Anal. Chem. 1996,67,1109-1114
Ferrocene-Conjugated Polyaniline-Modified Enzyme Electrodes for Determination of Peroxides in Organic Media Chia=LinWang and Ashok Mulchandani*
Chemical Engineering Department, College of Engineehng, University of Califomia, Riverside, Califomia 92521
The development and characteristics of a reagentless amperometric organic phase enzyme electrode (OPEE) employing covalently attached horseradish peroxidase and an electrochemically deposited ferrocene-modified polyaniline film on a glassy carbon electrode is reported, The covalent attachment of ferrocene to an electrochemically deposited insoluble polymer film provided a mechanism of preventing the leaching of ferrocene into the predominantly organic solvents, required for construction of reagentless OPEE. Hydrodynamic voltammetry studies showed that the response of the OPEE to hydrogen peroxides increased at higher cathodic potentials; however, interference due to molecular oxygen also increased. Interference of molecular oxygen was “ i z e d when the OPEE was operated at an applied potential of -50 mV (or less negative) vs AgIAgCl. The cathodic response of the OPEE was found to increase steeply when the aqueous buffer content of the acetonitrile was increased from 0% to 5%and then plateau with no further increase when the buffer content was increased to 30%. The dynamic properties of this enzyme electrode were exploited for the detection of micromolar concentrations of different peroxides in flow injection analysis where the sensitivitytrend was lauroyl peroxide > hydrogen peroxide > 2-butanone peroxide > cumene hydroperoxide > tert-butyl hydroperoxide. Applicability of the enzyme electrode for measurement of peroxide in real sample was demonstrated. The recent remarkable finding that enzymes can function as catalysts in organic solvents has attracted increased interest in organic synthesis and analytical The operation of bioanalytical devices based on enzymes, i.e., enzyme electrodes, in nonaqueous phase offer several advantages, such as, extended analyte range due to increased solubility of certain reactants and monitoring of many hydrophobic substrates, novel detectors as a result of changes in substrate specificity, prevention of undesirable side reactions and even change in reaction direction, improved operational stability due to increased thermal stability, and decreased microbial contamination and simplilied immobilization techniques. (1) Klibanov, A M. CHEMTECH 1986,16,354-359. (2) Dordick, J. S. Enzyme Microb. Technol. 1989,1 1 , 194-211. (3) Dordick, J. S. Biotechnol. Prog. 1992,8,259-267. (4) Wescott, C. R; Klibanov, A M. Biochim. Biophys. Acta 1994,1206,1-9. (5) Saini, S.; Hall, G. F.; Downs, M. E. A; Turner, A P. F. Anal. Chim. Acta 1991,249, 1-15. (6) Wang, J. Talanta 1993,40,1905-1909.
0003-2700/95/0367-1109$9.00/0 0 1995 American Chemical Society
The determination of peroxides, organic and hydrogen, is of practical importance in clinical, food, pharmaceutical, and environmental fields. In many of these applications, the peroxide to be monitored is present in an organic matrix that either is not soluble in aqueous medium or has very low solubility in aqueous media. A few of these examples are (1)determinationof hydrogen peroxide, used as disinfectant or bleach, in pharmaceutical and cosmetic formulations,7 (2) monitoring of lipid hydroperoxides, formed by peroxidation of lipids, in vegetable oils, baby foods, biological tissues, etc.,s-12(3) measurement of organic peroxides released in the environment from industrial processes and those produced during the ozonation reactions in air and process of ozonation of drinking water,13-15 and (4) analysis of hydrogen peroxide formed during the reaction of substrates with low aqueous phase solubility, such as cholesterol, with their oxidases.16-18 Horseradish peroxidase (HRF) is known to catalyze the reduction of hydrogen peroxide and certain organic peroxide^.