Anal. Chem. 1996, 68, 3344-3349
Hydrogen Peroxide Sensor Based on Coimmobilized Methylene Green and Horseradish Peroxidase in the Same Montmorillonite-Modified Bovine Serum Albumin-Glutaraldehyde Matrix on a Glassy Carbon Electrode Surface Chenghong Lei and Jiaqi Deng*
Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
A new approach to construct a second-generation amperometric biosensor is described. The classical dye methylene green as a probing-needle mediator and horseradish peroxidase as a base enzyme were coimmobilized in the same montmorillonite-modified bovine serum albumin (BSA)-glutaraldehyde matrix to construct a H2O2 sensor. The immobilization matrix was formed from the pretreated sodium montmorillonite colloid in which the enzyme and the cross-linker were dissolved. Immobilization of methylene green from the dye mother solution was attributed to the adsorption function of the montmorillonite, whereas immobilization of horseradish peroxidase was attributed to the cross-linking function of the BSA-glutaraldehyde as usual. Cyclic voltammetry and potentiostatic measurements indicated that methylene green efficiently mediated electrons from the base electrode to the enzyme in the matrix. The sensor responded rapidly to low H2O2 concentration and achieved 95% of the steady-state current in less than 20 s, with a detection limit of 4.0 × 10-7 M H2O2. Determination of hydrogen peroxide is of great importance, ascribable to both the facts that H2O2 is the product of the reactions catalyzed by a large number of highly selective oxidase enzymes and that H2O2 is essential in food, pharmaceutical, and environmental analyses.1-5 The sensors based on the mediatorless activation of electroreduction of H2O2 in the presence of peroxidase (POD) at carbon black,6 spectrographic graphite,7 nonplatinized activated carbon electrodes,8 pyrolytic graphite,9 polypyrrole,9 or carbon paste10 can be fabricated, but, in general, they
exhibit a lower sensitivity than those coupling peroxidase with a mediator.11-15 Such a mediated H2O2 sensor could hold a detection limit as low as 10-8-10-7 M by use of an electron transfer mediator such as [Ru(NH3)5py]2+,12 ferrocene derivatives,12,13 tetrathiafulvalene,14 or phenazine methosulfate.15 As far as mediated H2O2 sensors are concerned, many different solution-phase mediators could be used. These mediators have been used in solution in many cases. However, to fabricate an amperometric sensor for multiple use, it would be preferable to immobilize a soluble mediator or an insoluble one (often gradually leaching into the solution phase as reported14) on the electrode surface. If properly designed, such electrodes could provide a mediated or electrocatalytic response toward an inert redox species at the proper redox potential of the mediator, without requiring the actual addition of the mediator to the sample solution. Water-soluble dyes, such as methylene blue,16 methylene green,17 meldola blue,18 phenazine methosulphate,19 and other dye derivatives,20-23 can be attached to graphite electrode surfaces as electron transfer mediators to catalyze the redox reactions of biomolecules or to shuttle the electrons between the electrodes and some redox enzymes. However, their electroactivities decreased rather rapidly, and these dye-modified electrodes were seldom combined with peroxidase to construct amperometric biosensors previously. Sodium montmorillonite colloid, as a modifying material on the electrode, has the advantages of high chemical stability, good conductivity and penetrability attributed to its appreciable surface area, special structural features, and unusual intercalation proper-
(1) Kulys, J. J.; Pesliakiene, M. V.; Samalius, A. S. Bioelectrochem. Bioenerg. 1981, 8, 81-88. (2) Tatsuma,T.; Watanabe, T. Anal. Chim. Acta 1991, 242, 85-89. (3) Wang, J.; Lin, Y.; Chen, L. Analyst 1993, 118, 277-280. (4) Dunford, H. B.; Stillman, J. S. Coord. Chem. Rev. 1976, 19, 187-251. (5) Maehly, A. C. Methods Enzymol. 1955, 2, 801. (6) Yaropolov, A. L.; Malovik, V.; Varfolomeev, S. D.; Berrzin, I. V. Dokl. Akad. Nauk SSSR 1979, 249, 1399. (7) Jonsson, G.; Gorton, L. Electroanalysis 1989, 1, 465. (8) Ho, W. O.; Athey, D.; McNeil, C. J.; Hager, H. J.; Evans, G. P.; Mullen, W. H. J. Electroanal. Chem. 1993, 351, 185-197. (9) Wollenberger, U.; Bogdanovskaya, V.; Bobrin, S.; Scheller, F.; Tarasevich, S. M. Anal. Lett. 1990, 23, 1795-1808. (10) Wollenberger, U.; Wang, J.; Ozsoz, M.; Gonzalez-Romero, E.; Scheller, F. Bioelectrochem. Bioenerg. 1991, 26, 287-296.
