Fundamental Studies of Glucose Oxidase

Anal. Chem. 2002, 74, 362-367

Fundamental Studies of Glucose Oxidase Deposition on a Pt Electrode Norio Matsumoto,† Xiaohong Chen, and George S. Wilson*

Department of Chemistry, University of Kansas, Lawrence, Kansas 66045

The direct electrodeposition of glucose oxidase (EC 1.1.3.4) on a platinum electrode was investigated as a means for controlled immobilization. The presence of a nonionic detergent, Triton X-100, was found essential to produce a multilayered deposit. Moreover, to work properly, the detergent must be present above its critical micelle concentration. Under these conditions, a deposit of ∼50 enzyme layers (480 nm), with surface uniformity of (20 nm, was verified using an electrochemical quartz crystal microbalance and by atomic force microscopy. In the absence of detergent, a layer of 25 nm is formed. Contrary to most previous claims, the deposition, which is potential dependent but optimal at 1.3 V versus AgCl/ Ag electrode, is not electrophoretically driven, but is instead controlled by a lowering of the pH at the electrode surface due to concomitant oxygen evolution. Over the years, a large number of methods have been developed for the electrochemical deposition of enzymes on electrodes as part of the fabrication of electrochemically based biosensors. Four general approaches have been taken to this process: (1) electrochemically driven co-deposition of enzymes with other proteins such as collagen and bovine serum albumin (BSA);1,2 (2) co-deposition of enzyme (glucose oxidase, GOx) with a Pt salt to form a Pt black surface;3-6 (3) co-deposition of enzyme with monomers such as pyrrole, phenol, 1,3-diaminobenzene, or their derivatives7-14 to form an insoluble matrix in which the enzyme is entrapped. This category also includes the grafting of † Present address: Bio-Science Department, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko City, Chiba 270-1194 Japan. (1) Wang, S. S.; Vieth, W. R. Biotechnol. Bioeng. 1973, 15, 93-115. (2) Johnson, K. W.; Allen, D. J.; Mastrototaro, J. J.; Morff, R. J.; Nevin, R. S. ACS Symp. Ser. 1994, No. 556, 84-95. (3) Ikariyama, Y.; Yamauchi, S.; Yukiashi, T.; Ushioda, H. Anal. Lett. 1987, 20, 1791-801. (4) Wang, J.; Chen, L.; Hocevar, S. B.; Ogorevc, B. Analyst (Cambridge, U. K.) 2000, 125, 1431-1434. (5) Johnson, K. W. Sens. Actuators, B 1991, B5, 85-89. (6) Kim, C. S.; Oh, S. M. Electrochim. Acta 1996, 41, 2433-2439. (7) Fortier, G.; Brassard, E.; Belanger, D. Biosens. Bioelectron. 1990, 5, 473490. (8) Rishpon, J.; Gottesfeld, S. Biosens. Bioelectron. 1991, 6, 143-149. (9) Malitesta, C.; Palmisano, F.; Torsi, L.; Zambonin, P. G. Anal. Chem. 1990, 62, 2735-2740. (10) Bartlett, P. N.; Cooper, J. M. J. Electroanal. Chem. 1993, 362, 1-12. (11) Curulli, A.; Palleschi, G. Electroanalysis 1997, 9, 1107-1112. (12) Zhang, Z.; Liu, H.; Deng, J. Anal. Chem. 1996, 68, 1632-1638. (13) Sadik, O. A.; Brenda, S.; Joasil, P.; Lord, J. J. Chem. Educ. 1999, 76, 967970. (14) Vidal, J. C.; Garcia, E.; Castillo, J. R. Anal. Chim. Acta 1999, 385, 213222.