^^^^^ HRF-based enzyme electrodes for detection of HzOz and organic peroxides in organic phase have been reported in literat~re>l-~~ In many of these biosensors, the mediator was added to the electrochemical cell or the mobile phase flowing through the flow (7) Wang, J.; Lin,Y.; Chen, L. Analyst 1993,118, 277-280. (8) Mannino, S.; Cosio, M. S.; Wang, J. Anal. Lett. 1994,27,299-308. (9) Eun, J.-B.; Boyle, J. A; Heamsberger, J. 0.1. Food Sci. 1994,59, 251255. (10) Shantha, N. C.; Decker, E. A]. AOACInf. 1994,77,421-424. (11) Roozen, J. P.; Linssen, J. P. H. In Lipid Oxidation in Food St. Angelo, A J., Ed.; ACS Symposium Series 500; American Chemical Society: Washington, DC, 1992; Chapters 17, 18, pp 302-321. (12) Hageman, G.; Kikken, R; ten Hoor, F.; Kleinjans, J. Lipids 1989,24,899902. (13) IARC Monographs on the Evalution of the Carcinogenic Risk of Chemicals to Humans. Allyl Compounds, Aldehydes, Epoxides and Peroxides; IARC: Lyon, France, 1985; Vol. 36, pp 267-321. (14) Glaze, W. H. Enuiron. Sci. Technol. 1987,21,224-230. (15) Kok, G. L.; Holler, T. P.; Lopez, M. B.; Nachtrieb, H. A; Yuan, M. Enuiron, Sci. Technol. 1978,12,1072-1076. (16) Braco, L;Daros, J. A; de la Guardia, M. Anal. Chem. 1992,64,129-133. (17) Hall, G. F.; Turner, A. P. F. Anal. Lett. 1991,24,1375-1388. (18) Kazandjian, R Z.; Dordick, J. S.; Klibanov, A. M. Biotechnol. Bioeng. 1986, 28,417-421. (19) Maehly, A C. Methods EnLymol. 1955,2,801-813. (20) Campa, A In Peroxidase in Chemistry and Biology; Everse, J., Everse, K. E., Grisham, M. B., Eds.; CRC Press: Boca Raton, FL, 1991;Vol. 11, Chapter 2, pp 25-50. (21) Wang, J.; Lin,Y.; Chen, Q. Electroanalysis 1993,5, 23-28. (22) Wang, J.; Lin, Y.Anal. Chim. Acta 1993,271,53-58. (23) Wang, J; Naser, N; Kwon, H.-S; Cho, M. Y. Anal. Chim. Acta 1992,264, 7-12. (24) Schubert, F.; Saini, S.; Turner, A P. F. Anal. Chim. Acta 1991,245, 133138. (25) Wang, J.; Wu, L.-H.; Angnes, L. Anal. Chem. 1991,63,2993-2994.
Analytical Chemistfy, Vol. 67, No. 6, March 15, 1995 1109
cell detector.7~8~22~23 Use of mediator free in solution is not desirable for construction of reagentless biosensors aimed at in situ monitoring of environmental and industrial processes and in bienzyme electrodes, combining FADcontaining oxidase and peroxidase, because the oxidized mediator formed upon the electron transfer to HRP can be reduced by the FADHz centers of the oxidase and therefore diminish the cathodic current. These problems are expected to be alleviated by using mediator attached/immobilized to the electrode surface. By taking advantage of the low solubility of potassium hexacyanoferrate in organic solvents, Schubert et al." and Wang et al.25constructed reagentless OPEEs for determination of hydrogen and organic peroxides by retaining the hexacyanoferrate on the working electrode either by adsorption or entrapment by a poly(ester-sulfonic acid) coating. However, since the electrolyte used contained 10- 15% water or buffer, a slow leaching of the mediator into the solution can be anticipated. In order to overcome the problems associated with the mediator present in solution and leaching of the mediator from the electrode, recently we reported on the development of amperometric enzyme electrodes based on HRP and polyanilineconjugated ferrocene polymer film for the detection of hydrogen peroxide and organic peroxides in aqueous phase.26 Since ferrocene is attached covalently to the insoluble polymer film, the polyanilineconjugatedferrocene polymer is an ideal candidate for development of organic phase amperometric enzyme electrodes where leaching of the mediator is a significant problem. This paper reports the features and application of an organic phase enzyme electrode based on HRP and ferrocene-modified polyaniline film for the monitoring of peroxides. EXPERIMENTAL SECTION
