Anal. Chem. 1995, 67, 94-100
Amperometric Detection of Peroxides with Poly(ani1inomethylferrocene)=Modified Enzyme Electrodes Ashok Mulchandani,*lt Chia=LinWang,t and Howard H. Weetall*
Chemical Engineering Department, College of Engineering, Universiiy of Califomia, Riverside, Califomia 92527, and Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Malyland 20899
An amperometricenzyme electrode employing covalently attached horseradish peroxidase to glassy carbon elec-
trode and a ferrocene-modifiedp o m e hlm deposited by the electrochemical polymerization of N-(ferrocenylmethy1)aniline monomer is reported. The electrode responded rapidly to micromolar concentrations of peroxides, in accordance with the following trend hydrogen peroxide > cumene hydroperoxide > tert-butyl hydroperoxide. The effect of operational parameters, such as methods of enzyme immobilization,operatingpotential of the working electrode, pH, and effect of molecular oxygen, were explored for optimum analytical performance. The dynamic properties of this enzyme electrode were exploited for the detection in flow injection analysis. Applicability of the enzyme electrode for measurement of peroxide in real sample was demonstrated. The determination of hydrogen peroxide and organic peroxides is rapidly becoming of practical importance in clinical and environmental fields. Sensitive measurement of hydrogen peroxide formed during the reaction of many important chemicals in the presence of oxidases is necessary for the development of many bienzyme electrodes for clinical and environmental applications. Measurement of lipid peroxides in food products and biological tissues is of great medical signi6cance in establishing a relationship between diseases such as breast cancer and the level and type of fat in the diet1 Monitoring of organic peroxides released in the environment from many industrial processes? produced during the process of ozonation of the drinking water;l and ozonation reaction in the air? is desirable. Conventional methods for the determination of peroxides such as spectrophotometry? colorimetry,6 and chemiluminescence7involve complicated methods and suffer from various interferences. The need University of Califomia, Riverside. National Institute of Standards and Technology. (1) Rose, D.P.; Hatala, M. A; Connolly, J. M.; Raybum, J. CancerRes. 1993, 53,4686-4690. (2) 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. (3) Glaze, W. H.Enuiron. Sci. Technol. 1987,21, 224-230. (4)Kok, G. L.; Holler, T. P.; Lopez, M. B.; Nachtrieb, H. A; Yuan, M. Environ. Sci. Technol. 1978, 12, 1072-1076. (5) Sellers, R M. Analyst 1980,105, 950-954. (6)Ito, Y.; Tonogai, Y.; Suzuki, H.; Ogawa, S.; Yokoyama, T.; Hashmme, T.; Santo, H.; Tanaka, IC-I.; Nishigaki, IC;Iwaida, M.J-Rssoc. off:Anal. Chem. 1981,64,1448-1452. (7)Kok, G. L.; Holler, T. P.; Lopez, M. B.; Nachtrieb, H. A; Yuan, M. Enuiron. Sci. Technol. 1978,12, 1073. +
94 Analytical Chemistry, Vol. 67, No. 7, January 1, 7995
for simple, sensitive, selective, and rapid detection schemes for monitoring of peroxides has promoted much of the research in development of biosensors for these applications. Horseradish peroxidase (HRP) is known to catalyze the reduction of hydrogen peroxide and certain organic peroxides according to the following reaction8.9 ROOH
+ HRP-
ROH
+ HRP - I
