Anal. Chem. 1989,61, 2352-2355
2352
(8) (9) (10) (11) (12) (13)
Kovach, P. M.; Deakin, M. R.; Wightman, R. M. J . Phys. Chem. 1986, 90. 4812. Szabo, A.; Cope, D. K.; Tallman, D. E.; Kovach, P. M.; Wightman, R. M. J. Electrcenal. Chem. Interfeciel Electrochem. 1987, 277,417. Jaeger, J. C.; Clarke, M. A. R o c . R. Soc. Edinburgh, A 1942, 6 1 , 229. Jan, C.G.; McCreery, R. L. Anal. Chem. 1988, 56,2771. Jan, C.-C.; McCreery, R. L. J. Electroanel. Chem. InterfacialElectrochem. 1987, 220, 41. Deputy, A. L.; McCreery, R. L. J. Electroanal. Chem, InterfacialElectrochem. 1988, 257, 57. Wu, H. Ping; McCreery, R. L. J. Electrochem. SOC.1989, 736, 1375. Amatore, C. A,; Deakln, M. R.; Wightman. R. M. J. Electroanel. Chem. Interfecfal Elecb.ochem. 1988, 206. 23. Feldberg, S. I n ElectroenalyHcel Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1989; Vol. 3. p 199.
(14) Pletcher, D.; Josiin, T. J . Electroanat. Chem. InterfacielElectrochem. 1974, 49, 171. (15) Bard, A. J.; Fauikner, L. R. Electrochemical Methods; Wlley: New York. 1980; Chapter 5. (16) Wu. H. Ping Ph.D. Dissertation, The Ohio State University, 1989. (17) Mark, B. J.; Scheellne, A. Spectrochim. Acta €3 1987, 428, 1063. (18) Blades, M. W. Appl. Spectrosc. 1982, 37,371. (19) Young, S. J. J. Quant. Spectrosc. Radiat. Transfer 1981, 25,479. (20) Caudill, W. L.; Ewing, A. G.; Jones, S.; Wightman, R. M. Anal. Chem. 1983,55,1877.
RECEIVED for review May 10,1989. Accepted August 9,1989. This work was supported by the Chemical Analysis Division of the National Science Foundation.
Enzyme Monolayer- and Bilayer-Modified Tin Oxide Electrodes for the Determination of Hydrogen Peroxide and Glucose Tetsu Tatsuma, Yusuke Okawa,' and Tadashi Watanabe* Institute of Industrial Science, University of Tokyo, Roppongi, Minato-ku, Tokyo 106, Japan
An enzyme-bmed amperometrlc blosensor for hydrogen peroxide was devekprsd, employby a horscHadlsh peroxidase (HRP) monolayer covekntty attached to a tin oxide electrode and a dlsealved electron mediator. The 88nsoc can determine hydrogen peroxide at levels down to lod M and can be applied to a flow system. StaMlity ol the eiectrode, kinetics of theslwlaceprocess, andthe effldendes ofdfferenl mediators were studled. As an extendon, glucose oxidase (GOx) was chemically bound to the HRPmodllkd electrode. A OOx/HRP Mlayer+nodMd electrode thus obtalned exttibk much better performance In glucose detection limit, sensitivity, and r e sponse speed than previously reported GOx monolayerinodHied electrodes.
a one chip device sensor with reduced fabrication cost. Moreover, monolayer immobilization would provide sensors with reproducibility regarding the amount of enzyme molecules carried by each plate, on which each molecule would function effectively. Rapid removal of reaction products from the surface region, and simpler mode of mass transfer which facilitates kinetic analysis, are additional advantages. In the present work, development of an efficient hydrogen peroxide sensor was envisaged employing an immobilized horseradish peroxidase (HRP) monolayer coupled with a dissolved electron mediator. A hydrogen peroxide sensor can be applied to sensors for other substrates by coupling it with an enzymatic system which produces hydrogen peroxide (1,3,4). In view of this, GOx was utilized for constructing a GOx/HRP bilayer-modified electrode as a hydrogen peroxide based glucose sensor.