Chemicals. Acetonitrile (HPLC grade), hydrogen peroxide (30% in water), and glutaraldehyde (25%solution in water) were acquired from Fisher Scientifc (Tustin, CA). Horseradish peroxidase type VI-A, EC 1.11.1.7 (HRP, 1100 units mg-'), l-ethyl3-[3-(dimethylamino)propyllcarbodiimidehydrochloride (EDC, protein sequencing grade), 2-(N-Morpholino)ethanesulfonicacid monohydrate (MB), and tetrabutylammonium perchlorate ('EL4P) were purchased from Sigma Chemical Co. (St. Louis, MO). Cumene hydroperoxide, lauroyl hydroperoxide, 2-butanone peroxide, and tert-butyl hydroperoxide were acquired from Aldrich (Milwaukee, WI). Hair bleach aolen, CT) was purchased from a local pharmacy. All the chemicals were used without purification. Where ever required, double-distilled ultrapure water was used. Synthesis of N-(Ferrocenylmethy1)aniline Monomer (I). Monomer I was synthesized according to the reported protocol.261.n HRP Immobilization. HRP was immobilized on glassy carbon electrodes (GCE; Bioanalytical Systems, Lafayette, IN) by (1) adsorption and (2) covalent binding using carbodiimide chemistry. Before immobilization, electrodes were prepared by polishing the surfaces with 1pm diamond paste followed by 0.05 pm y-alumina particles (Buehler, Lake Bluff, IL). For adsorption, 5 pL of 10 mg mL-1 HRP in pH 6.4 phosphate buffer was put on the electrode surface to air-dry, followed by extensive washing with buffer to remove unbound enzyme. For covalent attachment of HRP, the polished GCE was first oxidized electrochemically (26) Mulchandani, A; Wang, C.-L.; Weetall, H. H. Anal. Chem. 1995,67, 94100. (27) Horwitz, C. P.; Dailey, G. C. Chem. Mater. 1990,2, 343-346.
1110 Analytical Chemistry, Vol. 67,No. 6, March 15, 1995
by holding the potential at 2.2 V (vs Ag/AgCl) for 10 s in a 2.5% bCrz07 solution in 10%HN03,%followed by activation of carboxyl groups by placing the electrode in 0.15 M EDC solution in 0.1 M pH 4.6 MES buffer with gentle stirring. Immobilization was achieved by adding HRP into the above buffer (0.5 mg mL-') and incubating overnight at room temperature. Electrochemical Polymerization. A polymer film, poly(anilinomethylferrocene) [designated as poly(Ah4Fc) ], of monomer I was deposited on polished GCE and HRP-modified GCE from an argon-deaerated electrolyte bath containing 5 mM I and 0.1 M TBAP in acetonitrile by cycling the electrode potential between 0 and 1.1-1.3 V vs Ag/AgC1 at 100 mV s-l for a total of five cycles. The electrode was rinsed with acetonitrile to remove entrapped monomer and TBAP and then stored dry at 4 "C in the refrigerator. Apparatus. Voltammetry studies were performed on either a Princeton Applied Research Model 263A potentiostat/galvanostat (EG&G, Princeton, NJ) interfaced to a 80486based personal computer or a Bioanalytical Systems voltammograph (Model CV 27) with a lowcurrent module (BAS, PA1 preampliiier) coupled to a flat-bed chart recorder (BD-112, Kipp and Zonen, Delft, Holland), Electropolymerization and cyclic voltammetry were performed under stationary conditions in a 10 mL electrochemical cell (BAS, VC-2) placed inside a Faraday cage (BAS, C-2). Amperometric measurements were made in batch and flow injection modes. Batch studies were performed in a 10 mL electrochemical cell (BAS, VG2) placed inside a Faraday cage (BAS, C-2) with constant stirring to provide convective transport. Flow injection analyses (FIA) were performed using a thin-layer flow cell (BAS, CC-5) with dual glassy carbon working electrodes in parallel. A precision flow peristaltic pump (EVA pump, Eppendorf North America, Madison, wr) was used for the delivery of mobile phase and sample. A 20 pL sample was injected into the mobile phase by a motorized injection valve (EVA inject valve, Eppendorf North America). All the experiments were performed with Ag/AgCl (3 M NaC1) reference and platinum auxiliary electrodes in a mixture of the appropriate organic solvent with 0.1 M TBAP and appropriate proportion of pH 5.5,0.05 M MES