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pyrrole monomers to the enzyme prior to polymerization to promote cross-linking of the enzyme with the growing polymer;15-17 (4) covalent attachment of enzyme to film formed on the electrode by electropolymerization.18,19 In any case, the purpose of these strategies is to immobilize enzyme in a highly active state and, at the same time, provide a membrane preferentially permeable to hydrogen peroxide over endogenous electroactive species such as ascorbate, urate, and the widely employed analgesic, acetaminophen. In general, however, electropolymerized membranes maintain their permselective and active enzyme characteristics for only a few days, making them unsuitable for in vivo monitoring. Accordingly, we have developed a procedure involving only the deposition of glucose oxidase that leads to films of high and stable enzymatic activity.20 The first step in this process is the deposition of the enzyme on the surface of a bright Pt electrode. The formation of a uniform enzyme layer has been monitored by an electrochemical quartz crystal microbalance (EQCM) and by atomic force microscopy to assess enzyme layer uniformity and thickness. EXPERIMENTAL SECTION Materials. Glucose oxidase (EC 1.1.3.4, 277 units/mg) was purchased from Biozyme International (San Diego, CA). Triton X-100 was obtained from Sigma (St. Louis, MO). β-D(+)-glucose, sodium hydrogen phosphate, and potassium dihydrogen phosphate were obtained from Fisher (Fair Lawn, NJ). Deionized water used in these studies was prepared using a Barnsted Nanopure II system. All other chemicals used in this study were analytical grade. GOx concentration can be expressed in terms of active flavin content with a differential molar absorptivity ∆450 nm ) 13.1 mM-1‚cm-1 for oxidized minus reduced enzyme21 yielding a concentration of ∼6 × 10-5 M for a 10 mg/mL solution of enzyme. Platinum disk microelectrodes were used to measure glucose response under a variety of deposition conditions. A Pt-Ir wire (90% Pt) obtained from Medwire (Mt. Vernon, NY) was cut to 1-cm length and washed with acetone under ultrasonication for (15) Yon-Hin, B. F. Y.; Smolander, M.; Crompton, T.; Lowe, C. R. Anal. Chem. 1993, 65, 2067-2071. (16) Yon-Hin, B. F. Y.; Lowe, C. R. J. Electroanal. Chem. 1994, 374, 167-172. (17) Cosnier, S. Appl. Biochem. Biotechnol. 2000, 89, 127-138. (18) Schuhmann, W.; Kittsteiner-Eberle, R. Biosens. Bioelectron. 1991, 6, 263273. (19) Situmorang, M.; Gooding, J. J.; Hibbert, D. B. Anal. Chim. Acta 1999, 394, 211-223. (20) Chen, X.; Matsumoto, N.; Hu, Y.; Wilson, G. S. Anal. Chem., following paper in this issue. (21) Weibel, M. K.; Bright, H. J. J. Biol. Chem. 1971, 246, 2734-2744. 10.1021/ac015536x CCC: $22.00