The reduction of HRP-I to HRP can then be achieved through two separate oneelectron steps8-"J using an electron transfer mediator AH or by direct electron transfer without a mediator from the electrode to the hemin of the HRP when the enzyme is in intimate contact with the conducting surface. HRP-I
+ AH (or e-) - HRP-I1 + Ao
HRP-I1
+ AH (or e-)
-
HRP
+ A'
Biosensorsfor detection of HzOz and organic peroxides using both direct e l e c t r ~ n and ~ ~ -mediator-assisted ~~ electron transfel-20-25 from HRP have been reported in the literature. In general, the (8) Maehly, A C. Methods Enzymol. 1955,2, 801-813. (9)Campa, A In Peroxidase in Chemistty and Biolom Everse, J., Everse IC E., Grisham, M. B., Eds.; CRC Press: Boca Raton, FL 1991;Vol. 11, Chapter 2, pp 25-50. (10)Yamada, H.;Yamazaki, I. Arch. Biochem. Biophy. 1974,165, 728-738. (11) Jonsson-Pettersson, G.; Gorton, L.Electroanalysis 1989, 1, 465-468. (12)Kulys, J.; Schmid, R D. Bioelectrochem. Bioe#erg. 1990,24,305-311. (13) Wollenberger, U.;Bogdanovskaya, V.; Bobrin, S.; Scheller, F.; Tarasevich, M. Anal. Lett. 1990,23,1795-1808. (14)Gorton, L.; Bremle, G.; Csoregi, E.; Jonsson-Pettersson, G.; Persson, B. Anal. Chim. Acta 1991,249,43-54. (15)Jonsson-Pettersson, G. Electroanalysis 1991,3, 741-750. (16)Wollenberger, U.; Wang, J.; Ozsoz, M.; Gonzalez-Romero, E.; Scheller, F. Bioelectrochem. Bioeneq. 1991,26,287-296. (17)Gorton, L.; Jonsson-Pettersson, G.; Csoregi, E.; Johansson, IC;Dominguez, E.;Marko-Varga, G. Analyst 1992, 117, 1235-1241. (18)Csoregi, E.; Gorton, L;MarkeVarga, G. Anal. Chim. Actu 1993,273,5970. (19)Csoregi, E.;Jonsson-Pettersson, G.; Gorton, L/.Biotechnol. 1993,30,315337. (20)Yao, T.; Sato, M.; Kobayashi, Y.; Wasa, T. Anal. Chim. Acta 1984, 165, 291-296. (21)Sanchez, P. D.;Blanco, P. T.; Alvarez, J. M. F.; Smyth, M. R; O'Kennedy, R Electroanalysis 1990,2, 303-308. (22)Schubert, F.; Saini, S.; Tumer, A P. F. AnalXhim. Acta 1991,245,133138. (23)Garguilo, M.G.; Huynh, N.; Proctor, A; Michael, A C. Anal. Chem. 1993, 65,523-528. (24)Ohara, T. J.; Vreeke, M. S.; Battaglini, F.; Heller, A Electroanalysis 1993, 5,825-831. (25)Wang, J.; Lin,Y.; Chen, L. Analyst 1993,118, 277-280. Q 1994 American Chemical Society 0003-2700/95/0367-0094$9.00/0
sensitivity for detection of peroxides was significantly improved EXPERIMENTAL SECTION Chemicals, Horseradish peroxidase type VI-A, EC 1.11.1.7 by using a mediator.l6 HexacyanoferrateUI),20,22*26-B o-phenylene (HRP, 1100 units mg-I), 1-ethyl-3-[3-(diethylamino)propyllcardian~ine,~Oal quinone,21*22 and f e r r ~ c e n e ~and ~ sits ~ derivativeB.33 bodiiiide hydrochloride (EDC, protein sequencing grade), 2-(Nin the dissolved form have been used as mediators in peroxidasemorpholiio)ethanesulfonic acid monohydrate (MES), and tetmodified electrodes. Use of mediator free in solution is not rabutylammonium perchlorate (l”I3AP) were purchased from desirable for construction of reagentless biosensors aimed at in Sigma (St. Louis, MO). Sodium phosphate monobasic monositu monitoring of environmental and industrial processes and in hydrate, sodium phosphate dibasic anhydrous, citric acid monobienzyme electrodes, combining oxidase and peroxidase, because hydrate, acetonitrile (HPLC grade), ethyl acetate, n-hexane, ethyl the oxidized mediator formed upon electron transfer to HRP can ether, sodium sulfate anhydrous, hydrogen peroxide (30% in be reduced by the FADHz centers of the oxidase and therefore