buffer. Measurement of Hydrogen Peroxide in Real Sample. HzOzconcentration in the real sample, creme hair bleach, was determined using the HRP/poly(AMFc) enzyme electrode and by an enzymatic method. A measured weight of the hair bleach was dissolved in 10 mL of 90%acetonitrile with 0.1 M TBAP and 10%pH 5.5,0.05 M MES solution. A three-point calibration curve was generated immediately prior to determini HzOz concentration. The Trinder reaction, in which the quinoimine dye (molar extinction coefficient 12700 M-l cm-l at 500 nm)Bwas produced by the reaction of HzOz with phenol and Caminoantipyrene in the presence of HRP, was used to determine HzOz concentration enzymatically. The assays were performed in 1000 pL of 0.1 M pH 6.5 phosphate buffer containing 250 p L each of 3 mM phenol and 3 mM Caminoantipyrene (prepared in 0.1 M pH 6.5 phosphate buffer), 0.25 unit of HRP in 10 pL of phosphate buffer, and 200 pL of the sample with the balance of 0.1 M pH 6.5 phosphate buffer. (28) Bourdillon, C.; Bourgeois, J. P.; Thomas, D.J Am. Chem. SOC.1980,102, 4231-4235. (29) Tamaoku, IC; Murao, Y.; Akiura, K; Ohkura, Y. Anal. Chim. Acta 1982, 136, 121-127.
8.550
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[H,OJ PM
-30.80
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I0.558
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O.=
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Flgure 2. Comparison of the response of enzyme electrodes prepared by different immobilization techniques: ( 0 )carbodiimideimmobilized HRP/poly(AMFc), (A) carbodiimide-immobilized HRP, and (+) adsorbed HRP-modified GCE to H202 in 95% acetonitrile and 5% water with 0.1 M TBAP. Operating potential, -50 mV vs Agl ASCI. Each point represents an average of measurements with two electrodes.
Potential (V vs. AglAgCI) Figure I. Cyclic voltammograms for repeated cycling (50 cycles) of the poly(AMFc)-coated GCE in (A) acetonitrile with 0.1 M TBAP and (B) 0.1 M, pH 6.5 phosphate buffer with 0.1 M NaC104; scan rate, 50 mV s-l. The number indicates the scan number.
RESULTS AND DISCUSSION Characterization ofthe Poly(anilinomethytferr0cene) Fh. Panels A and B of Figure 1show cyclic voltammographs for poly-
(AMFc)-coated GCE in organic and aqueous electrolytes, respectively. The polymer-modified electrodes exhibited a characteristic redox signal for ferrocene immobilized on a surface with peak potential separation of 50 and 72 mV (after the first cycle in CV), respectively, in organic and aqueous media. In contrast to organic medium, where the peak potential separation between the first and the fiftieth cycle remained practically unchanged, in aqueous buffer there was a large peak separation and a sharp anodic peak for the first cycle of CV, which later disappeared and the film behaved close to the one in organic medium but with significantly lower peak currents. This phenomenon is attributed to the fact that in organic medium the polymer film is swollen allowing a rapid transport of the counterions in the film, whereas in the aqueous medium there is a low ambient electrolyte inside the fresh polymer film during the first cycle due to the film compactness and requires breaking in of the film. Similar behavior has been observed when different redox couples were immobilized on the electrode using polymer films.3O When cycled repeatedly for 50 cycles between 0 and +650 mV at a scan rate of 50 mV s-l, there was a 28%and 6%(calculated from the second to the fiftieth cycle) decrease in the anodic peak current in the aqueous and organic media, respectively. The decrease of the peak current in acetonitrile observed in this study is similar to that reported (5%)for poly(AMFc) film27and for a ferrocene-siloxane film31deposited on platinum. This much smaller decrease in organic medium (30)Daum, P.: Murray, R W. J. Phys. Chem. 1981,85,389-396. (31)Lenhard, J. R:Murray, R W. J. Am. Chem. SOC.1978, 100, 7870-7875.