© 2002 American Chemical Society Published on Web 12/13/2001

10 min. After connecting with a conducting Cu wire (4 cm) using silver epoxy (Dotite, Type D-550, Fujikura Kasei Co., Ltd.), the Pt-Ir wire was inserted into a glass capillary and sealed by heating. After sealing, the tip of the electrode was polished with sandpaper and with Al powder (1.0 and 0.3 µm, Micropolish II, Buehler, Lake Bluff, IL) until all scratches on the electrode surface disappeared. The electrodes were cleaned by soaking in 1 M HClHNO3 (1:1) for 20 min and then rinsed with a large amount of deionized water. The diameter of the disk electrode was 170 µm. The uniformity of each electrode was checked by measuring the peak current corresponding to the cyclic voltammetric oxidation of 1 mM Fe(CN)64-. The current variation for the electrodes using this approach was less than 5%. Apparatus. All electrochemical measurements were made with a CH Instruments (Austin, TX) model CHI422 electrochemical analyzer connected to a Dell Dimension XPS B800r computer. This instrument was also interfaced to an EQCM provided by CH Instruments. The EQCM consisted of a Teflon cell 37 mm high and 35 mm in diameter with a total volume of 4 mL. Pt-coated AT-cut crystals (crystal diameter, 14 mm; oscillation frequency, 7.995 MHz; obtained from International Crystal Manufacturing, Oklahoma City, OK) were employed in this study except as noted. Each crystal had an etched (3-5 µm roughness) surface and Pt was directly deposited on it (thickness, 100 nm; electrode area, 0.196 cm2). A AgCl/Ag reference and a gold counter electrode (50 mm2) were employed. The system was allowed to equilibrate for ∼1 h while the crystal frequency became stable. The system was calibrated by deposition of Ag. To avoid silver chloride precipitation, a silver wire (diameter, 0.5 mm; length, 10 cm) was used as the reference instead of AgCl/Ag. The electrodeposition was performed by applying 0.0 V versus Ag in 1.0 mM AgNO3 (with 0.1 M KClO4) under Ar atmosphere for 30 s. From the relationship between frequency change and total charge from a cathode deposition of Ag, a sensitivity of 1.4 ng/ Hz was obtained. The maximum mass change measurable is ∼1500 ng. For the AFM studies of film thickness, Pt-quartz crystals (100-mm Pt film deposited on AT-cut polished quartz (frequency, 9.995 MHz)) were employed. The sensitivity is 0.9 ng/Hz. Atomic force microscopy (AFM) was carried out using a Nanoscope E (Digital Instruments, Santa Barbara, CA). A silicon nitride tip was used as a detection probe (Nanoprobe SPM tips, type DNP-20, spring constant, 0.06 N/m; cantilever length, 100 µm; cantilever configuration, V-shaped; tip radius of curvature, 20-60 nm; sidewall angles, 35° on all four sides). Each image was processed and analyzed through built-in software (NanoScope V4.1). Dynamic light scattering was conducted using a Brookhaven Instruments model BTC9865 (Holtsville, NY) system. The system components include a model BI-200SM goniometer, 532-nm diode laser, and a thermostated cell holder. The scattering angle was set at 90°. Postacquisition data evaluation was carried out using a BI9000AT digital autocorrelator. The system was calibrated using a polystyrene nanosphere standard (96 ( 3.1 nm) (Duke Scientific, Palo Alto, CA). ζ Potential and mobility analysis for enzyme in buffer solution was carried out using a zeta potential analyzer (BIC Zeta PALS, Brookhaven Instruments Co.). Data were calculated with software provided by the manufacturer.

Figure 1. Effect of Triton X-100 concentration on the electrodeposition of glucose oxidase as measured by the current response to 5 mM glucose (n ) 3). Electrodeposition was performed at 1.3 V vs AgCl/Ag for 30 min in a solution containing 10 mg/mL GOx.