(11) Chen, L.; Lin, M.; Hara, M.; Rechnitz, G. A. Anal. Lett. 1991, 24, 1-14. (12) Frew, J. E.; Harmer, M. A.; Hill, H. Allen O.; Libor, S. I. J. Electroanal. Chem. 1986, 201, 1-10. (13) Tatsuma, T.; Okawa, Y.; Watanabe, T. Anal. Chem. 1989, 61, 2352-2355. (14) Bifulco, L.; Cammaroto, C.; Newman, J. D.; Turner, A. P. F. Anal. Lett. 1994, 27, 1443-1452. (15) Qian, J.; Liu, Y.; Liu, H.; Yu, T.; Deng, J. J. Electroanal. Chem. 1995, 397, 157-162. (16) Ye, J.; Baldwin, R. P. Anal. Chem. 1988, 60, 2263-2268. (17) Chi, Q.; Dong, S. Anal. Chim. Acta 1994, 285, 125-133. (18) Gorton, L.; Torstensson, A.; Jaegfeldt, H.; Johansson, G. J. Electroanal. Chem. 1984, 161, 103-120. (19) Torstensson, A.; Gorton, L. J. Electroanal. Chem. 1981, 130, 199-207. (20) Huck, H. Fresenius Z. Anal. Chem. 1982, 313, 548-552. (21) Persson, B. J. Electroanal. Chem. 1990, 287, 61-80. (22) Persson, B.; Gorton, L. J. Electroanal. Chem. 1990, 292, 115-138. (23) Polasek, M.; Gorton, L.; Appelqvist, R.; Marko-Varga, G.; Johansson, G. Anal. Chim. Acta 1991, 246, 283-292.
3344 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
S0003-2700(96)00291-0 CCC: $12.00
© 1996 American Chemical Society
ties. In 1983, Bard and Ghosh reported the stable electrochemical behavior of several substances, such as Ru(NH3)62+, MV2+, Fe(bpy)32+, and TMA Fe(Cp)2+, incorporated in such a montmorillonite-modified membrane on a SnO2 electrode surface.24 Further research into the electrochemical behavior of other electroactive species incorporated in similar clay-modified membranes on SnO2 electrode surfaces, such as thionine,25 Cr(bipy)33+, and Co(bipy)33+,26,27 has been done. However, to our knowledge, the use of montmorillonite as one kind of material to immobilize the electron transfer mediator for enzyme electrodes has not been previously described. In this work, for the first time, the soluble mediator methylene green was successfully incorporated in the cross-linked montmorillonite-horseradish peroxidase (POD) hydrogel on a glassy carbon electrode surface using bovine serum albumin (BSA)glutaraldehyde immobilization matrix. The results indicated that methylene green molecules incorporated in this way stably and efficiently shuttled electrons on the wire of the H2O2-POD electrode. EXPERIMENTAL SECTION Reagents. Peroxidase from horseradish (POD, EC 1.11.1.7, type VI) was obtained from Sigma; methylene green (MG, not purified before use) was purchased from Fluka. The 25% glutaraldehyde was obtained from Merck. Bovine serum albumin (BSA), polyvinyl alcohol (PVA, average degree of polymerization, 1800 ( 100), chloroplatinic acid, collodion, and hydrogen peroxide (30% w/v solution) were purchased from Shanghai Chemical Reagent Co. (Shanghai, P. R. China). The concentrations of more diluted hydrogen peroxide solutions were determined by titration with cerium(IV) to a ferroin end point.28 All other chemicals were analytical grade reagents. All the solutions were prepared with doubly distilled water. Preparation of Sodium Montmorillonite Colloid. The mineral montmorillonite powder was obtained from Zhejiang Province, P. R. China. Twenty grams of the powder was added in 60 mL of 30% H2O2 solution and heated on a 60 °C water bath to oxidize some organic impurities. Next, about 10 mL of 0.1 M HCl was added to eliminate the carbonate compounds until no gas bubble was produced. The distilled water (200 mL) was then replenished. The obtained suspension was repeatedly decanted several times to remove some feldspar and other impurities, and 20 g of NaCl was dissolved in it. The system was stirred for 24 h at room temperature. After this, the upper transparent liquid layer was poured out. The deposit was stirred in 200 mL of 10% NaCl aqueous solution for 24 h again and then was transferred into a semipermeable packet (made from collodion by this laboratory). The packet was immersed in water to get rid of inorganic salts. The purified, type-changed montmorillonite