(over that in aqueous) makes this electrode much more suitable for application as an amperometric sensor. The surface coverage of ferrocene on the HRP-modifled and bare GCE, determined from the charge under the oxidation peak for ferrocene from the CV, was 7 x 10-10 and 8 x 10-9 mol cm-2, respectively. Enzyme Immobilization. One advantage of organic phase enzyme electrodes is reported to be the use of a simpler and faster enzyme immobilization scheme, like adsorption, due to the insolubility of enzyme in organic solvent compared to the covalent attachment required for an aqueous system. In the investigation of enzyme immobilization methods it was observed that the response of an organic phase enzyme electrode constructed by covalent immobilization using carbodiimide chemistry was significantly higher than that for the electrode constructed by adsorbing the enzyme on a polished GCE (Figure 2). Since the response of the covalently immobilized enzyme electrode was higher, attributed to the higher enzyme activity on the GCE surface, this method was selected for the study reported here. It should be pointed out that, in the present comparison, GCE was not treated prior to immobilization of HRP by adsorption (Wang and Enz2reported that a higher enzyme loading is achieved when the electrode surface is treated electrochemically before enzyme adsorption). Direct electron transfer from HRP immobilized covalently on carbon fibers and graphite powder has been reported in the l i t e r a t u ~ - e and ~ ~ -was ~ ~ also observed in this work (Figure 2). As in aqueous phase,26the cathodic current response of the HRP/ poly(AMFc) enzyme electrode in 95% acetonitrile and 5%water (32)Yaropolov, A I.; Malo&, V.: Varfolomeev, I.; Berezin, I. V. DoklAkad. Nauk. SSSR 1979,249,1399-1401. (33)Gorton, L:Jonsson-Pettersson, G.; Csoregi, E.: Johansson, IC;Dominguez, E.: Markc-Varga, G. Analyst 1992, 117,1235-1241. (34)Csoregi, E.; Jonsson-Pettersson, G.: Gorton, L.]. Biotechnol. 1993,30,315337. (35)Csoregi, E.;Gorton, L.; Markc-Varga, G. Anal. Chim.Acta 1993,273,5970.
Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
1111
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c t T
I
-300
-400
301
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300
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-100
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Potential (mV vs. AglAgCI)
Figure 3. Effect of operating potential on the response of the HRP/ poly(AMFc) electrode to (0)10 pM HZOZand (A)10 pL injection of acetonitrile in deaerated 95% acetonitrile and 5% water with 0.1 M TBAP. Each point represents an average of three measurements, and the error bars represent &1 standard deviation.