Methodology. Enzyme electrodeposition was carried out by potential step chronoamperometry (I-t curve mode). In each trial, the initial potential was set at 0.3 V versus AgCl/Ag. After 10 s of initial holding, the potential was stepped to the defined value. Current data were taken every 0.2 s. The Pt-Ir disk electrode was placed in a 50 mM phosphate buffer solution, pH 7.0, containing variable amounts of GOx and Triton X-100. The volume of each solution was 0.5 mL, a AgCl/Ag electrode was used as a reference, and a Pt-Ir wire was used as the counter electrode. After the electrodeposition was carried out, the electrode was removed from solution and dried in air for 10 min. The electrode was then immersed in deionized water and washed by agitation. The response of sensors to glucose was established by immersing each sensor in 10 mL of phosphate-buffered saline (PBS, 0.1 M, pH 7.4). A potential of 0.65 V versus AgCl/Ag electrode was then applied to the sensor. After the background current became stable (at least 10 min), glucose was added to produce sequential 5 mM increases in concentration. For AFM measurements, the x, y, and z axis were calibrated using a calibration standard of inverted pyramids (5-µm length, 200-nm depth, Digital Instruments). The enzyme was deposited on platinum quartz crystals (AT-cut polished quartz; total diameter, 14 mm; electrode area, 0.196 cm2; electrode thickness, 100 nm using the same cell as for the EQCM measurements). Crystals were rinsed with acetone before electrodeposition, which was carried out at 1.3 V versus Ag Cl/Ag for 1 h in 50 mM phosphate buffer (pH 7.0) containing 10 mg/mL GOx with and without 0.8 mM Triton X-100. After electrodeposition, each electrode was carefully washed with deionized water and stored under dry conditions in the refrigerator. AFM measurements were made on the dry film 24 h later. RESULTS AND DISCUSSION Effect of Triton X-100 on Electrodeposition. Figure 1 shows the dependence of current response to 5 mM glucose at a Pt-Ir electrode on which GOx has been deposited in the presence of variable concentrations of Triton X-100. It will be noted that in the absence of the nonionic detergent the response is extremely low and that the optimal response occurs between about 0.32 and 3.2 mM detergent. No enhancement of deposition is observed if a cationic (cetyltrimethylammonium chloride) or anionic detergent (sodium dodecyl sulfate) is employed. Figure 2, obtained from Analytical Chemistry, Vol. 74, No. 2, January 15, 2002

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Figure 2. EQCM mass changes during electrodeposition of GOx at different Triton X-100 concentrations. Conditions the same as for Figure 1. Triton X-100 concentration was 0 (×), 0.8 (O), and 8 mM (]).

Figure 4. EQCM mass changes during electrodeposition of GOx at different potentials. Conditions the same as for Figure 3. Applied potential was 1.0 (O), 1.2 ([), 1.3 (4), and 1.4 V (b) vs AgCl/Ag. Broken line in the figure corresponds to the response at the rest potential.

Figure 3. Effect of applied electrodeposition potential on current response to 5 mM glucose (n ) 3). Electrodeposition was performed for 30 min in a solution containing 10 mg/mL GOx and 0.8 mM Triton X-100.

EQCM measurements, further supports the notion that the deposition of enzyme is strongly influenced by the presence of detergent as noted previously.2 Some deposition can occur in the absence of detergent so that after 60 min ∼450 ng is deposited. If the detergent concentration is too high, the amount of electrodeposited enzyme is significantly reduced. It is important to note that the critical micelle concentration (cmc) of Triton X-100 is 0.2 mM22 and that efficient enzyme deposition occurs above that value. The significance of this observation will be discussed subsequently. Effect of Potential on Sensor Response. Figure 3 shows the effect of the applied electrodeposition potential on the apparent activity of the immobilized enzyme. As indicated, the maximum current is obtained at 1.3 V versus AgCl/Ag reference, suggesting that this is the potential at which the maximum amount of enzyme is deposited. On the contrary, Figure 4 shows that, above an applied potential of 1.2 V, the rate of deposition of enzyme increases rapidly. Figure 5 shows a voltammogram in which the potential dependence of enzyme deposition is further illustrated. From Figure 3 one must conclude either that some of the enzyme deposited at higher potentials is not active or that the response as measured from the evolving hydrogen peroxide is not correct. We believe the latter to be the case. Hall23 has demonstrated that the oxidation of hydrogen peroxide is electrocatalytic and depends (22) Tiller, G. E.; Mueller, T. J.; Dockter, M. E.; Struve, W. G. Anal. Biochem. 1984, 141, 262-266. (23) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1997, 43, 579588.

