Polypyrrole-Based Optical Probe for a Hydrogen Peroxide Assay

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Anal. Chem. 2007, 79, 240-245

Polypyrrole-Based Optical Probe for a Hydrogen Peroxide Assay Haiyan Wang† and Su-Moon Park*

Department of Chemistry and Center for Integrated Molecular Systems, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang, Gyeongbuk 790-784, Korea (ROK)

An optical sensing probe has been developed by taking advantage of the polypyrrole (PPy) chromophore. The absorbance of the oxidation product of pyrrole, i.e., solubilized PPy colloids, is shown to be directly proportional to the concentration of hydrogen peroxide, when H2O2 is used as an oxidant for pyrrole in the presence of a surfactant, sodium dodecyl sulfate, and Fe(II) in a slightly acidic aqueous solution. Based on this result, a new optical sensing method has been developed for the determination of H2O2. The probe has also been applied to optical sensing of ethanol by biocatalyzed generation of H2O2 in the presence of O2, ethanol, and alcohol oxidase. The novel methodology is expected to provide a general protocol for the determination of H2O2 as well as for numerous other oxidase-based reactions producing H2O2 as a product. Hydrogen peroxide is an important intermediate species in many biological and environmental processes. It has been shown to be present in the atmospheric and hydrospheric environments.1,2 It is widely used in industry for bleaching and cleaning, as well as disinfection, and is released to the environment in large quantities. In addition, H2O2 is a major reactive oxygen species in living organisms and plays an important role as a second messenger in cellular signal transduction. Oxidative damages resulting from the cellular imbalance of H2O2 and other reactive oxygen species are connected to aging and severe human diseases such as cancers and cardiovascular disorders.3,4 Furthermore, H2O2 is one of the products of reactions mediated by almost all oxidases.5 Therefore, molecular probes for H2O2 are of broad interest to the research community in environmental sciences and biochemistry as well as in clinical assays and screening.6 The determination of H2O2 has been conducted by electrochemical * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +82-54-279-2102. Fax: +82-54-279-3399. † Present address: College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, P.R. China. (1) Price, D.; Worsfold, P. J.; Mantoura, R. F. C.; Fauzi, C. Trends Anal. Chem. 1992, 11, 379. (2) Anglada, J. M.; Aplincourt, Ph.; Bofill, J. M.; Cremer, D. ChemPhysChem 2002, 3, 215. (3) Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2004, 126, 15392-. (4) Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2005, 127, 16652. (5) Barman, T. E., Ed. The Enzyme Handbook; Springer: Berlin, 1974. (6) Wolfbeis, O. S.; Durkop, A.; Wu, M.; Lin, Z. H. Angew. Chem., Int. Ed. 2002, 41, 4495.