was redispersed into 100 mL of water and centrifuged at 8000 rpm for 0.5 h. Thus, sodium montmorillonite colloid (SMC), whose content was about 2 g/L, was obtained. Then, 100 µL of such a colloid was mixed with 10 µL of colloid Pt liquid and 5 µL of 3% PVA aqueous solution.24,29 Such a pretreated sodium montmoril(24) Ghosh, P. K.; Bard, A. J. J. Am. Chem. Soc. 1983, 105, 5691-5693. (25) Kamat, P. V. J. Electroanal. Chem. 1984, 163, 389-394. (26) Fitch, A.; Lee, S. A. J. Electroanal. Chem. 1993, 344, 45-59. (27) Fitch, A.; Krzysik, R. J. J. Electroanal. Chem. 1995, 379, 129-134. (28) Hurdis, E. C.; Romeyn, H., Jr. Anal. Chem. 1954, 26, 320. (29) Hirai, H.; Nakao, Y.; Toshima, N. J. Macromol. Sci.-Chem. 1979, A13, 727750.
lonite colloid (PSMC) was used throughout this paper. One of its advantages was that water-soluble enzyme could be dissolved in it. Construction of H2O2 Sensor. A glassy carbon electrode (GCE, 3 mm in diameter, First Carbon Factory, Shanghai, P. R. China) was polished to a mirror-like finish with fine emery papers and rinsed thoroughly in deionized water between each polishing step. Next, the polished electrode was sonicated in 1:1 nitric acid, acetone, and doubly distilled water consecutively and dried in air before use. Aliquots (4 µL) of PSMC, or aliquots (6 µL) of the liquid mixture formed from 2.5 mg of POD, 4 mg of BSA, 50 µL of PSMC, and 5 µL of 5% glutaraldehyde aqueous solution, were deposited on such a pretreated GCE surface and allowed to dry under ambient conditions for 3 h. Thus, the montmorillonitemodified membrane (MM) or the montmorillonite-modified enzyme immobilization matrix (MEM) on the GCE surface was obtained. The modified electrode was immersed into 1 × 10-4 M MG aqueous solution for 2 h; in this period, the dye was automatically adsorbed and incorporated in MM or MEM. Then, the immersed electrode was rinsed with doubly distilled water and successively immersed into the blank 0.1 M phosphate buffer (pH 6.5) to get rid of the non-firmly adsorbed dye molecules two or three times until the electrochemical response given by the dye molecules was stable. Thus, the MG-incorporated montmorillonite-modified membrane electrode (MG/MME) and the H2O2 sensor based on MEM were obtained. MG/MME and the H2O2 sensor were kept in 0.1 M phosphate buffer (pH 7.0) at 4 °C in a refrigerator when not in use. Apparatus. Cyclic voltammetry and amperometric measurement were done with a three-electrode system comprising the H2O2 sensor (MG/MME or GCE) as a working electrode, a saturated calomel reference electrode, and a platinum wire auxiliary electrode. The electrodes were connected to a FDH 3205 cyclic voltammetry apparatus (Scientific Equipment Co., Fudan University, Shanghai, China) in line with a type 3086 x-y recorder (Yokogawa Hokushin Electric, Tokyo, Japan). All electrochemical experiments were carried out in 5 mL of 0.1 M phosphate buffer (pH 6.5, apart from the pH parameter experiments) containing 0.1 M Na2SO4 in a thermostated and stirred electrochemical cell at 30.0 ( 0.2 °C. All experimental solutions were thoroughly deoxygenated by bubbling nitrogen through the solution for at least 10 min. In the potentiostatic experiments, the current-time data were recorded after a constant residual current had been established and successive additions of stock H2O2 solution in the buffer were done. The sensor response was measured as the difference between total and residual current. UV spectra were done on a UV-240 spectrometer (Shimadzu, Tokyo, Japan) at room temperature. RESULTS AND DISCUSSION Visible Absorption Spectrum of MG Dissolved in Sodium Montmorillonite Colloid. It is known that sodium montmorillonite colloidal particles (SMCP) have an appreciable surface area, special structural features, unusual intercalation, and cation exchange properties.24-27 Water-soluble dye MG under the present research conditions was the oxidized form and held the positive charge. Due to electrostatic forces and adsorption properties of SMCP, MG would strongly interact with SMCP, resulting in changes of the electron states of MG and immobilization of the dye. Its absorption peaks would obviously move in Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