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Figure 5. Current (above) and mass change profiles (bottom) obtained from cyclic voltammetry using the EQCM system. Conditions the same as for Figure 3. Scan rate, 10 mV/s. Table 1. Effect of Potential Application in a 50 mM Phosphate Buffer for 30 min on Current Response to H2O2 at Pt-Ir Electrode (n ) 3) applied potential (V vs Ag/AgCl)

response to 50 µM H2O2 (nA)a

SD

% ratio against control

control 1.2 1.3 1.4 1.5 1.8

10.22 2.37 1.30 0.66 0.54 0.09

1.89 0.56 0.08 0.13 0.05 0.02

100 23.1 12.8 6.5 5.3 0.9

a

Applied potential, 0.65 V vs Ag/AgCl.

on an electrode surface with Pt(OH)2 functionalities. The applied potential region for enzyme deposition corresponds to the region of water oxidation, a point that will also be important in understanding the deposition process. It is a region in which platinum oxide is formed which is not beneficial to peroxide oxidation. This is illustrated by the results shown in Table 1. The control sample corresponds to the current obtained at a Pt-Ir electrode for a 50 µM peroxide solution at an applied potential of 0.65 V. This is the effective concentration that would be measured if peroxide were generated from the enzyme-catalyzed reaction. If, however, a potential of greater than 1.0 V is applied to the electrode as required for enzyme electrodeposition and then returned to 0.65 V, the response to peroxide is much lower. We have also shown

Figure 6. Effect of GOx concentration on the electrodeposition as reflected in the current response to 5 mM glucose (n ) 3). Triton X-100 concentration in the solution was 0.8 mM.

Figure 7. EQCM mass changes during electrodeposition at varied GOx concentrations. Electrodeposition was performed at 1.3 V vs AgCl/Ag in a solution containing 0.8 mM Triton X-100. The GOx concentration was 0 (broken line), 2 (O), 5 (4), 7 (]), and 10 mg/mL (×). Dashed line in the figure from 0 mg/mL GOx without Triton X-100.

that platinum oxides deposited on Pt electrodes tend to be stabilized by the presence of adsorbed films.24 Effect of Enzyme Concentration on Electrodeposition. As shown in Figure 6, the current due to the immobilized enzyme increases rapidly with increasing GOx concentration. At very high enzyme concentrations, the amount of immobilized enzyme actually decreases. A possible reason for the decrease in amperometric signal for GOx concentrations higher than 20 mg/mL may have to do with the stoichiometry of GOx and Triton X-100. In Figure 6, the mean current for 40 mg/mL GOx is 2.5 nA for a detergent concentration of 0.8 mM. If this concentration is increased to 1.6 mM, the mean current doubles to 5.0 nA. The uptake of enzyme onto the electrode is similarly reflected in the EQCM results shown in Figure 7. Effect of Electrodeposition Time on Sensor Response. Figure 8 shows the relationship between the sensor response current as a function of deposition time under otherwise optimized conditions. It will be noted that the current increases linearly with deposition time up to 1 h. However, no further current increase was observed beyond this point. The current response to 50 µM H2O2 on a bare Pt-Ir disk electrode, after 2 h of polarization at 1.3 V versus AgCl/Ag, was 1.13 nA (data not shown), which was almost the same as that for 30-min polarization under the same (24) Zhang, Y.; Wilson, G. S. J. Electroanal. Chem. 1993, 345, 253-271.

Figure 8. Effect of electrodeposition time on current response to 5 mM glucose (n ) 3). Each electrodeposition was performed at 1.3 V vs AgCl/Ag in the solution containing 10 mg/mL GOx and 0.8 mM Triton X-100.