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reduction using a Clark7 as well as other modified electrodes,8 or by measuring the intensities of chemiluminescence,9 electrochemiluminescence,10 luminescence of a metal ion,6 etc., resulting from the excitation of luminescers such as luminol or lanthanide ions via their reactions with H2O2. The optical detection of biological processes using metal and semiconductor nanoparticles (NPs) has received a great deal of attention. Semiconductor quantum dots have been widely used as fluorescence labels in sensing biological molecules.11 The surface plasmon absorbance of metal NPs including gold NPs, particularly the interparticle-coupled plasmon absorbance of aggregated NPs, has been extensively employed to follow molecular and biomolecular recognition processes such as DNA hybridization or antigen-antibody complex formation.12 Recently, Willner et al.13,14 have reported on a biosensing protocol, which relies on the catalytic growth of gold NPs by reducing AuCl4- with the enzymatically liberated H2O2 or NADH and subsequent optical detection of the corresponding plasmon absorbance. Conducting polymers such as polyaniline and polypyrrole (PPy) have attracted considerable attention for a variety of reasons.15-18 Due to their unique electrical, optical, and chemical properties, they have found a variety of applications such as corrosion inhibitors, batteries, organic electronics, electrochromic devices, and sensors.15 Spatially extended π-bonding systems in conjugated polymers give rise to electronic transitions in the UVvisible region, particularly in doped states.15,17 Just like the plasmon absorbance of NPs, the optical properties of the conjugated polymers may find potential applications in various chemical analyses. In fact, the color change of the polyaniline film due to (7) See, for example, Enke, C. G. The Art and Science of Chemical Analysis; John Wiley & Sons, Inc.: New York, 2001. (8) Miah, Md. R.; Ohsaka, T. Anal. Chem. 2006, 78, 1200. (9) DeLuca, M. A.; McElroy, W. D. Bioluminescence and Chemiluminescence; Academic Press: New York, 1981. (10) Fa¨hnrich, K. A.; Prawda, M.; Guilbault, G. G. Talanta 2001, 54, 531. (11) Gao, X. H.; Nie, S. M. Trends Biotechnol. 2003, 21, 371. (12) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849. (13) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519. (14) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21. (15) Nalwa, H. S., Ed. Handbook of Organic Conductive Molecules and Polymers; John Wiley & Sons Ltd.: West Sussex, England, 1997; Vol. 3. (16) (a) Park, S.-M. in ref 15. (b) Park, S.-M.; Lee, H.-J. Bull. Korean Chem. Soc. 2005, 26, 697. (c) Malinauskas, A. Polymer 2001, 42, 3957. (17) (a) Stilwell, D. E.; Park, S.-M. J. Electrochem. Soc. 1989, 136, 427. (b) Shim, Y.-B.; Stilwell, D. E.; Park, S.-M. Electroanalysis 1991, 3, 31. (c) Hoier, S. N.; Park, S.-M. J. Phys. Chem., 1992, 96, 5188. (d) Hong, S.-Y.; Jung, Y. M.; Kim, S. B.; Park, S.-M. J. Phys. Chem. B 2005, 109, 3844. (18) Liu, Y.-C.; Chuang, T. C. J. Phys. Chem. B 2003, 107 (45), 12383. 10.1021/ac0608319 CCC: $37.00

© 2007 American Chemical Society Published on Web 11/23/2006

the presence of oxygen has been taken advantage of for use as an oxygen sensor.17b In the present paper, we take advantage of the fact that an analyte, H2O2, can be used as an active oxidant for the generation of PPy in the presence of Fe(II) and demonstrate that the optical properties of properly stabilized and solubilized PPy colloids correlate well with the concentration of H2O2 under appropriate experimental conditions. Our strategy for the optical detection of H2O2 relies on Fe(II)-mediated generation of PPy colloids by oxidation with H2O2. As an example of the application of the methodology, we also report an optical biosensing system for ethanol based on the biocatalyzed generation of H2O2 in the presence of O2, ethanol, and alcohol oxidase; the reaction provides a protocol for studies of numerous other oxidase-based biosensing systems. EXPERIMENTAL SECTION Pyrrole (Aldrich) and sodium dodecyl sulfate (SDS; Fluka) were used as received. Hydrogen peroxide and alcohol oxidase (AOx; EC 1.1.3.13, from Pichia pastoris) were obtained from Sigma. UV-visible spectra were obtained with an HP-8453 UVvisible spectrophotometer. Chromatographic analysis was performed using a Shimadzu 14B gas chromatograph. The solution for the polymerization of pyrrole was optimized to have 0.010 M pyrrole, 0.020 M SDS, 1.0 × 10-5 M FeCl2, and 1.0 × 10-3 M HCl, along with various concentrations of H2O2, different concentrations of ethanol, or both in the presence of 0.1 unit/mL AOx. The absorbance spectra of all solutions were recorded after a fixed reaction time of 15 min. For measurements of ethanol contents in a local kaoliang liquor, wine, and beer, the samples were diluted properly before they were subjected to the chemical analysis. RESULTS AND DISCUSSION Determination of H2O2. H2O2 is a powerful oxidizing agent and can be applied to oxidation of pyrrole, aniline, and thiophene to their corresponding oligomers/polymers with its reduction product of only H2O, simplifying the postreaction treatment.13,14 The reaction between H2O2 and pyrrole (Py) can be expressed by the following reaction in the presence of Fe(II),

nPy + 2nxH2O2 + 4nxH+ f f f PPy + 4nxH2O (1)

with Fe(II) acting as a catalyst or mediator (vide infra). Here x represents the fraction of PPy doped, 0.25-0.3, meaning that about one of three or four pyrrole molecules in the PPy chain are in an oxidized state.15 After adding H2O2 to an aqueous solution containing appropriate amounts of pyrrole, SDS as a surfactant, FeCl2 as a catalyst, and HCl, two characteristic absorption bands located at around 466 and 900 nm are observed. The band at 466 nm has been assigned to the π-π* transition and the one at 900 nm to the doping-induced band of PPy, respectively,18 indicating that PPy has indeed been formed. At the same time, the solution changed from colorless to green after 15 min of reaction. Figure 1 displays the evolution of the absorbance spectra recorded from the reaction mixture in the presence of various concentrations of H2O2 (a) and the corresponding calibration curve for the absorbance changes

