Anal. Chem. 2002, 74, 1322-1326
Detection of Hydrogen Peroxide at Mesoporous Platinum Microelectrodes Stuart A. G. Evans,† Joanne M. Elliott,‡ Lynn M. Andrews,† Philip N. Bartlett,† Peter J. Doyle,§ and Guy Denuault*,†
Department of Chemistry, The University of Southampton, Southampton SO17-1BJ, U.K., Department of Chemistry, The University of Reading, P.O. Box 224, Whiteknights, Reading RG6 6AD, U.K., and Port Sunlight Laboratory, Unilever Research, Quarry Road East, Bebington, Wirral, L63-3JW, U.K.
Mesoporous (HI-ePt) platinum microelectrodes electrodeposited from the hexagonal (HI) lyotropic liquid crystalline phase are shown to be excellent amperometric sensors for the detection of hydrogen peroxide over a wide range of concentrations. Good reproducibility, high precision, and accuracy of measurements are demonstrated. Mesoporous microelectrodes retain the high rates of mass transport typical of conventional microelectrodes, and their high real surface area greatly enhances their catalytic activity. This unique combination of properties overcomes the limitations of previous amperometric hydrogen peroxide sensors and yields outstanding qualitative and quantitative results. An accurate and reliable method for the determination of hydrogen peroxide is of interest to many fields but particularly in biosensing because it forms the diagnostic response for several medical sensing devices such as blood glucose monitors.1 In one of the first reports of a biosensor,2 Clark described a two-electrode system that could be used to monitor glucose concentrations via amperometric detection of hydrogen peroxide produced by the action of glucose oxidase upon glucose in the presence of oxygen. Since then, many investigations have been undertaken using in vivo glucose sensors.3 Other fields of interest include the bleaching industry4 and now the waste treatment industry (namely, municipal wastewater applications, industrial waste/wastewater applications, and air applications of H2O2).5 Hydrogen peroxide concentrations typically range from micromolar for in vivo conditions and residual levels in foodstuff and drinking water to tens of millimolar for bleaching applications and molar for waste treatment applications. Current spectrophotometric analytical methods based on peroxidase enzyme6 or the cobalt-bicarbonate reaction7 cover the range 0.6-6 µM while commercial test strips and kits for field measurement of residual hydrogen peroxide work down to ∼15 µM (e.g. †
The University of Southampton. The University of Reading. § Unilever Research. (1) ThomeDuret, V.; Reach, G.; Gangnerau, M. N.; Lemonnier, F.; Klein, J. C.; Zhang, Y. N.; Hu, Y. B.; Wilson, G. S. Anal. Chem. 1996, 68, 3822-3826. (2) Clark, L. C., Jr. Membrane polarographic electrode system and method with electrochemical compensation. U.S. Patent 3539455, 1965. (3) Armstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 2623-2645. (4) Westbroek, P.; Van Huate, B.; Temmerman, E. Fresenius J. Anal. Chem. 1996, 354, 405-409. (5) Fagan, M.; Walton, J. R. U.S. Peroxide 1999, 949, 661-671. ‡
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Merckoquant peroxide test, EM Science, division of EM Industries, Inc.). In contrast, permanganate titration is suitable for measuring aqueous solutions of H2O2 ranging from millimolar to molar. To date it has been difficult to detect hydrogen peroxide accurately with amperometric techniques because its electrode reactions are very irreversible and voltammograms tend to be irreproducible at the majority of electrode surfaces. Different electrode materials have been investigated, for example, carbon fiber,8 glassy carbon, gold, tin, nickel, zirconium, titanium, palladium, and platinum;4 all, however, exhibit the aforementioned problems. Although some electrodes have shown linear responses over a small concentration range (1-20 mM), the responses obtained are still relatively poor and very unstable.9 Strategies to circumvent this irreproducibility and poor linear response have included modification of electrode surfaces with enzymes10,11 and Prussian Blue (PB)-type analogues.12 Using these methods, it has been possible to obtain high sensitivity (1.5 A M-1 cm-2) and very low detection limits (10 nM),13 but once more, their upper limit of detection is quite small (up to 12 mM) and their long-term stability drops by up to 85% with repeated use.14 A mechanism for the oxidation of hydrogen peroxide at platinum electrodes has recently been discussed in detail and provides an adequate explanation for the lack of linearity observed in the electrode response. Hall et al., in a series of papers,15 reported that the current response due to hydrogen peroxide (6) Chin, H. S.; Cortes, A. Determination of Hydrogen Peroxide: A Comparison Between the Potentiometric Titration Method and an Enzyme Catalyzed Procedure, Unpublished Draft, National Food Processors Assn., 1950 Sixth St., Berkeley, CA 94710, 1982. (7) Masschelen, W. Water Sewage Works 1977, 124 (Aug), 69. (8) Aoki, K.; Ishida, M.; Tokuda, K.; Hasebe, K. J. Electroanal. Chem. Interfacial Electrochem. 