with 0.1 M TBAP electrolyte was higher (approximately 2-fold) when compared to the direct electron transfer from covalently immobilized HRP to the electrode (Figure 2). This demonstrated an advantage of the HRP/poly(AMFc) electrode over the electrode based on direct electron transfer from HRP. Hydrodynamic Voltammetry. Figure 3 shows the hydrodynamic response plot of the HRP/poly(AMFc) electrode for injections of 10 pM HzOz and 10p L injections of pure acetonitrile in deaerated acetonitrile with 5% water and 0.1 M TBAP. The profile of the electrode response to HzO2 is very similar to that reported by others for the HRP electrode with the mediator in organic media7,22and for a similar electrode in an aqueous p h a ~ e . As ~ ~in, ~the ~ aqueous phase, at potentials negative of 0 mV, there was a measurable cathodic response when acetonitrile was injected into the deaerated electrolyte. This cathodic response,which was attributed to the reduction of molecular oxygen, was a function of the applied potential and increased as more negative potential was applied. Additionally, when HRP/poly(AMFc) electrodes were used in a nondeaerated electrolyte at potentials more negative of -50 mV, the baseline drifted for a long period before a constant but a significantly higher residual current was recorded. On the basis of the above results, -50 mV was selected as the working potential for the study reported here. An advantage of using this potential was that the mobile phase in flow injection analysis could be used without deaeration. Buffer Content in Organic Phase. In order to be active in a predominately organic phase, enzymes require a critical amount of water. The amount of water required for optimum catalysis is dependent on the enzymes, the organic solvent, and the affinity of the enzyme support for water.2,3,5s37-42 Using enzyme lyophilized from di€ferentpH buffers, Zaks and Kliban0v4~showed that the (36) Wang, J.; Frieha, B.; Naser, N.; Romero, E. G.; Wollenberger, U.; Ozsoz, M.; Evans, 0. Anal. Chim. Acta 1991,254,81-88. (37) Ryu, IC;Dordick, J. S. Biochemistry 1992,31,2588-2598. (38) AMeck, R; Xu, Z.-F.; Suzawa, V.; Focht, IC; Clark, D. S.; Dordick, J. S. Proc. Natl. Acad. Sci. U.S.A. 1992,89,1100-1104. (39) Ryu, K; Dordick, J. S . Resour. Consew. Recycl. 1990,3, 177-185.
1112 Analytical Chemistty, Vol. 67, No. 6,March 75, 7995
0
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%MES Buffer in CH,CN Figure 4. Effect of buffer (0.05 M, pH 5.5 MES with 0.1 M NaC104) content on the response of the HRP/poly(AMFc) electrode to 50 pM H202 in mobile phase (acetonitrile with 0.1 M TBAP with appropriate amount of buffer) using flow injection. Operating potential, -50 mV vs Ag/AgCI; flow rate, 0.5 mL min-I. The points represent the mean of nine analyses, and the error bars represent f l standard deviation.
enzyme in organic media depended on the pH from which it was lyophilized. This would mean that the operating electrolyte for the enzyme electrode could be made up of organic phase and water. An electrolyte mixture containing organic solvent and water mixture has been found to be adequate for HRP and tyrosinasebased organic phase enzyme electrode~.~.~~~22 However, recently, Halling and Valivety44 showed that, depending on the substrate and product pH, the effective pH of the enzyme in a predominately organic medium changes. Therefore, in this work it was decided to replace water with 0.1 M,pH 4.0 citrate/ phosphate with 0.1 M NaC104 buffer [this buffer was selected on the basis of our previous work with HRP/poly(AIvlFc) electrode in aqueous medialz6in the electrolyte mixture. However, while making different organic to aqueous ratio mixtures to determine the optimum electrolyte, it was noticed that some of these mixtures formed emulsions rather than clear solutions. This problem was found to be caused by the high concentration of inorganic salts in the aqueous solution which caused phase separation. In order to overcome this problem, it was decided to use an organic buffer, MES,instead of the citrate/phosphate. Since 5.5 is the lowest effective pH for the MES buffer, pH 5.5 buffer was selected for the reported study. Figure 4 shows the effect of the buffer content on the response of the HRP/poly(AMFc) electrode to HzOz. Unlike the response of an organic phase enzyme electrode prepared by adsorption of HRP and the mediator on graphite foil, which exhibited a maximum response for H2Oz in a mixture of 30%buffer and 70% the response of the (40) Ryu, IC; Stafford, D. R; Dordick, J. S. In Biocafalysis in Agricultural Biotechnology; Whitaker, J . R, Sonnet, P. E., Eds.; ACS Symposium Series 389; American Chemical Society: Washington, DC, 1989 Chapter 10, pp 141-157. (41) Zaks, A; Klibanov, A M. 1.Biol. Chem. 1988,263,8017-8021. (42) Aldercrreutz, P. Eur. 1.Biochem. 1991,199,609-614. (43) Zaks, A; Klibanov, A M. J Bid. Chem. 1988,263,3194-3201. (44)Halling, P. J.; Valivety, R H. In Biocatalysis in Non-ConventionalMedia. Progress in Biotechnology;Tramper, J., Vermiie, M. H., Beeftink, H. H., von Stockar, U., Eds.; Elsevier: Amsterdam, 1992; Vol. 8, pp 13-21.