conditions (Table 1). Thus, the decrease of sensor activity was not strongly dependent on the electrodeposition time. The current decrease observed at 2 h of electrodeposition in Figure 8 probably resulted from the loss of enzyme activity suffered from exposure to low local pH conditions for an extended time. Morphology of the Enzyme Layer. The thickness and roughness of the enzyme layer created by electrodeposition on a platinum electrode was estimated using AFM. Figure 9 shows AFM images for the enzyme layer after 1 h of electrodeposition in GOx solution with and without 0.8 mM Triton X-100. Panel A shows the polycrystalline platinum surface without enzyme present. When electrodeposition is carried out in the absence of detergent (panel B), the AFM of the dry film reflects the features of the underlying substrate, with a correspondingly smooth film. By contrast, panel C is seen to be apparently rougher. Figure 10 shows the results of depth profiling of the electrodeposited films. Panel A indicates a thickness of the enzyme layer of ∼25 nm, corresponding to deposition of the film in the absence of detergent. By contrast, the thickness of the enzyme layer where detergent is used (panel B) is ∼480 nm. The dimensions of glucose oxidase, taken from the X-ray structure, are reported as 60 Å × 52 Å × 77 Å.25 Based on these dimensions, the thickness of a monolayer would be ∼10 nm. Thus, in the absence of detergent, about two equivalent monolayers are formed. If the area occupied by a single GOx molecule is assumed to be 8 × 10-13 cm2, the total weight of a monolayer on the electrode should be ∼66 ng. However, from Figure 2, the total mass change is ∼450 ng. Some of the mass gain is due to uptake of water and counterions. Rickert26 noted that a mass increase due to protein adsorption monitored by QCM was 2-4 times larger than the respective mass for the dry protein. We are unable to measure the total mass gain in the presence of detergent because it exceeds the capacity of the QCM (the mass change should be at least 3168 ng (66 × 48). It must be emphasized that the film thickness was estimated on dry films. Such protein films will swell significantly on hydration so that the operating thickness could increase by as much as 100-200%. Solution Properties of GOx and Triton X-100 Micelles in Solution. To investigate the interactions between GOx and Triton (25) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153-172. (26) Rickert, J.; Brecht, A.; Gopel, W. Anal. Chem. 1997, 69, 1441-1448.

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Figure 10. AFM images for enzyme membrane layers created by electrodeposition at 1.3 V vs AgCl/Ag for 1 h in a solution containing 10 mg/mL GOx without Triton X-100 (a) and with 0.8 mM Triton X-100 (b). For these images, samples were scored with a razor blade to expose the electrode surface. The line through the image indicates where the profile was measured.

Figure 9. AFM electrode surface images: (A) bare platinum electrode; (B) electrodeposition without Triton X-100; (C) electrodeposition with 0.8 mM Triton X-100.

Figure 11. Hydrodynamic diameter spectra (DLS) for a solution containing 10 mg/mL GOx (top), 10 mg/mL GOx + 0.8 mM Triton X-100 (center), and 0.8 mM Triton X-100 (bottom) in 50 mM phosphate buffer (pH 7).

X-100 in solution, dynamic light-scattering (DLS) experiments27 were carried out. Prior to measurements, the 50 mM phosphate buffer solution was filtered through a 0.22-µm filter to remove dust particles. As shown in Figure 11, the hydrodynamic diameter of GOx is ∼10 nm, consistent with the X-ray results. Triton X-100 is observed to have a similar diameter at concentrations above its cmc, nearly equal to the reported value of 8-9 nm at 25 °C.28 When the GOx and detergent are mixed together, a somewhat

broader distribution is observed but with approximately the same peak maximum. There appears also a peak at ∼4.5 nm, and since it does not appear in the spectrum of either the enzyme or detergent alone, it must be the result of detergent-enzyme interactions.29 A possibility is the dissociation of the enzyme into subunits although direct interactions between proteins and nonionic detergents are believed to be weak.30

(27) Brown, W., Ed. Light Scattering: Principles and Development; Oxford University Press: New York, 1996. (28) Streletzky, K.; Phillies, G. D. J. Langmuir 1995, 11, 42-47.

(29) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Kragel, J.; Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko, O. V. Adv. Colloid Interface Sci. 2000, 86, 39-82.

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Figure 12. Schematic of the enzyme layer profile measured by AFM: (a) bare electrode; (b) no Triton X-100; (c) Triton X-100.