1988, 251, 63-71. (9) Nowall, W. B.; Kuhr, W. G. Electroanalysis 1997, 9, 102-109. (10) Horrocks, B. R.; Schmidtke, D.; Heller, A.; Bard, A. J. Anal. Chem. 1993, 65, 3605-3614. (11) Ruzgas, T.; Cso ¨regi, E.; Emne´us, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1996, 330, 123-138. (12) Karyakin, A. A. Electroanalysis 2001, 13, 813-819. (13) Ferapontova, E. E.; Grigorenko, V. G.; Egorov, A. M.; Bo¨rchers, T.; Ruzgas, T.; Gorton, L.; Biosens. Bioelectron. 2001, 16, 147-157. (14) Garjonyte, R.; Malinauskas, A. Sens. Actuators, B 1999, 56, 93-97. (15) (a) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1998, 43, 579-588. (b) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1998, 43, 2015-2024. (c) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1999, 44, 2455-2462. (d) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1999, 44, 4573-4582. (e) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 2000, 45, 3573-3579. 10.1021/ac011052p CCC: $22.00
© 2002 American Chemical Society Published on Web 02/14/2002
oxidation is under mixed kinetic and diffusion control and that the mechanism for the oxidation of hydrogen peroxide can be explained in terms of Michaelis-Menten-type kinetics. The oxidation of hydrogen peroxide is further complicated by two additional reactions. The first is the competitive adsorption of dioxygen onto the Pt surface sites and the second is the protonation of the adsorbed hydrogen peroxide complex. A lack of available Pt surface sites will therefore limit the reaction and will result in a depression of response for higher concentrations of hydrogen peroxide.15 Electrochemistry at electrodes of micrometer dimensions represents one of the important advances of modern electrochemical science.16 Microelectrodes have significantly improved the quality and ease of analysis of experimental data and enabled a range of new experiments to be performed.17 Their simple mode of operation is combined with a useful amperometric response where the diffusion-controlled limiting current is directly proportional to the concentration of the electroactive analyte. Unfortunately, the use of unmodified microelectrodes to amperometrically measure hydrogen peroxide concentrations has so far been unsuccessful; the hydrogen peroxide reaction appears to be kinetically limited, probably because of the very small surface area. One way to address this limitation is to modify the microdisk to increase its electroactive area while retaining diffusional properties typical of a microelectrode. A recent technique reported by Attard et al. has enabled the fabrication of high surface area platinum microelecrodes from the deposition of mesoporous platinum layers18 onto conventional microdisk electrodes to form so-called “mesoporous microelectrodes”. The preparation utilizes the inherent three-dimensional structure of lyotropic liquid crystal phases, which form a template for the electrodeposition of the platinum film (Figure 1A). Transmission electron micrographs of the typical films reveal a highly porous structure consisting of 2.5-nm-diameter cylindrical holes arranged in a hexagonal lattice18 and separated by 2.5-nmthick walls of platinum (Figure 1b). The specific surface area of the mesoporous platinum films (22 ((2) m2 g-1) was found to compare reasonably with literature values for the specific surface area of platinized platinum films (between 2 and 30 m2 g-1) and platinum black powders (35 m2 g-1).18a In this paper, we report for the first time the use of a mesoporous microelectrode amperometric sensor for the detection of hydrogen peroxide. We compare the response of mesoporous platinum microelectrodes with that of conventional microelectrodes and present, with an example, the application of these electrodes toward detection of hydrogen peroxide. EXPERIMENTAL SECTION Solutions. All solutions were prepared using Milli-Q reagent water (resistivity g18 MΩ cm), and all compounds were used as received without further purification. Hydrogen peroxide (30%, unstabilized, ACS., Fluka) solutions were prepared by dilution (16) Montenegro, M. I., Queiro´s, M. A., Daschbach, J. L., Eds. Microelectrodes: Theory and Applications; NATO ASI series E 197; Kluwer Academic Press: Dordrecht, The Netherlands, 1991. (17) Bond, A. M. Analyst 1994, 119, R1-R21. (18) (a) Attard, G. S.; Bartlett, P. N.; Coleman, N. R. B.; Elliott, J. M.; Owen, J. R.; Wang, J. H. Science 1997, 278, 838-840. (b) Elliott, J. M.; Birkin, P. R.; Bartlett, P. N.; Attard, G. S. Langmuir 1999, 15, 7411-7415. (c) Birkin, P. R.; Elliott, J. M.; Watson, Y. E. Chem. Commun. 2000, 17, 1693-1694.