water), and glutaraldehyde (25% solution in water) were from diminish the cathodic current. ‘These problems are expected to Fisher Scientific (Tustin, CA). Aniline, silica gel (230-400 mesh, be alleviated by using mediator attached/immobilized to the 60 A), cumene hydroperoxide, and tert-butyl hydroperoxide were electrode surface. Mediators have been immobilized on electrode acquired from Aldrich (Milwaukee, WI). [ (N,NDimethylamino)surface by covalently attaching them to monomers which can then methyllferrocene methiodide was obtained from Strem Chemicals be polymerized chemically or electr~chemically.~~-~~ Osmium (Newburyport, MA). All the chemicals were used without bipyridine mediator conjugated to poly(viny1pyridine) polymer, puritication. Double distilled ultrapure water was used for which forms a hydrogel, was used to “electrically wire” HRP for preparation of the buffers and standards and for all the electromonitoring HzO2 in bienzyme electrode^.^^ chemistry work. Electropolymerization offers the advantage of providing an Synthesis. N-(Ferrocenylmethy1)aniline monomer 0 was ultrathin (nanometers to micrometers thick) uniform coating over synthesized according to the following p r o c e d ~ r e . ~In~ brief, .~ small areas of complex geometry. The method deposits film with 15 mL of aniline was mixed with 5 g of [(N,N-dimethylamino)few gross pinholes because the monomer reacts rapidly at the methyllferrocenemethiodide in water and refluxed for 3 h, when exposed surface to fill the pinholes. Depending on the choice of a reddish orange viscous oil was formed. After cooling, the water the monomer and the polymerization conditions, conducting or was decanted and the oily layer extracted in ethyl ether. This nonconducting polymer films are formed. Pyrrole-ferrocene layer was then dried overnight on sodium sulfate and chromatomonomer that can be deposited on an electrode surface by graphed on silica gel using a 1:l mixture of ethyl acetate and hexane. The first band containing the desired product was electrochemical polymerization has been used to construct a collected, and after evaporating the solvent, the residue was glucose biosensor.34 In such a biosensor, glucose oxidase was dissolved in petroleum ether and the product crystallized by entrapped in the redox polymer film deposited by electrochemical cooling in the refrigerator to 4 “C. polymerization of pyrrole. Recently, a novel ferrocene-modiiied Enzyme-Electrode Construction. A glassy carbon elecaniline polymer, deposited by electrochemical polymerization of trode (GCE; Bioanalytical Systems, Lafayette, IN) surface was an aniline-ferrocene monomer, whose thicknesses can be conprepared for immobilizationby polishing with 1pm diamond paste trolled by the electrical charge passed, has been r e p ~ r t e d . ~ ~ - ~ ~ followed by 0.05 pm y-alumina particles (Buehler, Lake Bluff, IL). The polymer has interesting electronic conductivity and a redox Electrodes were rinsed with water and ultrasonicated for 2-5 min hopping charge transfer property, which makes it an ideal after each polishing step. candidate for constniction of mediated amperometric biosensors. HRP was immobilized on prepared GCE by (1) adsorption, (2) In this paper we report on the construction, characterization, cross-linking using a bifunctional group, and (3) covalent binding. and application of a biosensor based on HRP and ferroceneAdsorption of HRP on the electrode was achieved by allowing 5 modified polyaniline film deposited on the electrode by electrop L of 10 mg mL-’ HRP in pH 6.4 phosphate buffer on the electrode chemical polymerization of N-(ferrocenylmethy1)anilinemonomer. surface to air dry followed by extensive washing with the buffer to remove unbound enzyme. To immobilize HRP by cross-linking, (26) Kulys, J. J.: Pesliakiene, M. V.; Samalius, A S. Bioelectrochem. Bioenerg. 