Mechanism of Electrodeposition and the Role of the Detergent. Although enzyme can chemisorb on the electrode surface, the dramatic influence of applied potential is quite evident as it produces much more extensive and compact enzyme deposit. Although the enzyme is negatively charged at pH 7, and the electrode positively charged, our view is that electrophoretic (migration) effects in 50 mM phosphate buffer are rather unimportant. The ζ potential of GOx in this medium was measured as -0.4 mV, and this corresponds to an enzyme mobility of 3.5 × 10-2 (µm/s)/(V/cm). This mobility was much smaller than that for HPO42- or H2PO4- (3.4 (µm/s)/(V/cm)), which were dominant anion species in the solution.31 The ion mobility of the enzyme is 2 orders of magnitude lower than the supporting electrolyte. When the relative concentrations are taken into account, we can conclude that the electrophoretic migration of the enzyme is negligible. Thus, we concur with the suggestion of Im and co-workers32 that the driving force for immobilization is the precipitation of enzyme on the electrode surface as a consequence of a local pH decrease created by the evolution of oxygen (oxidation of water). The linear increase in mass of deposited enzyme is further consistent with evolution of protons. It is probably not an accident that Triton (30) Saito, S.; Anghel, D. F. In Polymer-Surfactant Systems; Kwak, J. C. T., Ed.; Marcel Dekker: New York, 1998; pp 357-404. (31) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; McGraw-Hill: New York, 1999. (32) Im, D. M.; Jang, D. H.; Oh, S. M.; Striebel, C.; Wiemhoefer, H. D.; Gauglitz, G.; Goepel, W. Sens. Actuators, B 1995, B24, 149-155. (33) Rharbi, Y.; Li, M.; Winnik, M. A.; Hahn, K. G. J. Am. Chem. Soc. 2000, 122, 6242-6251.

X-100 is effective in promoting the deposition. The micelles are the same size as the GOx molecules and therefore can suppress aggregation of enzyme and may easily substitute in a pseudolattice formed as the enzyme is deposited. It is possible that enzyme molecules eventually replace many of the detergent micelles in the deposited structure; however, we do not know how much detergent remains in the final structure. There is a clear contrast between deposition of enzyme in the presence and absence of detergent. In the latter case, about two monolayers of protein are formed as depicted in Figure 12. The surface features of the substrate are preserved. The deposition process thus appears to be self-limiting. If too much detergent is added, then the deposition process is inhibited. The detergent micelles, established as necessary for promotion of deposition, minimize aggregation of enzyme and probably initially shield it from the very high electric field gradient at the electrode/solution interface. The detergent may also serve to slow the deposition and make it more uniform, performing the analogous function of surfactants in electroplating. It is also possible that the micelles once deposited are in an environment below the cmc in which case each micelle should break up into over 100 individual detergent molecules.27,33 The enzyme layer deposited in the presence of the detergent is almost 25 times thicker (Figure 12) and shows surface irregularities equivalent to almost two enzyme molecules. It is also important to keep in mind that the presence of detergent on the electrode surface does not interfere with either the oxidation of water or the oxidation of hydrogen peroxide. In each case, a small molecule must diffuse through the deposited enzyme layer. In conclusion, we have demonstrated the electrochemically mediated deposition of enzyme on an electrode promoted by the presence of a nonionic detergent. Under these conditions, a uniform and biologically active film is formed. It is then possible, using electropolymerization of small organic molecules through the already deposited enzyme layer, to generate a stable permselective film. The resulting sensor has the highest reported response to glucose, and we attribute this to the initial deposition of a dense, active enzyme layer.20 ACKNOWLEDGMENT We thank Russ Middaugh for use of the light-scattering instrument and helpful discussions. The support of the National Institutes of Health (Grant DK55297) and the Centers for Disease Control (Grant CCR017796) is gratefully acknowledged. Received for review June 20, 2001. Revised manuscript received September 19, 2001. Accepted November 8, 2001. AC015536X

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