Figure 1. (A) Fabrication of mesoporous platinum films onto platinum microdisk electrodes. Initially, the surfactant molecules aggregate into rods which orientate to form a three-dimensional template. Upon application of the potential, platinum is electrodeposited within the aqueous domains surrounding the rods. Removal of the surfactant by soaking in water reveals a film with a hexagonal array of cylindrical pores. (B) TEM image of a typical film (deposited at +0.1 V vs SCE at 25 °C and with a charge density of 6.4 C cm-2) shows 2.5-nm pores separated by 2.5-nm walls.
from a buffered stock solution (0.1 M, pH 7 phosphate buffer) after standardization using a sodium thiosulfate (Aldrich) titration. The liquid crystalline plating mixture consisted of 42% (w/w) octaethylene glycol monohexadecyl ether (98% purity, Fluka), 29% (w/w) water, and 29% (w/w) hexachloroplatinic acid hydrate (99.9% purity, Aldrich). D-Glucose (Aristar, BDH, Merck, Lutterworth, U.K.) solutions were prepared 24 h before each experiment and stored at room temperature to allow complete equilibration of the anomers. The buffer solution was prepared from 0.2 M Na2H2PO4‚H2O (AnalaR, BDH, Merck) and 0.2 M Na2HPO4‚12H2O (AnalaR, BDH, Merck). The pH of the buffer solution was measured using a Mettler Toledo 320 pH meter. Solutions for the glucose oxidase immobilization consisted of 25 mM phenol (98% purity, Adrich), 0.1 M, pH 7 phosphate buffer, and 5 µM glucose oxidase (Sigma). Microelectrodes were characterized in 5 mM ruthenium hexaamine trichloride (Strem) solutions with 0.1 M KCl (Aristar, Merck) and in 2 M sulfuric acid (AristaR, BDH, Merck). Procedures. Voltammetric and chronoamperometric measurements were undertaken using a two-electrode configuration, with either a polished or mesoporous platinum microdisk as working electrode and either a saturated calomel electrode or Hg/ Hg2SO4, saturated K2SO4 electrode as reference. The potential of the working electrode was controlled using a HiTek PPR1 waveform generator, and the current was recorded on a homebuilt current follower. The temperature of the cell was set at 25 °C for all voltammetric and chronoamperometric experiments. All solutions were thoroughly deoxygenated with argon (BOC) before recording the voltammetric response. Scanning electrochemical microscopy (SECM) measurements were undertaken using piezoelectric micropositioners (Inchworm motors, model IW-710) mounted on a three-dimensional translation stage (models TS100 and TS300) and controlled by an Inchworm controller (model 6200), all from Burleigh instruments. A personal computer fitted with a digital input/output card (Advantech PCL-724) and an acquisition card (Advantech PCLAnalytical Chemistry, Vol. 74, No. 6, March 15, 2002
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818L) was used to control the tip movement and acquire its response. The tip potential was controlled using a HiTek PPR1 waveform generator whereas its current was measured using a home-built current follower. No precautions were taken to thermostat the SECM cell, but all experiments were undertaken with an ambient temperature between 19 and 23 °C. Electrodes. Platinum microelectrodes (25-µm diameter) were prepared by sealing platinum wire (25-µm diameter, Goodfellows) in soda glass as described previously.19 The electrodes were then polished with alumina (25-, 