates in the deglycosylated enzyme, the immobilization procedure with APBA would substantially augment (orders of magnitude) the probability of retaining the enzyme through this portion. However, if the observed effect depended on any of the sugars that were removed, then the APBA immobilization effect should have completely disap-

3.4. Evidence from electrodes with enzymes modified with electron relays Another attempt to increase the enhancement of electron transfer was based on the use of enzymes modified with electron relays. It has been shown that these enzymes can transfer electrons directly, but only if a specific zone of the enzyme surface points towards the electrode [38]. If the effect that we have observed is due to the fact that the enzyme has been immobilized with a particular orientation (independent of the carbohydrates) and if the function of these modified enzymes is dependent on their orientation, a large increase in the signal or its complete disappearance should be expected. The absence of a significant change in the anaerobic response (electrodes prepared with this enzymes had responses to glucose that were 14%, 71% (two) and 209% of the average response) provide no evidence that enzymes are immobilized differently from their more natural arrangement, i.e., randomly. 3.5. Use of highly purified enzyme Immobilization of a purified enzyme was also investigated to ensure that there were no other substances present that could influence the enhancement of electron transfer. No significant difference (26 percent of the average response) was observed with this enzyme. 3.6. Use of urea to open the enzyme structure during immobilization We attempted to increase the size of the anaerobic signals by another modification of the immobilization procedure. This attempt was based on the work of Degani and Heller [6], who used different concentrations of urea to open reversibly the structure of the enzyme while it was modified, and the work of Szucs et al. [241, who reported a strong attraction of FAD to graphite. Because the structure of GOx was not yet known, we attempted to open the enzyme structure and then to use the attraction between FAD and graphite to reduce the distance between FAD and the

M. Aluarez-Icaza, RD. Schmid / Direct electron transfer from glucose oxidase

electrode to a minimum and hence facilitate the electron transfer. Native enzyme in different concentrations of urea (0.5, 1 and 3.0 M) was immobilized to six new electrodes. This new immobilization procedure did not increase the response under anaerobic conditions. In contrast, no response to glucose was detected in five of the electrodes, and a response of only 0.03 PA was observed for an electrode prepared in a 1 M urea solution; the average response in aerobic conditions was 7.3 + 0.6 PA. Even at the low urea concentrations (0.5 M) that normally produce reversible opening of the enzyme structure, the combination of urea plus graphite inhibited the electron transfer phenomenon under anaerobic conditions. Opening of the structure probably helped the graphite to pull the redox centres (FAD) out of some enzymes. Two groups of enzymes could be distinguished on the surface. One was connected with the direct electron transfer observed using APBA, which immobilization with urea denatured, and other remained functional but had greater difficulty in transferring electrons directly to the electrode. 3.7. A possible mechanism to enhance electron transfer Using the previous information, we can postulate that APBA promotes the direct transfer of electrons, remaining sterically as an intermediate group between the enzyme and the graphite electrode. The presence of AF’BA prevents the detachment of FAD from enzymes which are well situated and/or oriented to transfer electrons to the electrode. This protective mechanism has also been observed for the promotion of electron transfer of smaller proteins, for which almost reversible electrochemistry has been reported 1391. In the latter case, in addition to protecting the protein and its prosthetic group from deleterious adsorption at the electrode, the promoters orient the enzyme to favour the transfer of electrons. 3.8. Evidence that APBA prevents FRD from being detached from the enzyme

Using the same procedure of electrochemically oxidizing the graphite surface, we prepared two sets of electrodes: one with and one without AF’BA. Enzyme was attached to the electrodes from solutions containing, in addition to the enzyme (10 mg ml-‘), different concentrations of urea (0, 0.5, 1, 2 and 3 M). The electrodes were then analysed for the presence of adsorbed FAD using cyclic voltammetry. After enzyme immobilization and rinsing in water for 30 min, the electrodes were immersed in the measuring cell in 40 mM phosphate buffer (pH 7). The potential was scanned for at least 1 h between 0 and -800 mV at a rate of 1 V s-i in order to remove weakly adsorbed

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material. The scan rate was then lowered to 10 mV s-i and the cyclic voltammograms recorded when they became stable. Figure 5A shows the cyclic voltammogram of an electrode that was immersed in a solution containing 1 mg ml-’ FAD instead of an enzyme solution, which was used as a control. As reported by Gorton and Johansson [401, oxidation and reduction peaks were observed at E”’ = -450 mV. No peaks were observed for a control electrode which was tested after oxidation of its surface and immersion into a solution containing 3 M urea for the same amount of time that the other electrodes were immersed in the FAD or enzyme solutions. Electrodes with enzyme (Fig. 5B) also showed oxidation and reduction peaks at E”’ = -447 mV, a potential that compares favourably with the peaks observed for FAD. The observed peaks should be due to FAD adsorption. In addition, the height of the peaks was proportional to the urea concentration of the enzyme solutions (Fig. 61, in agreement with the fact that the more open the enzyme structure, the easier it should be to remove the FAD. However, for the same urea concentrations, the peaks corresponding to electrodes prepared with APBA were consistently lower than the peaks of electrodes prepared without this compound. This demonstrates that APBA has a function opposite to that of urea i.e. it helps to prevent the removal of FAD. An interesting observation was that we could detect FAD adsorption not only for the electrodes prepared without APBA, but even for electrodes prepared without using any urea. This confirms the fact that graphite