Figure 1. (a) Absorbance spectra recorded from solutions containing optimized concentrations of reactants, i.e., 0.010 M pyrrole, 0.020 M SDS, 1.0 × 10-3 M HCl, and 1.0 × 10-5 M FeCl2, upon reaction for 15 min with different concentrations of H2O2: (1) 0, (2) 1.0 × 10-5, (3) 4.0 × 10-5, (4) 1.0 × 10-4, (5) 2.0 × 10-4, (6) 4.0 × 10-4, and (7) 6.0 × 10-4 M, respectively. (b) Calibration curve for the absorbance at λ ) 466 nm versus H2O2 concentration.

at λ ) 466 nm versus the concentration of H2O2 (b). As the H2O2 concentration increases, the absorbance increases as well. The absorbance signal increases linearly with the H2O2 concentration ranging from 1.0 × 10-5 to 1.2 × 10-3 M (r2 ) 0.996), with a detection limit of 1.0 µM. A blank experiment (shown in Figure 1a, curve 1) reveals that no absorbance is detected from PPy without H2O2, indicating that H2O2 is essential to the observation of the polymerization product of pyrrole. This set of experiments was carried out after the optimization experiments decscribed below had been conducted. Effect of SDS. Figure 2 shows the effect of the SDS concentration on the absorbance. Figure 2a shows that the absorbance, i.e., the amount of oligomer or polymer products, depends on the SDS concentration at a fixed pyrrole concentration. Figure 2b shows that the absorbance increases dramatically at both 466 and 900 nm as the SDS concentration increases up to 0.020 M. A further increase in the SDS concentration accompanied an increase in the absorbance at 466 nm but a slight decrease in the 900-nm band, suggesting that PPy may not be fully doped at higher SDS concentrations. Therefore, 0.020 M SDS was used Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

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Figure 2. (a) Absorbance spectra obtained from solutions containing 0.010 M pyrrole, 0.10 mM H2O2, 1.0 × 10-3 M HCl, and 1.0 × 10-5 M FeCl2 at different concentrations of SDS: (1) 0, (2) 0.0050, (3) 0.020, and (4) 0.050 M. (b) Plot of absorbance at λ ) 466 nm (9) and λ ) 900 nm (b) vs the SDS concentration.

for the subsequent experiments. Naoi et al.19 reported a similar effect of SDS on the electropolymerization reaction of pyrrole. Since the critical micelle concentration (cmc) of SDS is ∼8 mM in aqueous solutions,19 the increase in the absorbance above the cmc can be explained by increased micellar catalysis20 and also by an increase in the solubility, as well as the stability, of pyrrole oligomer or polymer colloids via inclusion inside the micelles. In aqueous solutions of surfactants, where a large number of micelles exist above their cmc, the pyrrole molecules are preferentially dissolved and concentrated inside the micelles because of their hydrophobic nature. From such a high monomer concentration in micelles, an efficient supply of monomers and dopants would occur during the oxidative polymerization process, which results in an increased rate of polymerization as polymerization proceeds.19 In addition, we note that the shape of the absorption band at 466 nm became sharper when the SDS concentration increased up to 0.020 M, suggesting less effective doping at high SDS (19) Naoi, K.; Oura, Y.; Maeda, M.; Nakamura, S. J. Electrochem. Soc. 1995, 142, 417. (20) Krivan, E.; Peintler, G.; Visy, C. Electrochim. Acta 2005, 50, 1529.