1.0-, and 0.3-µm grades) on felt (Buelher, Ltd., Lake Bluff, IL). The platinum microelectrodes were then electrochemically cleaned by cycling between -0.2 and +1.2 V versus SCE at 200 mV s-1 in 2 M sulfuric acid. The preparation of mesoporous electrodes followed the procedure previously described.18 The platinum microelectrodes were immersed in the plating mixture and the potential was stepped from +0.6 to -0.1 V versus SCE at 25 °C until a charge density of 6.4 C cm-2 had been passed. The microelectrodes were then removed from the plating mixture and soaked in copious quantities of water in order to remove the surfactant. Platinum substrate electrodes were prepared by sealing platinum wire (500-µm diameter, Goodfellows) in soda glass. The electrodes were then polished with alumina (25-, 1.0-, and 0.3-µm grades) on felt (Buelher, Ltd.) and electrochemically cleaned in 2 M sulfuric acid before use. Enzyme Immobilization. Glucose oxidase was immobilized within a polyphenol film according to the procedure described by Hodak and co-workers.20 The electropolymerization of the film was undertaken by cycling the potential of a 500-µm-diameter platinum disk electrode between 0 and +0.8 V versus SCE at 10 mV s-1 until the electrode was passivated (typically 8-10 cycles). The electrode was then removed from the phenol solution and rinsed with 0.1 M, pH 7 phosphate buffer to remove unbound enzyme. RESULTS AND DISCUSSION Electrode Characterization. Cyclic voltammetric analysis in dilute sulfuric acid media was used to characterize the mesoporous structure by proton adsorption/desorption methods.21 The results indicate that the electrode mesopores are highly accessible and yield a high internal surface area ∼100 times larger than that of the original electrode. Due to their high surface area, mesoporous microelectrodes also exhibit a larger double-layer capacitance although the charging time is still relatively fast, on the order of 4 ms. The ability to obtain a steady-state response with such a high surface area electrode was verified by recording linear sweep voltammograms with outer-sphere one-electron redox couples (e.g., 5 mM Ru(NH3)6 with 100 mM KCl). In all cases, the mesoporous microdisk yielded a steady-state limiting current equal ((5%) to that at the bare electrode. This indicates that the characteristic nonuniform current density distribution to the microdisk has not been affected by the presence of the film. The combination mesoporous film-microdisk yields a microelectrode with unique properties, namely, a very high rate of steady-state diffusion associated with a very high electroactive surface area. (19) Denuault, G. Chem. Ind. 1996, 18, 678-680. (20) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708-2716. (21) Bard, A. J. Electroanalytical Chemistry, Dekker: New York, 1976.
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Figure 2. Linear sweep voltammograms for a polished (dotted line) and mesoporous 25-µm-diameter platinum microdisk (solid line), in a pH 7 phosphate-buffered, 1.1 mM H2O2 solution at 25 °C. The scans were started at -0.6 V vs Hg/Hg2SO4, saturated K2SO4 reference electrode and recorded with a scan rate of 1 mV s-1. (A) plateau for the reduction of H2O2, (B) the zero current point where both oxidation and reduction of H2O2 occur at similarly high rates, and (C) plateau for the oxidation of H2O2.