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can remove FAD from enzymes even under normal immobilization conditions. Our results indicate that APBA protects the FAD in the enzyme and apparently does not contribute to the orientation of enzyme molecules. Therefore only the molecules that are in the appropriate orientation and/or position after random immobilization will participate in the transfer of electrons. It has also been shown that enzyme molecules which are able to transfer electrons directly appear to be more prone to denaturation by the removal of their FAD. A modification of the immobilization technique which, in addition to the protection of FAD, promotes enzyme orientation may produce an increase of one or two orders of magnitude in the rate of electron transfer. It should also be noted that the rate k,, of electron transfer for long distances can be described by [41] k,, = r~& exp[ -P(d

- &)I

Xexp[ -(AG0+A)‘/4ART]

(5)

where v” and r. are constants and their product is equal to lOi s-i, p is the rate of the decrease in electronic coupling with distance (for proteins it has an approximate value of 9.1 nm-’ [41]), d is the distance for electron transfer, do is the van der Waals contact distance (ca. 0.3 nm), AGO is the reaction free energy (driving force), h is the reorganization energy (approximately 0.65 eV [42]), R is the gas constant and T is the temperature. From the crystallographic information about the enzyme structure [43] we know that the minimum distance from the active centre (N(5) in the isoalloxazine ring) to the surface of the protein is approximately 1.9 nm. Thus we expect (from the first exponential term in eqn. (5)) that the rate of electron transfer will be greatly reduced. However, this reduction can be par-

tially compensated by increasing the second exponential term by modifying the driving force until AGO + A = 0, i.e. until the “inverted region” is reached. At this point the driving force equals the reorganization energy and the second exponential reaches its maximum value. In our experiments we can change the driving force by modifying the electrode potential since AGO = ne(E - Eof> [44], where n is the number of electrons and Ear is the standard potential, and hence there is an overpotential for which the rate of electron transfer reaches a maximum. The standard potential of the FAD/FADH, couple in its position within GGx is difficult to establish. Stankovich et al. [45] determined oxidation-reduction potentials for GGx of -63 mV and -65 mV for the first and second electron at pH 5.3, and of -200 mV and - 240 mV at pH 9.3 (against a standard hydrogen electrode). However, because these values were determined using redox titration by reduction with dithionite and equilibration with indicating dyes, the molecules of these solutions were capable of penetrating the enzyme structure, making it difficult to determine the influence of the position of the FAD in the electron transfer characteristics as observed from a solid electrode. Therefore, in this analysis, we take the standard potential of FAD/FADH, as that already measured in this work for FAD in solution ( - 450 mV>, and approximate the electron transfer characteristics using eqn. (5) and assuming that there are two electrons involved in the reaction, the reorganization energy and the distance separating the active site from the enzyme surface. We expect to see the “inverted region” at approximately 850 mV. This prediction agrees with the experimental results presented in Fig. 4 where no further increase in the signal from glucose with respect to the background was observed for potentials above 800 mV. Two facts are important for sensor development. First, the distance of 1.9 nm between the active centre of the functional enzyme and the electrode is large enough to require an “investment” in driving force to give observable rates of electron transfer. For example, in our experiments we were not able to observe any signal from the oxidation of glucose for potentials below 200 mV; this potential was necessary to compensate for the distance between the active site and the electrode, even for enzymes which were well situated and oriented, and to compensate for the relatively few functional molecules in the appropriate conditions to transfer electrons. Second, 1.9 nm is short enough to allow rates of electron transfer (4.75 X lo6 s-i in the “inverted region”) well in excess of the 200 s-l required by the turnover of GGx [38] to manufacture practical sensors. Therefore the results that we present

M. Alvarez-Icaza, RD. Schmid / Direct electron transfer from glucose oxidase

here show that, although rate of electron transfer observed in this work is too low for practical applications, there is a possibility of achieving practical electron transfer rates with the enzyme in its native form. The questions that require to be answered are as follows. Would it be possible, and practical, to find the right immobilization technique (to protect, place, and orientate the enzyme) to achieve the requirements for electron transfer to fabricate functional glucose sensors (a higher rate than that using oxygen or other soluble mediators which is 105-lo6 M-‘s-l [42])? Even with optimal immobilization, will some modification of the enzyme structure be required? Is there any possibility, using immobilization technology to compete with the redox polymers, that in addition to producing conductive pathways, they will also protect the FAD? Knowledge of the enzyme structure will certainly help to answer these questions. However, from the observations in this work, it is clear that electron transfer between an enzyme and an electrode cannot be treated as an isolated event. The interaction between the enzyme and the electrode is of foremost importance for the final result.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Acknowledgements 27

We want to thank Dr. H.M. Kalisz for providing us with deglycosylated enzyme and for illuminating discussions about the possible electron transfer mechanism. We are also grateful to Dr. H.J. Hecht for enlightening discussions about the GOx structure and Dr. M. Sokolov for allowing us to test the electrodes in the anaerobic chamber. Finally, we thank Mrs. Brigitte Kappinos for invaluable technical assistance. References

28 29 30 31 32 33 34

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