5 p L of 10 mg mL-I HRP in pH 6.4 phosphate buffer containing 1981,8,81-88. (27) Kulys, J. J.; Laurinavicius, V.4. A; Pesliakiene, M. V.; Gureviciene, V. V. 1%glutaraldehyde was applied to the electrode surface, allowed Anal. Chim. Acta 1983,148,13-18. to air dry for 2 h, and rinsed thoroughly with the buffer. For (28) Olsson, B.; Markc-Varga, G.; Gorton, L.; Appelqvist, R: Johansson, G. Anal. covalent attachment of HRP, the polished GCE was first oxidized Chim. Acta 1988,206, 49-55. (29) Cosgrove, M.; Moody, G. J.; Thomas, J. D. R Analyst 1988,113,1811electrochemically by holding the potential at 2.2 V (vs Ag/AgCl) 1815. for 10 s in 10%HN03 and 2.5%&Cr2O7, followed by activation of (30) Wang, J.; Frieha, B.; Naser, N.: Romero, E. G.; Wollenberger, U.;Ozsoz, carboxyl groups by placing the electrode in 0.15 M EDC solution M.;Evans, 0. Anal. Chim. Acta 1991,254,81-88. (31) Wang, J.: Wu, L.-H.; Angnes, L. Anal. Chem. 1991,63,2993-2994. in 0.1 M pH 4.6 MES buffer with gentle stirring. Immobilization (32) Sanchez, P. D.; Ordieres, A J. M.; Garcia, A C.; Blanco, P. T.Electoanalysis was achieved by adding HRP into the above buffer (0.5 mg mL-’) 1991,3, 281-285. and incubating overnight at room temperature. (33) Tatsuma, T.; Okawa, Y.; Watanabe, T. Anal. Chem. 1989,61,2352-2355. (34) Foulds, N. C.; Lowe, C. R Anal. Chem. 1988,60,2473-2478. A polymer film, poly(anilinomethylferrocene) [designated as (35) Hale, P. D.; Inagaki, T.; Karan, H. I.; Okamoto, Y.; Skotheim, T. A]. Am. poly(AMFc)], of monomer I was deposited on the above prepared Chem. SOC.1989,111,3482-3484. surface from an electrolyte bath containing 5 mM I and 0.1 M (36) Hale, P. D.: Boguslavsky, L. I.; Inagaki,T.;Karan,H. I.; Lee, H. S.; Skotheim, T.A;Okamoto, Y. Anal. Chem. 1991,63,677-682. TBAP in acetonitrile, which was deaerated with argon prior to (37) Horwitz, C. P.; Dailey, G. C. Chem. Mater. 1990,2, 343-346. electropolymerization, by cycling the electrode potential at 100 (38) Honuitz, C. P.; Suhu, N. Y.; Dailey, G. C. J Electroanal. Chem. Interfacial mV between 0 and 1.1-1.3 V vs Ag/AgCl for a total of 10 Electrochem. 1992,324,79-91. (39) Rudzinski, W. E.; Walker, M.; Horwitz, C. P.; Suhu, N. Y. 1.Electroanal. Chem. Interfacial Electrochem. 1992,335,265-279.
(40) Lombardo, A; Bieber, T. I.]. Chem. Educ. 1983,60,1080-1081.
Analytical Chemistry, Vol. 67, No. 1, January I , 1995
95
c.
t f
1
1
I
1
I
1.lW
8.908
0.788
0.500
c
1.388
I
1.10~
1
0.380
8.188
I
0.908
8.780
0.508
8.388
8.188
E (V vs. Ag/AgCI)
Figure 1. Cyclic voltammograms for the electropolymerization from a 5 mM solution of I in deaerated acetonitrile with 0.1 M TBAP on bare GCE (A) and HRP-modified GCE (B); scan rate 100 mV s-l. Cyclic voltammograms of poly(AMFc)-coated GCE (C) and HRP/poly(AMFc) GCE (D) in 0.1 M deaerated phosphate buffer with 0.1 M NaC104; scan rate 50 mVs-l. Arrows indicate the trend of the CV waves during the electropolymerization process.