This situation can therefore be exploited advantageously to study reactions with slow surface kinetics such as the oxidation of H2O2. Figure 2 shows the cyclic voltammogram obtained for a mesoporous platinum microelectrode in a phosphate buffer containing H2O2. The voltammogram presents well-defined plateaus for hydrogen peroxide oxidation (region C) and reduction (region A). In region B, the current potential curve crosses the zero current axis with a very steep gradient. When the experimental current is zero, the oxidation and reduction of the hydrogen peroxide are occurring with equal and high rates and the platinum surface is therefore decomposing hydrogen peroxide to water and oxygen at a significant rate. The voltammetric response obtained was stable and highly reproducible. However, over a period of several hours, the reduction current slowly increases by ∼5%. Oxygen reduction occurs over the same voltage range as hydrogen peroxide reduction. However, we believe that the cell and the argon blanket used did not permit the re-entry of oxygen and that this increase is probably due to the gradual production of oxygen in situ, during the oxidation of hydrogen peroxide. A qualitative comparison with the cyclic voltammogram obtained at a conventional microelectrode (the same size microdisk electrode but without a mesoporous film) (Figure 2) shows the tremendous improvement in the catalytic properties of the mesoporous electrode for hydrogen peroxide decomposition. The voltammogram obtained at the conventional microdisk electrode does not exhibit well-defined plateaus, and the overall poor shape and low currents indicate that the response is not diffusion controlled but under mixed kinetic and diffusion control. Similarly, the chronoamperometric response for the oxidation of hydrogen peroxide on the bare microelectrode is very poor (Figure 3) and decays by ∼30-40% over 1 h while the mesoporous microdisk yields a higher and stable limiting current. Both the voltammetric and chronoamperometric responses therefore indicate that the high surface area of the mesoporous electrode effectively increases its catalytic activity toward the decomposition of hydrogen peroxide and makes it possible to obtain reliable steady-state responses for both oxidation and reduction processes.
Figure 3. Chronoamperograms for the polished (dotted line) and mesoporous microdisks (solid line) for a step from the open circuit potential to +0.2 V vs Hg/Hg2SO4, saturated K2SO4 reference electrode. Other conditions as in Figure 2.
Figure 4. Comparison between the amperometric response of (b) polished and (O) mesoporous 25-µm-diameter platinum microdisks for the oxidation of hydrogen peroxide. Each data point is the average of four current values taken at +0.6 V vs SCE at 25 °C.
A quantitative analysis of the mass transport limiting current observed at the two types of microelectrode was undertaken to assess the potential applications of mesoporous microdisks. Measurements were obtained under steady-state conditions in the oxidation plateau, region C, to avoid any problems due to capacitive charging and to negate the effects of possible oxygen contamination. Figure 4 compares calibration data for a mesoporous platinum microelectrode and a conventional platinum microelectrode over the range 0.02-100 mM. The response of the conventional platinum microelectrode is found to be unstable (as indicated by the error bars, which are significantly larger than those observed for the mesoporous electrode); moreover, the limiting current is not proportional to the concentration of hydrogen peroxide beyond 1 mM.22 Above this concentration, the limiting current reaches a plateau as the binding sites become saturated. In comparison, the mesoporous microelectrode response is linear up to ∼40 mM. Over this range, the limiting current at the mesoporous electrode follows the theoretical steadystate diffusion-controlled current at a microdisk.16 Ultimately, at very high concentrations, the response at the mesoporous mi(22) Zhang, Y.; Wilson, G. J. Electroanal. Chem. 1993, 345, 253-271.
Figure 5. Calibration curve of steady-state limiting currents measured at +0.6 V vs SCE against hydrogen peroxide concentration at 25 °C. Each data point is the average of four measurements carried out with different 25-µm-diameter mesoporous platinum microdisks. The solid line is the best fit for the data up to 40 mM.