HRP/poly(AMFc) electrode increased steeply and reached a plateau. This desirable characteristic of the HRP/poly(AMFc) electrode, which allows electrode operation in a wide range of media, is attributed to the fact that both enzyme and ferrocene are attached permanently on the GCE and do not desorb even when the aqueous phase content is increased. Using HRP immobilized on glass beads as a catalyst for polymerization of phenol and its derivatives in dioxane/aqueous buffer mixtures, Dordick and his associates found that the catalytic turnover for phenol with hydrophobic substituents, such as alkyl phenols, showed a maximum in high organic content media (i.e., 80%dioxane).3gt40 However, for phenol with hydrophilic substituents, the catalytic turnover exhibited a continuously decreasing trend when the organic content increased. Since the behavior observed in the study reported here (Figure 4) was different, an experiment was designed to determine whether the observed difference was because of the poly(AMFc) film or due to the alteration in the behavior of HRP as a result of a different immobilization method (covalent attachment instead of adsorption) and immobilization support (glassy carbon instead of glass beads). HRP was covalently immobilized on GCE, and the effect of buffer content on the response of this electrode was evaluated in batch mode. The profile of the response vs buffer content curve of this electrode was similar to that observed for the HRP/poly(AMFc) electrode, i.e., a steep increase from 0% to 5%buffer and then a plateau between 5%and 30%buffer, leading to the conclusion that the poly(AMFc) film was not altering the electrode response characteristics (data not shown). A close look at the data in Figure 4 shows that there was a cathodic response to HzOz even in 100% acetonitrile. This is contrary to literature reports that HRP is inactive in pure organic solvents. In order to determine the plausible reasons for this observation,control experiments (in batch mode) were performed to determine whether there was any cathodic response to HzOz in 100%acetonitrile with (1) bare GCE, (2) covalently attached HRP on GCE, and (3) poly(AMFc)coated GCE as working electrodes. The results indicated that while there was no significant cathodic current response at the bare GCE and HRPm o d ~ e dGCE, there was a significant cathodic response for HzOz at the poly(AMFc)coated GCE at an applied potential of -50 mV in pure acetonitrile (data not shown). Due to the fact that a cathodic response was obtained for HzOz on a poly(AMFc)-modified GCE in 100% acetonitrile, it was considered important to determine whether it was indeed necessary at all to have the enzyme on the electrode. In order to investigate this, experiments were done in batch mode with (1) covalently attached HRP, (2) poly(AMFc) film, and (3) HRP/poly(AMFc)-modified GCE in 10%buffer and 90% acetonitrile. The results showed that the HRP/poly(AMFc) electrode had a significantly higher sensitivity compared to the enzyme and poly(AMFc)-modified electrodes and indeed there was an advantage of using HRP/poly(AMFc) electrode for analysis of peroxides (Figure 5). Biosensor Characterization. Figure 6 and Table 1 shows the calibration graph and analytical characteristic of the HRP/ poly(AMFc) electrode for various peroxides determined in acete nitrile with 10%MES buffer using FIA. When compared to the operation in aqueous media, the use of enzyme electrode in predominantly organic media did not offer any advantage for the determination of compounds such as hydrogen peroxide, cumene
50 -
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[HzOzJ PM Figure 5. Cathodic response of ( - - - ) bare, (A) carbodiimideimmobilized HRP, (W) poly(AMFc)-modified, and (0)HRP/poly(AMFc)modified GCE in 90% acetonitrile with 0.1 M TBAP and 10% 0.05 M, pH 5.5 MES buffer in batch mode at -50 mV vs Ag/AgCI. The points represent an average of measurements with two electrodes. 10
0-
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[Conc.] FM Figure 6. Calibration plots for (A)lauroyl peroxide, (0)H202, (+) 2-butanone peroxide, (H) cumene hydroperoxide, and (v)fert-butyl hydroperoxide using flow injection. Mobile phase, 90% acetonitrile with 0.1 M TBAP and 10% 0.05 M, pH 5.5 MES buffer: operating potential, -50 mV vs Ag/AgCI; flow rate, 0.5mL min-'. The points represent the mean of five analyses, and the error bars represent f l standard deviation.