cycles. The electrode was rinsed with acetonitrile to remove entrapped monomer and TBAP and then stored dry at 4 "C in the refrigerator. Instrumentation. Cyclic voltammetry was performed on a Princeton Applied Research Model 263A potentiostat/galvanostat (EG&G, Princeton, NJ) interfaced to a 80486-based personal computer. Constant-potential measurements were performed on a Bioanalytical Systems voltammograph (Model CV 27) coupled to a low-current module (BAS, PA1 preamplifier), and the signal was recorded on a flat-bed chart recorder (BD-112, Kipp and Zonen, Delft, Holland). Electropolymerization and cyclic voltammetry was performed under stationary conditions in a 10 mL electrochemical cell (BAS, VC-2) placed inside a Faraday cage (BAS, (2-2). Amperometric measurements were made in either batch or flow injection mode. In the former, measurements were made under stirred conditions, to provide convective transport, in a 10 mL electrochemical cell (BAS, VC-2) placed inside a Faraday cage (BAS, C-2). Flow injection analysis (FIA)were performed using a thin-layer flow cell (BAS,CC-5) with a dual glassy carbon working electrode. A precision flow peristaltic pump (EVA Pump, Eppendorf North America, Madison, WI) was used for the delivery of mobile phase and sample. A 20 yL 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/AgC1(3 M NaC1) reference and platinum auxiliary electrodes in 0.1 M phosphate or citrate-phosphate buffer with 0.1 M sodium perchlorate. Measurement of Hydrogen Peroxide in Real Sample. HzOz concentration in the real sample was determined using the HRF'/poly(AMFc) enzyme electrode and by enzymatic method. A 2.1 mg sample of the blue gel part of Mentadent toothpaste 96 Analytical Chemisfry, Vol. 67, No. 7, January 7, 7995
was dissolved in 10 mL of the appropriate buffer, pH 4.0, 0.1 M citrate-phosphate buffer with 0.1 M NaC104 for the enzyme electrode and pH 7.0,O.l M phosphate buffer for the enzymatic assay. For the enzyme electrode, a three-point calibration curve generated immediately prior to sample analysis was used to determine HzOz concentration. Enzymatically H202 was determined by the Trinder reaction in which the quinoneimine dye produced by the reaction of HzOz with phenol and 4aminoantipyrene in the presence of HRP was monitored at 500 nm by a spectrophotometer (Cary lE, Varian, Melbourne,Australia). The reaction cocktail contained 250 yL each of 3 mM phenol and 3 mM 4aminoantipyrene, 10 pL of buffer containing a total of 2.5 units of HRP, and 200 yL of the sample in 200 yL of 0.1 M pH 7.0 phosphate buffer, the total volume being 910 yL. The HzOz concentration in the sample was calculated by use of the molar extinction coefficient of 12 700 M-' cm-' for the dye. RESULTS AND DISCUSSION
Characterizaton of the Poly(anilinomethyEemene)Fi. Panels A and B of Figure 1 show the cyclic voltammograms for the electropolymerizationof I on a bare GCE and a HRP-moditied GCE, respectively. On both these electrodes, a growth of the polymer film containing the ferrocene moiety was observed in the form of increasing anodic current corresponding to the ferrocene oxidation potential. However, the ferrocene reduction peaks on the two electrodes were different. While on the bare GCE the cathodic current peak corresponding to ferrocene increased with repeated cycling, as was observed by Horowitz and Daile~,3~ the peak current corresponding to ferrocene decreased after each cycle on the HW-modified electrode. The reason for this decrease is currently being investigated in our
50
40
1f
25
st
10
0
20 15
3
1
10
5
1 0
0
50
100
150
200
[H,Od PM Figure 2. Comparison of the response of enzyme electrodes prepared by different immobilization techniques: (*) carbodiimideimmobilized HRP/poly(AMFc)-, (0)glutaraldehydecross-linked HRP/ poly(AMFc)-, and (0) adsorbed HRP/poly(AMFc)-modified GCE to H202 in 0.1 M pH 6.4 deaerated phosphate buffer with 0.1 M NaC104. Operating potential -100 mV vs Ag/AgCI.