3345
larger ∆Ep values (the differences between the reduction and the oxidation peak potentials) resulting from the interaction of MG with SMCP. These values produced by MG/MME were less than 50 mV, indicating that there were fast charge transfers through the membrane as well as charge transfers from the membrane to the electrode. The linear relationship of peak current to the square root of scan rate indicated that the electrode reaction initiated by MG in the working buffer was mainly controlled by diffusion of MG in the solution, whereas that initiated by MG incorporated in MM was under the control of transport of protons in the solution since the electrode reaction was accompanied by protonation and no redox reactant transport occurred (see the following results and discussion).17,24,25 The formal potential E°′, estimated from the midpoint of the anodic and cathodic peaks of MG/MME versus pH, gave a straight line from pH 1.0 to 5.4, with a slope of approximately 60 mV/pH unit. Another straight line with a slope of approximately 30/pH unit was obtained from pH 5.4 to 9.0 (Figure 3). Such a pH dependence was in accordance with the following reaction scheme:17,18 Figure 1. Visible absorption spectrum of 2 × 10-5 M MG dissolved in water (a) and in 0.8 g/L of SMC (b).
the spectrum. Figure 1 shows the difference of the visible absorption spectrum of MG dissolved in water from that of MG dissolved in SMC. The absorption peaks of MG observed at 662 and 609 nm were shifted to 760, 665, and 578 nm on addition of SMC to the dye aqueous solution. The addition of SMC also changed the blue-green solution to a deep blue one. Although the nature of the interaction of the dye with SMCP under the present conditions has not fully been investigated yet, this result indicated that such an interaction had altered the photochemical and photophysical properties of the dye. Electrochemical Characteristics of MG Incorporated in Montmorillonite-Modified Membrane on GCE Surface. Typical cyclic voltammograms of 1 × 10-4 M MG in 0.1 mM phosphate buffer at GCE and those of MG/MME in the blank buffer at different scan rates are shown in Figure 2. Under the experimental conditions, two rather different quasi-reversible waves were obtained. The latter displayed more negative peak potentials but
pH > 5.4
MG+ + H+ + 2e f MGH
pH < 5.4
MG+ + 2H+ + 2e f MGH+
A pKa value of 5.4 for adsorbed MG in MM was indicated by the intersection of the lines in E°′ versus pH plot in Figure 3. The literature pKa value of MG incorporated into carbon paste was 6.5.17 The difference should be ascribed to the intensive interaction of MG with montmorillonite. The violent blue color in MM firmly attached to the surface of the electrode indicated that MG was incorporated and enriched in it. The electrochemical response of MG/MME obviously did not decrease after 3 weeks in storage. The stable electroactivity of the dye demonstrated that MG was immobilized in MM from its mother solution. In this work, both colloidal Pt (containing PVA) and additional PVA were found to be critical for the modified electrode to obtain a stronger and more uniform membrane and an enhanced and stable electrochemical activity as the reference reported.24 Due to the different adsorption sites ascribed to the
Figure 2. (a) Cyclic voltammograms of 1 × 10-4 M MG dissolved in 0.1 M phosphate buffer (pH 6.5). (b) Cyclic voltammograms of MG incorporated in MM in 0.1 M phosphate buffer (pH 6.5) at different scan rates (from inner to outer): 5, 15, 35, 55, 85, and 125 mV/s. 3346
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
Figure 3. Formal potentials for adsorbed MG as a function of pH.