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Figure 3. (a) Absorbance spectra obtained from solutions containing 0.010 M pyrrole, 0.10 mM H2O2, and 1.0 × 10-5 M FeCl2 at different pH: (1) 7.0, (2) 6.0, (3) 5.0, (4) 4.0, (5) 3.0, and (6) 2.0. (b) Plot of the absorbance at λ ) 466 nm vs pH for H2O2. The point with an arrow is the pH obtained by using 1.0 × 10-3 M HCl.

concentrations as noted above due perhaps to the limited supply of the dopants inside the micelles. It has also been reported that the concentration of SDS has a significant effect on the morphology of the electropolymerized PPy film; the PPy films prepared in the presence of SDS below its cmc showed a smooth and compact structure, while those formed above the cmc exhibited perpendicularly oriented structures.19 Here, we believe the oligomer and polymer particles or their aggregates are isolated from each other as they are included within the micelles, leading to less extensive conjugation than through longer chains and, thus, to sharper absorption bands. Effect of HCl. The acidity of the solution also exhibited a significant effect on the polymerization reaction. It was found that the absorbance was very low when H2O2 was added to the pyrrole solution at a pH value higher than 6. As shown in Figure 3, the absorbance at both 466 and 900 nm decreases as the pH of the solution increases. In addition, we note that the shape of the

Figure 5. Absorbance spectra of a solution containing 0.010 M pyrrole, 0.020 M SDS, 1.0 × 10-3 M HCl, 0.1 unit/mL AOx, and 1.0 × 10-5 M FeCl2, upon reaction for 15 min with a diluted alcoholic beverage.

Figure 4. (a) Absorbance spectra from solutions containing 0.010 M pyrrole, 0.020 M SDS, 1.0 × 10-3 M HCl, 0.1 unit/mL AOx, and 1.0 × 10-5 M FeCl2 upon reaction for 15 min with different concentrations of ethanol: (1) 0, (2) 2.0 × 10-5, (3) 6.0 × 10-5, (4) 1.0 × 10-4, (5) 2.0 × 10-4, and (6) 6 × 10-4 M. (b) Variation in the absorbance intensity at λ ) 468 nm as a function of the ethanol concentration. Inset: corresponding calibration curve.

absorption band at 466 nm becomes sharper as the pH decreases, indicating that PPy has a pH-dependent absorbance and thus structure.21 This observation is consistent with those reported in previous studies, in which the polymerization of pyrrole was difficult and depressed in a neutral aqueous solution.22-25 This is because of the competition between the polymerization and degradation reactions of the oxidized pyrrole species at higher pHs owing to the presence of a more aggressive nucleophile, OH-, and also because of poorer conjugation of oxidized species formed (21) Marcos, S. de; Wolfbeis, O. S. Sens. Mater. 1997, 9, 253. (22) Wernet, W.; Monkenbusch, M.; Wegner, G. Mol. Cryst. Liq. Cryst. 1985, 118, 193. (23) Couves, S. L. D.; Porter, J. Synth. Met. 1989, 28, 761. (24) Pei, Q.; Qian, R. J. Electroanal. Chem. 1992, 322, 153. (25) Hong, S.-Y.; Park, S.-M. J. Phys. Chem. B 2005, 109, 9305.

at high pHs.13,25 It was reported that the addition of protons to an aqueous solution promoted polymerization of pyrrole; an acidic environment was favored for pyrrole electropolymerization in aqueous solutions.26,27 In the present work, when the HCl concentration is increased from 1.0 × 10-5 to 1.0 × 10-3 M, the absorbance increased dramatically. A further increase in the HCl concentration did not lead to an increase in absorbance. When 1.0 × 10-3 M HCl was used, the pH of the pyrrole solution was measured to be 3.52 due to the protonation of pyrrole and the absorbance approached that at pH 3 (see Figure 3b, the point with an arrow). For ethanol, it showed a pH dependency very similar to that with H2O2, indicating that the effect of pH on the activity of alcohol oxidase was negligible. Higher HCl concentrations were found to decrease the enzyme activity as could be seen by a slight decrease in PPy absorbance when ethanol was analyzed (not shown). Thus, an HCl concentration of 1.0 × 10-3 M was used for the subsequent experiments. Zhou and Heinze28 reported that the addition of a low concentration of HCl (10-5-5 × 10-5 M) to an acetonitrile electrolyte solution promoted the formation of what they called PPy(II); this is because HCl becomes a stronger acid in acetonitrile due to poor solvation of protons than in water. At higher concentrations of HCl (>10-4 M), generation of what was called PPy(III), a partially conjugated polymer formed through protonation, was observed in aqueous media. Therefore, the PPy formed by oxidation with H2O2 is likely to be mostly PPy(III). Since the oxidation potential of PPy is lower than that of the monomer,29 the polymer itself would be in an oxidized or doped state during polymerization.30 The charge carriers generated along the chains by oxidation, together with the positively (26) Kim, Y. T.; Collins, R. W.; Vedam, K.; Allara, D. L. J. Electrochem. Soc. 1991, 138, 3266. (27) Zinger, B. Synth. Met. 1989, 28, 37. (28) Zhou, M.; Heinze, J. J. Phys. Chem. B. 1999, 103 (40), 8443. (29) Diaz, A. F.; Crowley, J.; Bargon, J.; Gardini, G. P.; Torrance, J. B. J. Electroanal. Chem. 1981, 121, 355. (30) Piletsky, S. A.; Panasyuk, T. L.; Piletskaya, E. V.; Sergeeva, T. A.; El’skaya, A. V.; Pringsheim, E.; Wolfbeis, O. S. Fresenius J. Anal. Chem. 2000, 366, 807.