croelectrode also becomes nonlinear due to limited diffusion down the pores or perhaps saturation of available binding sites. We expect that an increased surface area, e.g., by deposition in a bicontinuous liquid crystal phase,23 would extend the linear calibration curve significantly. The quality of the calibration data obtained for the mesoporous microelectrode is remarkable. In Figure 5, the data points are the average of measurements performed with four different 25-µmdiameter mesoporous Pt microdisks, yet a linear response is observed over more than 3 orders of magnitude. Weighted regression of the data (r2 ) 0.999 over the range 0.02-40 mM using the error bars as weights) yields a diffusion coefficient for H2O2 (1.46 × 10-9 m2 s-1) within the range ((0.66-2.20) × 10-9 m2 s-1) of previously quoted values.15 The sensitivity determined using the slope of the calibration plot between 0.02 and 40 mM was found to be 2.8 A M-1 cm-2. This is a real improvement when compared to that obtained on transition metal hexacyanoferrate (1.5 A M-1 cm-2)12 and peroxidase modified electrodes (1.0 A M-1 cm-2).11 The detection limit, taken as the limiting current for H2O2 3 times greater than the standard deviation of the blank, was found to be 4.5 µM. This value is larger than that obtained with the most sensitive methods, flow injection analysis (10 nM)24 and chemiluminescent biosensor techniques, which utilize immobilized horseradish peroxidase (0.025 nM),25 but could be improved by depositing the film onto smaller microelectrodes. The accuracy and precision of the data are excellent, and it is now possible to quantitatively monitor H2O2 amperometrically. This suggests the potential use of this kind of sensor in many different fields, ranging from the very low levels of hydrogen peroxide produced in vivo due to oxidative cell stress26 to the millimolar levels found in some industrial processes. In fact, the range of linearity obtained covers more than that offered by commercially available field strips.27 In the remaining section, we illustrate the application of mesoporous microdisks with an experiment carried out with a (23) Elliott, J. M.; Attard, G. S.; Barlett, P. N.; Owen, J. R.; Ryan, N.; Singh, G. J. New. Mater. Electrochem. Syst. 1999, 2, 239-241. (24) Domı´nguez Sa´nchez, P.; Tun ˜on Blanco, P.; Ferna´ndez Alvarez, J. M.; Smyth, M. R.; O’Kennedy, R. Electroanalysis 1990, 2, 303. (25) Rubtsova, M. Y.; Kovba, G. V.; Egorov, A. M. Biosens. Bioelectron. 1998, 13, 75-85. (26) Amatore, C.; Arbault, S.; Bruce, D.; De Oliveira, P.; Erard, M.; Vuillaume, M. Faraday Discuss. 2000, 116, 319-333.
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SECM.28 In this technique, an electrochemical probe is very slowly scanned toward or over a sample surface to monitor 1D or 2D concentration profiles. The success of the experiments depends on the quality of the amperometric tip response. The latter must be quantitative and stable over at least 1 h as the experiments can be rather long. Figure 6 shows an image obtained when a 25-µm-diameter mesoporous platinum microelectrode operating in the SECM generation/collection mode28 was scanned above a monolayer of glucose oxidase immobilized with polyphenol. The tip detected H2O2 produced by the enzyme, and the tip current image reflects local variations in enzyme activity. To create a less active region, the enzyme was locally disrupted using the technique described by O’Brien and co-workers.29 The tip was brought close to the enzyme surface using the improved amperometric response for O2 reduction18c and then used to electrochemically generate OH-. The local increase in pH severely lowered the activity of the enzyme beneath the tip and significantly decreased the flux of H2O2 from this region of the sample. This can clearly be seen as a bright circular spot in the upper right corner of the image. With a conventional microdisk, the SECM image is not reliable because the tip current for H2O2 oxidation decays while the image is recorded and it is very difficult to link the tip response to the enzyme activity. With a mesoporous microdisk, the tip current was found to be highly sensitive to small variations in H2O2 concentration and the activity of submonolayer enzyme coverage was detected. CONCLUSION Thanks to a unique combination of quasi-hemispherical diffusion and large electroactive area, mesoporous microdisk electrodes significantly improve the amperometric detection of hy(27) (a) EM Science, EM Industries, Inc., Gibbstown, NJ. (b) Lab Safety Supply, Janesville, WI. (c) Scientific Products, Baxter Diagnostics, McGaw Park, IL. (d) CHEMetrics Inc., Calverton, VA. (e) Hach Co., Loveland, CO. (f) LaMotte, Inc., Chestertown, MD. (28) (a) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. Electroanal. Chem. 1994, 18, 243, (b) Bard, A. J.; Mirkin, M. V. Scanning Electrochemical Microscopy; Marcel Dekker: New York, 2001. (29) O’Brien, J. C.; Shumaker-Parry, J.; Engstrom, R. C. Anal. Chem. 1998, 70, 1307-1311.
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Figure 6. Scanning electrochemical microscopy image showing the variation in enzyme activity as a 25-µm-diameter mesoporous platinum SECM tip (held at +0.6 V vs SCE) was scanned above a glucose oxidase monolayer immobilized with an electropolymerized polyphenol film. Modification of the GOx activity was accomplished by electrochemically generating hydroxide at the SECM tip (Etip ) -1.8 V vs SCE for 18 min) and resulted in the region of lower current in the upper right-hand corner of the image. The solution was a pH 7 phosphate-buffered, 9.9 mM glucose solution at 20 °C, and the image was recorded with a scan rate of 7.5 µm s-1 and a tipsubstrate separation of