laboratory. Both the electrodes when transferred to aqueous medium, 0.1 M pH 6.4 phosphate buffer with 0.1 M sodium perchlorate, exhibited the characteristic ferrocene redox signal (Figure 1C,D) with large redox peak separation and a sharp anodic wave of ferrocene moieties in the polymer film. This large redox peak separation and the sharpening of the anodic peak is similar to that reported in the l i t e r a t ~ r e ~for l - ~other ~ redox polymer films in aqueous phase and is attributed to the resistance of the nonpolar compact polymeric film resulting from the slow movement of counterions into the polymeric film and the phaselike behavior of the redox site in the film during their oxidation in water, respectively. For all the polymer films, the first anodic scan in the CV showed a higher overpotential, from 40 mV and up, of the ferrocene moieties in the polymer film than in the successive scans. The anodic potential scan after the second anodic scan became consistent. These observations were similar to that observed for other redox polymer films and is attributed to the large resistance due to the low ambient electrolyte inside fresh polymer film.41344 The surface coverage of ferrocene for the bare and covalently immobilized HRP-modified electrodes, determined from the charge under the oxidation peak for ferrocene from the CV, was 2 x and 1 x mol cm-2, respectively. Enzyme Immobilization. Three different methods of enzyme immobilization, i.e. adsorption,cross-linking with glutaraldehyde, and covalent attachment using carbodiimide chemistry, were evaluated for immobilization of HRP in the construction of the enzyme electrodes (Figure 2). The response to HzO2 of the HRP/ poly (AMFc) enzyme electrodes constructed using cross-linking and covalent methods were identical and significantly higher to (41) Daum, P.;Murray, R W. J. Phys. Chem. 1981,85,389-396. (42) Willman, IC W.; Rocklin, R D.;Now& R; Kuo, IC-N.;Schultz,F. A; Murray, R W. J. Am. Chem. Soc. 1980, 102, 7629-7634. (43) Daum, P.; Murray, R W.J. Electroanal. Chem. Interfacial Electrochem. 1979, 103, 289-294. (44) Kaufman, F. B.; Schroeder, A. H.; Engler, E. M.: Kramer, S. R; Chambers, J. 8.J. Am. Chem. Soc. 1980, 102,483-488.
0
20
40
60
80
100
120
[H202l PM Figure 3. Response of (0)carbodiimide-immobilized HRP/poly(AMFc) and (m) carbodiimide-immobilizedHRP-modified GCE to H202 in 0.1 M pH 6.4 deaerated phosphate buffer with 0.1 M NaC104. Operating potential -50 mV vs Ag/AgCI.
the response of the sensor constructed using HRP immobilized by adsorption. Since the covalent technique for enzyme immobilization can be easily applied to ultramicroelectrodes and electrodes with complex geometry with better control, this method was selected for HRP immobilization in the present work. Direct electron transfer from HRP immobilized covalently on carbon fibers and graphite powder has been reported in the literature.11-lg Therefore, it was deemed necessary to determine whether there was any advantage of using the ferroceneconjugated polymer for electron transfer from HRP to the GCE. A comparison of the cathodic currents of the two enzyme electrodes (Figure 3) shows that the HRP/poly(AMFc) enzyme electrode produced approximately 5fold higher cathodic current than the covalently immobilized HRP electrode for HzOz. Hydrodynamic Voltammetry. Figure 4 shows the response of the HRP/poly(AMFc) electrode to 10 pM HzOz prepared in deaerated water and 10 p L of nondeaerated water as a function of the applied potential studied between the potentials of +0.2 and -0.2 V under deaerated conditions. While the hydrodynamic voltammogram for HzOz was similar to that reported in the literature,25330 i.e. a very small response between potentials of 0.2 and 0.0 V and then a rapid increase at more negative potentials, there is no documentation in the literature for the results obtained in this study for water. For water injections, while no response was observed for potentials between 0.2 and -0.05 V, a cathodic response at potentials of -0.1 and -0.2 V due to the reduction of molecular oxygen was observed. Furthermore, when the HRP/ redox polymer-modified electrode was poised at potentials of -0.1 and -0.2 V in nondeaerated medium, the background was significantly high and there was a continuous drift of the baseline. Based on the above results, an operating potential of -0.05 V was selected for future studies for the following reasons: (1) This potential will not require deaeration of the mobile phase and the samples. (2) If this HRP/redox polymer-modified sensor has to be converted into a bienzyme electrode using oxidase, which requires oxygen for the first step of the detection scheme to produce HzOz, then the effects of oxygen on both the background and signal will be eliminated. Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
97
2.5 I
2.0
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1
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0
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-100
-200
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Potential (mV vs. AglAgCI) Figure 4. Effect of operating potential on the response of the HRP/ poly(AMFc) electrode to (0)10 pM H202 and (m) 10 pL injection of nondeaerated water in (10 mL) 0.1 M pH 6.4 deaerated phosphate buffer with 0.1 M NaC104.