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Table 1. Comparison between Other Method and Our Method methods

advantages

chemiluminescence fluorescence

high sensitivity moderate sensitivity high selectivity low sensitivity moderate sensitivity moderate sensitivity and selectivity, low cost, simple instrumentation

electrochemical nanoparticles our method

drawbacks

charged segments originated from protonation, are to be compensated by the counter anions,15,31 which contribute to the high absorbance of the doping-induced band at ∼900 nm in the spectrum. Effect of Fe(II). The reaction between Fe(II) and H2O2 is a well-known Fenton reaction, in which a hydroxyl radical (•OH) is generated according to the reaction,32

Fe(II) + H2O2 f Fe(III) + OH- + •OH

(2)

The Fenton-type reaction plays an important role in the oxidative destruction of toxic chemicals in aqueous solutions.33 The ferrous ion, Fe(II), has been used as a catalyst for chemical polymerization of aniline in an acidic solution with H2O2 as an oxidant34 due to the high oxidation potential of the hydroxyl radical produced in the reaction. It was found that Fe(III) can also oxidize pyrrole leading to its polymerization albeit slowly.15 We found, however, that Fe(II) does not oxidize pyrrole in air without H2O2 present for a period of a day. We varied the Fe(II) concentration from 0 to 200 µM in order to find an optimum concentration. The absorbance increased linearly as the concentration of Fe(II) increased from 0 to 10 µM (not shown); a further increase in Fe(II) did not lead to a further increase in absorbance signals. This indicates that Fe(II) is regenerated upon oxidation of pyrrole by Fe(III), and both Fe(III) and ‚OH are continuously supplied for the reaction until all H2O2 is completely used up. We thus used 10 µM Fe(II) for the subsequent experiments. Effect of the Reaction Time. The absorbance showed an increase as a function of the reaction time until it levels off at a certain point. However, the polymer was precipitated at a high H2O2 concentration when the reaction time was as long as a few hours, which caused a decrease in absorbance. For this reason, a reaction time of 15 min was chosen. Determination of Ethanol. While the method developed can be used for the determination of hydrogen peroxide, it may also be applied to the analysis of other analytes if they undergo an enzyme (oxidase)-mediated reaction with oxygen to produce hydrogen peroxide as a product. It is well known that numerous oxidases catalyze the oxidation of the respective substrates by molecular oxygen leading to the production of H2O2. This indicates that the polymerization reaction of pyrrole described above can (31) Li, Y.; Qian, R. J. Electroanal. Chem. 1993, 362, 267. (32) Fukushima, M.; Tatsumi, K.; Morimoto, K. Environ. Sci. Technol. 2000, 34, 2006. (33) Walling, C. Acc. Chem. Res. 1975, 8, 125. (34) Sun, Z. C.; Geng, Y. H.; Li, J.; Wang, X. H.; Jing, X. B.; Wang, F. S. J. Appl. Polym. Sci. 1999, 72, 1077.