special structure and cation exchange function of montmorillonite, and in agreement with previous work,24,25 only a fraction of the incorporated dye was found to be electroactive in MM. A successful electron transfer mediator immobilized at the sensor must meet several demands: (1) firm immobilization, (2) stable electroactivity, (3) reversible or quasi-reversible electrode reaction, and (4) proper peak potentials. Hence, MG immobilized in MM could be a good electron shuttle between the redox enzyme and the base electrode. The fine response of the H2O2 sensor based on coimmobilization of MG with POD proved this. Amperometric Biosensing of H2O2 through the H2O2 Sensor. In the preliminary experiment for the sensor construction approach, we found that the cross-linked POD layer coated on MG/MME was easily peeled off, and such a probe displayed poor response to H2O2. Fortunately, the water-soluble enzyme could easily be dissolved in PSMC used in this paper. Thus, the mediator and the enzyme were coimmobilized in the same immobilization matrix through dipping PSMC containing the enzyme and cross-linker onto the polished electrode surface. Here, the mediator MG was automatically adsorbed and incorporated in MEM from its mother solution. Accordingly, the H2O2 sensor based on MEM was obtained (as described in the Experimental Section). Indeed, coimmobilization of MG and POD in the same matrix did not lead to enzyme layer dropping because the coimmobilization layer could be directly and firmly attached to the polished electrode surface due to the atmospheric pressure and/or the strong bonding force between the layer and such a pretreated surface. Also, a more sensitive response of the sensor to H2O2 was obtained because MG incorporated in this way could disperse within all over MEM, so that there were more mediator molecules communicating with the enzyme active center. In other words, the mediator molecules could more efficiently mediate the electrons from the base electrode to the enzyme than those where the mediator layer was sandwiched between the enzyme layer and the electrode (the cross-linked POD layer was coated on MG/ MME). In the blank buffer solution, the H2O2 sensor (in the presence of POD) showed stable electrochemical behavior similar to that of MG/MME (in the absence of POD) but exhibited more positive peak potentials (Figures 2b and 4). The detailed mechanism for this shift relative to the immobilized enzyme has not been clear. The small ∆Ep values demonstrated that the sensor displayed the characteristics of a kinetically fast redox couple attached to the electrode surface. In the absence of H2O2, the enzyme and other
Figure 4. Cyclic voltammograms of the H2O2 sensor at a scan rate of 25mV/s in the absence of H2O2 (a) and in the presence of 1.0 mM H2O2 (b).
substances did not contribute to the response, and only the immobilized MG in MEM showed the electrochemical behavior under the control of diffusion since a linear plot of peak current vs square root of scan rate (ip/V1/2) was observed.24,25 In fact, since the protons had to be exchanged with the solution for the electrode reaction (MG+ + H+ + 2e f MGH, at pH 6.5) and no immobilized dye could be transported, it might be the proton transport that led to all the “diffusion-limiting” electrode reactions at the electrode surface.17,24,25 In addition, at scan rates beyond 100 mV/s, this linear relationship did not continue, indicating that the electrode reaction had been hysteretic. With the addition of H2O2, the obvious catalytic reaction appeared, accompanied by a dramatic increase of the reduction current and an almost complete disappearance of the oxidation current in the meantime. These phenomena demonstrated that MG incorporated into MEM could effectively shuttle electrons from the base electrode to the enzyme in the coimmobilization matrix. A typical response of the H2O2 sensor to H2O2 with a scanned potential between -0.40 and +0.10 V is shown in Figure 4. At first, POD did reduce the H2O2 diffusing from the solution:
H2O2 + POD(red) f H2O + POD(ox) Then, the oxidized POD oxidized methylene green (MGH) to MG+:
POD(ox) + MGH f POD(red) + MG+ This overall rereduction reaction of POD (ox) included two separate steps:4,5,30,31
POD(I) + MGH f POD(II) + MG•
(1)
POD(II) + MG• f POD(red) + MG+
(2)
Where one electron was donated at a time, POD(I) was POD(ox) and MG• represented the free radical formed during the (30) Durliat, H.; Courteix, A.; Comtat, M. Bioelectrochem. Bioenerg. 1989, 22, 197-209. (31) Danner, D. J.; Brignac, P. J., Jr.; Arceneaux, D.; Patel, V. Arch. Biochem. Biophys. 1973, 156, 759.
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
3347
Figure 5. Cyclic voltammograms of the H2O2 sensor in the presence of 0.6 mM H2O2 at several scan rates: (a) 15, (b) 55, and (c) 125 mV/s.