Effect of pH. Panels A and B of Figure 5 are plots of current against pH for HzOz and cumene hydroperoxide. In the pH range of 3.5-7.5, the cathodic current for both the substrates increased as the pH of the solution decreased without a clear optimum. S i c e this profile was different from the optimum between 6 and 8, reported in some of the l i t e r a t ~ r e ~ 5 Jfor ~ sboth ~ ~ ~ direct ~ and mediated electron transport from the enzyme to the electrode for both HzOz and other organic peroxides, it was decided to investigate whether this difference was due to the poly(AMFc) film. For this study, the effect of pH on the response of the enzyme electrode prepared with covalently immobilized HRP was evaluated. As is shown in Figure 5C, the pH profile of such an electrode was identical to that of the HRP/poly(AMFc) electrode without a clear optimum. This demonstrated that the poly(AMFc) film was not responsible for the modified pH profile for an HRPmodified electrode but rather tbe physicochemical properties of the immobilization support, GCE, may be contributing to the changed pH profile. Effect of Flow Rate. The effect of the buffer flow rate on the peak current during the flow injection analysis of HzOz and cumene hydroperoxide is shown in Figure 6. As the flow rate increased the response decreased steeply until a flow rate of 1.5 mL min-' for both HzOz and cumene peroxide and reached a plateau. These results are in accordance with the theoretical prediction for FIA systems with negligible mass transfer resistance in the bulk solution45and the experimental observation of many r e s e a r c h e r ~ . ~The ~ t ~response ~ * ~ ~ to cumene hydroperoxide was lower than that for HzOz by a factor of at least 2, indicating that HzOz is a preferred substrate over cumene peroxide/organic peroxides. This was also co&med by the calibration plots for different organic peroxides evaluated in this study (Figure 7). Biosensor Characterization. The calibration graphs and the analytical features of the HRP/poly(AMFc) electrode for the
peroxides evaluated are shown in Figure 7 and Table 1, respectively. As expected, the sensitivity was the highest for HzOz followed by cumene hydroperoxide and tert-butyl hydroperoxide. When the relative standard deviation (RSD) for HzOz and cumene hydroperoxide was evaluated, it was observed that the RSD for cumene hydroperoxide was 3.6 times larger than for HzOz. This high RSD can be caused by the deactivation of enzyme or/and the degradation of the mediator. The latter was suspected because of reports in the literature that there was a slow decay in the cyclic voltammetric response when ferrocene covalently immobilized to platinum electrode was cycled between ferrocene and ferricinium ~tates.4~8~ To investigate the cause of the decline in the cathodic current response of the HRP/poly(AMFc) electrode, the poly(AMFc) film was evaluated by performing CV before and after 50 repeated measurements of 100 pM cumene hydroperoxide. The results showed that compared to a 54% decrease in the cathodic current response to cumene hydroperoxide there was only an 8.5%decrease in the peak anodic current for ferrocene after 50 repeated measurements. This may indicate
(45) Olsson, B.; Lundback, H.;Johansson,G.;Scheller, F.;Netwig, J.Ana1. Chem. 1986, 58, 1046-1052. (46) Elmgren, M.; Magnus, N.; Lindquisf S.-E. Anal. Eiochem. 1993,215,261265.
(47) Lenhard, J. R; Murray,R W. J. Am. Chem. SOC. 1978, 100, 7870-7875. (48) Szenhimay, R; Yeh, P.;Kuwana, T.In Electrochemical Studies of Biological Sptems; Sawyer, D. T.,Ed.; ACS Symposium Series 38; American Chemical Society: Washington D C, 1977; pp 143-169.
3
No. 7, January 1, 7995
7
6
8
PH Figure 5. Effect of pH on the response of carbodiimide-immobilized HRP/poly(AMFc)-modified GCE to 10 pM H202 (A) and 10 pM cumene hydroperoxide (B) and the response of carbodiimideimmobilized HRP-modified GCE to 10 pM H202 (C) in 0.1 M deaerated citrate-phosphate buffer with 0.1 M NaC104. Operating potential -50 mV vs Ag/AgCI. n = 3.