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low selectivity, pH >8 expensive instrumentation and reagents, trained operators complex chemistry, precise operation complex reaction, precise operation

refs 9, 10 6 8 14 -

be applied to optical biosensing of a number of other substrates including ethanol, glucose, and cholesterol. In the present work, the H2O2 detection method was employed for the optical sensing of ethanol with an alcohol oxidase used for the O2/ethanol reaction as a H2O2 generating system.35 Figure 4a shows the spectral changes of the solution containing pyrrole, SDS, and AOx, upon addition of different amounts of ethanol. The spectra were recorded after a reaction period of 15 min. The absorbance values increased at both 468 and 900 nm as the ethanol concentration was increased. The corresponding calibration curve is shown in Figure 4b. The absorbance versus the ethanol concentration plot shows a characteristic feature of a typical enzyme-catalyzed reaction due to the saturation of the reaction sites of the enzyme by the substrate, which is similar to the Michaelis-Menton behavior.7 The linear ethanol concentration range extends from 2.0 × 10-5 to 2.0 × 10-4 M (r2 ) 0.999) (inset of Figure 4b), with a detection limit of 1.0 × 10-5 M. Control experiments indicated that, without addition of ethanol or AOx, or in the absence of O2, no polymerization of pyrrole took place within the given reaction time. This result suggests that the biocatalytically formed H2O2 can also induce the polymerization of pyrrole and the presence of enzyme does not interfere with the determination of alcohol. As the ethanol concentration increased, the concentration of H2O2 generated increased also from the above reaction leading to more polymerization of pyrrole. We applied the new analytical method to the determination of ethanol contents in a local liquor called kaoliang spirit, wine, and beer, which also contains a few additives such as L-asparagine amide as an alcohol detoxicant. Figure 5 shows the absorbance spectra of the PPy colloid solution upon reaction with the diluted wine for 15 min. The shape and position of the absorbance spectra were identical to that of authentic ethanol. Our method yielded ethanol contents of 53.6 (( 0.86)% for the kaoliang spirit, 11.6 (( 0.31)% for the wine, and 3.59 (( 0.086)% for the beer from five spectrometric measurements, while gas chromatography gave a value of 53.9 (( 1.6)% for the kaoliang spirit, 11.8 (( 0.050)% for the wine, and 3.61 (( 0.045)% for the beer from five measurements. The standard t-tests indicated that there is no significant difference in three sets of values at a confidence level of 95%. We found, however, that addition of an efficient reductant such as ascorbic acid lowers the apparent amount of H2O2 due to the consumption of H2O2 via its oxidation of ascorbic acid. Thus, the method is not applicable when an efficient reductant is present. Our method for the determination of H2O2 is compared favorably with other recently published methods as summarized (35) Salgado, A. M.; Folly, R. O. M.; Valdman, B.; Cos, O.; Valero, F. Biotechnol. Lett. 2000, 22, 327.

in Table 1. For assaying ethanol via an enzyme-catalyzed reaction, our method also compares well with a recently reported method,35 in which another coloring agent, e.g., 4-aminophenazone, was used with its detection limit of ∼1.1 mM. CONCLUSION A novel method for detecting H2O2 based on the H2O2-mediated growth of PPy has been developed. The results clearly demonstrate that the absorbance changes correlate well with the concentration of H2O2 and can be used for the optical assaying of H2O2. The detection limit of the method is slightly lower than those reported recently.6,8 However, our demonstration that the sensing probe can be applied to other analytes, e.g., ethanol, indicates that the method also provides reliable results for analysis of H2O2 in a biocatalytic reaction mixture containing reactants and additives. We thus believe that the new methodology can be extended to a host of other oxidase-based reactions to determine

biologically important substrates/analytes including cholesterol, lactate, glucose, xanthine, etc. Further, the method offers a convenient way to assay H2O2 during studies on the kinetics of many enzyme-catalyzed reactions as long as H2O2 is produced. Work is currently underway to extend the method to the analysis of other biologically important analytes as well as to the studies of oxidase-catalyzed reactions. ACKNOWLEDGMENT This work was supported by a grant from KOSEF (grant R112000-070-070010) through the Center for Integrated Molecular Systems at POSTECH. The BK-21 program provided a part of the postdoctoral fellowship for H.y.W. Received for review May 4, 2006. Accepted October 23, 2006. AC0608319

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