Figure 6. Effect of pH at 30 °C when [H2O2] ) 0.8 mM in 0.1 M Na2HPO4-KH2PO4 buffer solution.
reaction. Then, oxidized MG was reduced to MGH at the sensor, resulting in the cathodic catalytic current:
MG+ + H+ + 2e f MGH The disappearance of oxidation peaks indicated that the oxidation rate of MGH by the oxidized POD was rather fast. However, when the scan rate increased to some extent, the remaining MGH could still be directly oxidized at the sensor, and the oxidation peak rose again (Figure 5). This indicated that the oxidation rate of MGH by the oxidized POD was also rather definite. All these phenomena further proved the overall mechanism process described above. The effect of pH on the H2O2 sensor response lay in two main aspects: one was that pH affected the activity of POD, and the other was that pH affected the peak potentials of MG. Therefore, both the oxidation and reduction reactions of the POD catalyzed cycle were influenced by the pH of the working buffer. An optimum pH between 6.0 and 7.5 was considered for both of these aspects. The effect of pH was studied at different pH values in 0.1 M Na2HPO4-KH2PO4 buffer at 30 °C (Figure 6). It can be seen that the optimal response was obtained at pH 6.5. This result indicated that the rereduction of the oxidized POD (the reduction reaction) by MGH and the following electrode reaction, MG+ + H+ + 2e f MGH, occurred at an optimum pH of 6.5, which was 3348 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
Figure 7. Potential dependence of the steady-state current when [H2O2] ) 0.8 mM.
critical for the proper peak potentials of MG. This pH value also guaranteed the high activity of POD for both the oxidation and reduction reactions of the catalyzed cycle. Hence, the buffer solution of pH 6.5, 0.1 M Na2HPO4-KH2PO4 was used throughout this paper. The response signal of the H2O2 sensor increased as the temperature varied from 15 to 45 °C. But at temperatures lower than 20 °C, the activity of the enzyme in MEM was rather lower and the response time was relatively longer. On the other hand, at temperatures higher than 40 °C, the activity of the enzyme would decrease rapidly, and the lifetime of the sensor would be shortened. Taking both the lifetime and the response time into consideration, 30 °C was the selected temperature for this work. The potential dependence of the steady-state current is shown in Figure 7. Electroreduction of H2O2 was observed already at approximately -0.10 V, and the steady-state current slowly increased, with the applied potential decreasing from -0.19 to -0.27 V. However, it is preferable to control the lower working potential to avoid or decrease the interference caused by some electroactive species at the sensor. As a matter of fact, at the lower working potential of -0.21 V, molecular oxygen could be still reduced. Hence, the deaerated solutions were used for this work. At this applied potential, the calibration plot of the sensor was linear from 2 µM to 3 mM. The sensor achieved 95% of the steady-state current within 3-20 s. At a signal-to-noise ratio of 3, the detection limit of H2O2 was found to be 4.0 × 10-7 M. The recoveries of six H2O2 samples with concentrations of 1.0 × 10-51.0 × 10-3 M were determined by a calibration curve method. The average recoveries of the H2O2 sensor were in the range 96.8-103%. The prepared H2O2 sensor was stored in the 0.1 M phosphate buffer (pH 7.0) in a refrigerator when not in use. During 2 weeks, the decrease of the response sensitivity was less than 5%. In the following days, the sensor response decreased somewhat faster. After 40 days, the sensor maintained only 60% of the response values at the beginning of the experimental period. As for MG/ MME, the H2O2 sensor had a stable electrochemical response of the mediator MG up to 3 weeks under proper storage conditions. Owing to the light sensitivity and the chemical instability of dyes, most dye-modified electrodes (mainly graphite electrodes) showed stable electroactivity over a very short period of time. The stability of the cation dye incorporated in MM or MEM should be attributed to the specific binding and immobilization properties of SMCP resulting from its cation exchange ability and special structure.
CONCLUSIONS That the spectral and electrochemical characteristics of MG adsorbed by SMCP differred from those of MG in the solution demonstrated the intensive interaction of MG molecules with this kind of montmorillonite colloidal particles. It was this interaction that led to the binding and immobilization of MG as the electron transfer mediator. That MG could stably and efficiently shuttle electrons from the base electrode to the oxidized peroxidase was attributed to the electrochemical stability of MG incorporated in MM or MEM. This novel and efficient strategy, that is, successful coimmobilization of methylene green as a probing-needle mediator with peroxidase as a base enzyme for the construction of the H2O2 sensor, opens up a new approach to construct amperometric
biosensors, owing to the multiformity of available solution-phase mediators and redox enzymes. ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Science Foundation of China and the Electroanalytical Chemistry Open Laboratory of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Received for review March 25, 1996. Accepted July 3, 1996.X AC960291N X
Abstract published in Advance ACS Abstracts, August 15, 1996.
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
3349