~
98 Analytical Chemistry, Vol. 67,
5
4
~~~~
~
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~
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Table I.Characteristics of Calibration Graphs for the HRPiPoly(AMFc) Electrode
characteristic
hydrogen peroxide
linear range
4-90
slope (nA/pM)
0.349
intercept (d)
0.8
corr coeff
0.9982
cumene tert-butyl hydroperoxide“ hydroperoxide 10-80 160-400 0.0524 0.0222 1.05 4.78 0.9955 0.9949
40-240 0.0149 1.32 0.9993
c)
6j f
%e calibration data for cumene hydroperoxide were fitted by two straight lines.
6
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30
Flow Rate (mumin)
Figure 6. Effect of flow rate on the flow injection response of carbodiimide-immobilized HRP/poly(AMFc)-modified GCE to (A) 80 p M H202 (20 pL injection loop) and (B) 100 pM cumene hydroperoxide ( 5 0 p L injection loop). Mobile phase 0.1 M pH 4.0 citrate-phosphate buffer with 0.1 M NaC104; operating potential -50 mV vs Ag/AgCI. 50
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C
d
Flgure 8. (A) Flow injection response to (a) 15, (b) 30, and (c) 60 p M H202 and (d) to 2.1 mg of Mentadent fluoride toothpaste with baking soda and peroxide dissolved in 10 mL of buffer. (B) Threepoint calibration (0) using the data in (A) superimposed with the response to the toothpaste sample (0).Mobile phase 0.1 M pH 4.0 citrate-phosphate buffer with 0.1 M NaC104; operating potential -50 mV vs Ag/AgCI; injection loop 20 pL; carrier flow rate 0.5 mL min-I.
400
(Conc.]pM Flgure 7. Calibration plots for (0)H202, (B) cumene hydroperoxide, and (+) fert-butyl hydroperoxide. Mobile phase 0.1 M pH 4.0 citratephosphate buffer with 0.1 M NaC104; operating potential -50 mV vs Ag/AgCI; injection loop 20pL; carrier flow rate 0.5 mL min-I. n = 5.
that enzyme deactivation may be the primary reason for the response decrease. Analysis of Real Sample. The HRP/poly(AMFc) electrode was then used to measure HzOz concentration in a peroxidecontaining toothpaste. A three-point calibration curve for HzOz was constructed (correlation coefficient 0.99791, as shown in Figure 8, and the hydrogen peroxide concentration was determined to be 0.5 & 0.01% (w/w) (n = 7) in the blue peroxide gel
of the Mentadent toothpaste. This value was in very good agreement with the value of 0.44 f 0.054%(w/w) (n = 3) HzOz determined by the enzymatic method. CONCLUSIONS
The currently used “bulk technology” approach for the construction of enzyme electrodes is not conducive for the construction of ultramicroelectrodes required for in vivo monitoring and electrodes of complex three-dimensional geometry of small dimensions necessary for on-line bioprocess monitoring applications. To solve these problems, all-chemical methods of construction which use “molecular technology” instead of bulk Analytical Chemisfry, Vol. 67, No. 1, January 1, 1995
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technology are required.49 In this report we have presented a novel “integratedmolecular assembly” approach for the construction of an amperometric enzyme electrode. This new approach uses allchemical modification techniques to incorporate the necessary components of an amperometric enzyme electrodeenzyme by covalent attachment to a modified glassy carbon electrode and the redox mediator and a permselective membrane by electrochemical polymerization of the ferrocene-conjugated aniline monomer. These techniques can be easily adapted to the ~
~
~~
construction of ultramicroelectrodes and electrodes of complex three-dimensional geometry. ACKNOWLEDGMENT
This work was financially supported by the National Science Foundation Grant BCS 9309741. Received for review July 13, 1994. Accepted October 19,
1994.B AC940703X
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(49) Stoecker, P. W., Yacynych, k M. Sel. Electrode Reu. 1990, 12, 137-160.
100 Analytical Chemistry, Vol. 67, No. 1, Januaty 7 , 7995
@Abstractpublished in Advance ACS Abstracts, November 15, 1994.