hydroperoxide, and tert-butyl hydroperoxide, which have good solubility in aqueous phase. The sensitivity of detection for these compounds was significantly lower in predominantly organic phase Analytical Chemistty, Vol. 67, No. 6, March 15, 1995
1113
Table 1. Characterirtlcsof Callbratlon Graphs for the HRP/Poly(AMFc) Electrode.
linear range analyte lauroyl peroxide hydrogen peroxideb
(UM)
0.2-6 6-50 70-140 2-butanone peroxide 40-160 cumene hydroperoxide 64-300 fed-butyl hydroperoxide 100-400
sensitivi (nAyM-7
intercept (nA)
72
0.56 0.08 0.044 0.016 0.002 0.0001
1.9 0.7 2.8 0.62 0.008 -0.048
0.9999 0.9987 0.9865 0.9987 0.9989 0.9979
n = 5. The calibration data for hydrogen peroxide was fitted by two straight lines.
than in aqueous media, probably due to the significantly lower activity of HRP in organic media. However, on the other hand, the use of the enzyme electrode in organic medium allowed the determination of a water-insolublehydrophobic compound, such as lauroyl peroxide, which was not possible in aqueous media. The trend of detection of the various peroxides tested were lauroyl peroxide =. hydrogen peroxide > 2-butanone peroxide > cumene hydroperoxide > tert-butyl hydroperoxide. The trend of the electrode sensitivity to hydrogen peroxide, 2-butanone peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide is similar to that observed with the HRP/poly(AMFc) electrode and for other HRP-based enzyme electrodes in aqueous phase. However, the extremely high sensitivity for lauroyl peroxide detection (even higher than hydrogen peroxide) was rather unusual and not in agreement with the literature report that the sensitivity for lauroyl peroxide is lower than for 2-butanone peroxide in chloroform (saturated with buffer) .22 The higher sensitivity for lauroyl peroxide compared to HzOz, the preferred substrate for HRP, in acetonitrile with 10%MES buffer was also observed for a HRPmodified GCE (data not shown). The higher sensitivity for lauroyl peroxide than HzOz is probably due to a change in substrate
1114 Analytical Chemisfry, Vol. 67, No. 6,March 15, 1995
selectivityand specificity in organic media as has been evidenced with other enzymes operating in organic media.3337339.40 Real Sample Analysis. In order to demonstrate the application of the HRP/poly(AMFc) electrode to real sample, the HzOz concentration in a peroxidecontaining creme hair bleach were measured and compared to that determined spectrophotometrically. The hydrogen peroxide concentration in the creme hair bleach, determined from a three-point calibration curve prepared using standard HzOz constructed prior to the measurement, was found to be 4.83 f 0.30% (w/w) (for n = 4). This value was in very good agreement with the 5.01 f 0.05% (w/w) (for n = 3) determined spectrophotometrically and 5%(w/w) quoted by the manufacturer. CONCLUSIONS The currently used technology for construction of enzyme electrodes for analyses of peroxides in organic media has limitations. They are not amenable for in situ monitoring and construction of ultramicroelectrodes, electrodes of complex threedimensional geometry, and bienzyme electrodes. In order to overcome these problems, we have presented a method of incorporating the mediator by covalently attaching it to an insoluble polymer film. Additionally, we have presented a “molecular assembly approach” for the construction of OPEE in which chemical modification techniques are used to incorporate the necessary components-enzyme, redox mediator, and permselective membrane-of amperometric enzyme electrodes. ACKNOWLEWYENT This work was financially supported by the National Science Foundation Grant BCS 9309741. Received for review September 20, 1994. Accepted January 4, 1995.@ AC940941S Abstract published in Advance ACS Abstracts, February 1, 1995.