INTRODUCTION The determination of hydrogen peroxide is of practical importance in chemical, biological, clinical, and many other fields. However, conventional methods for the determination of hydrogen peroxide do not satisfy, a t the same time, sensitivity, reliability, and operational simplicity. Enzyme electrodes have been reported as the sensor for hydrogen peroxide (1-8). Among these, the amperometric sensors based on electron transfer between an enzyme and the electrode (3-8) are promising in fabricating sensitive and linearly responding devices. Though a direct electron transfer is possible between an electrode and a peroxidase catalyzing the reduction of hydrogen peroxide (4-6,8), this is generally a slow process on common electrode materials. An appropriate electron donor can mediate the electron transfer between peroxidase and an electrode (3,7),and hence such a mediator is expected to improve the performance of a peroxidase-based hydrogen peroxide sensor. In recent papers we described fabrication of glucose sensors modified with a glucose oxidase (GOx) monolayer (9-11). Chemical immobilization of an enzyme as a monolayer gives
EXPERIMENTAL SECTION A tin(1V) oxide coated glass plate (Nippon Sheet Glass) was chosen as the electrode for its chemical (12) and electrochemical (13)stability and the ease in chemically modifying the surface with functional groups (14). The plate was treated successively with a 10% aqueous solution of (3-aminopropy1)triethoxysilane (APTES) for 1 h at 50 "C, then with a 2.5% glutaraldehyde aqueous solution (Wako Pure Chemical, practical grade, unless otherwise noted) for 30 min at room temperature, and finally with a pH 5.9 citrate-buffered solution of horseradish peroxidase (EC 1.11.1.7,Sigma Chemical Co., type VI) for 15 min at room temperature, to obtain an HRP-modifiedelectrode. For comparison, a cross-linked HRP membrane-modifiedelectrode was prepared by casting a HRP/glutaraldehyde mixed solution onto a tin oxide electrode pretreated with APTES. A GOx/HRP bienzyme electrode was obtained by further treatments of an HRP monolayer-modified electrode with glutaraldehyde (15min) and GOx (EC 1.1.3.4,Boehringer Mannheim GmbH, Grad I) (15min) solutions. These enzyme electrodes were stored in the working buffer under refrigeration (below 10 "C). The sensor signal was obtained as a cathodic current with a conventional three-electrodesystem in a 0.1 M citrate buffer (pH 5.9) containing an electron mediator at 30 "C. Ag/AgCl and Pt-black were employed as reference and counter electrodes, respectively. Ferrocenemonocarboxylicacid, ferrocenedicarboxylic acid, ferrocenecarboxaldehyde (Aldrich Chemical Co.), and potassium ferrocyanide (Junsei Chemical co.)were used as electron
Present address: Department of Image Science and En 'neering, Faculty of Engineering, Chiba University, Yayoi-cho, Cfiba 260, Japan.
0003-2700/89/0361-2352$01.50/0
0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989
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2353
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Flgm 1. Typical time response of the HRP-modlfied electrode at H202 concentratkn of 5.0 X lo-' M. Concentration of ferrocenecarboxylic acid is 0.2 mM. Potential applled Is +150 mV vs Ag/AgCI.
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[HzOZI/M Figure 2. Cathodlc current increase vs H202 concentration profiles for the HRP-modlfled electrode (1-3) and a bare tin oxide electrode (4-6). Concentration of ferrccenecarboxyllc ackl is 1.0 mM (1, 4),0.2 mM (2, 5), or 0.05 mM (3, 6). Electrode potential is +150 mV vs Ag/AgCI.
meditors without further purification. Flow injection analysis (FIA) was performed with a conventional system (10). A 31% hydrogen peroxide aqueous solution was determined by titration with a potassium permanganate standard solution. Calibration curves for batchwise measurement are obtained through successive injections of diluted peroxide standard solutions with Eppendorf pipets. A calibration wrum 'PreciFlo" was purchased from Boehringer Mannheim GmbH.
RESULTS AND DISCUSSION Response Properties of the Hydrogen Peroxide Sensor. Figure 1 shows a typical time response of the HRP-modified electrode to a 5 X lo-' M hydrogen peroxide solution containing ferrocenemonocarboxylic acid (0.2 mM) as an electron mediator. The applied potential was +150 mV vs Ag/AgCl. The first peak current was adopted as the output signal, and its dependence on the concentration of hydrogen peroxide is depicted in Figure 2. The second peak current noted in Figure 1 generally did not bear a linear relation to the hydrogen peroxide concentration, and occasionally, was not observed. Figure 2 shows that the sensor output exhibits saturation corresponding to an upper detection limit which depends on the mediator concentration. As the mediator concentration increases, the upper limit increases, but concomitantly the fluctuation of the output current becomes greater. A kinetic analysis described below substantiates the observed relationship between the mediator concentration and the upper
Figure 3. Time courses for the surface coverage of HRP (0)and the sensor response to a 1.0 X lo-' M hydrogen peroxide solution (A), both normalized to 1.O at the lnltiil state. The response of the sensor
employing glutaraldehyde from Sigma is also depicted (A).
limit. Good linearity of the calibration curves in the region below the upper limit indicates superior reliability of the sensor. The three sets of data were acquired with independently fabricated electrodes; these data are good of reproducibility in performance of the enzyme monolayer-carrying sensor. In Figure 2 are also plotted the cathodic currents for reduction of hydrogen peroxide on a bare tin oxide electrode. The dependence of the magnitude of these currents on the mediator concentrationsuggests that ferrocenemonocarboxylic acid can directly reduce hydrogen peroxide. These currents are however at least an order-of-magnitude smaller than the catalytic current at the HRP-modifiedelectrode, so that signal correction was practically unnecessary. Cross-linked HRP membrane-carryingelectrodes exhibited only 4-5 times as high response as the monolayer-modified one, in spite of a roughly @-fold higher surface density of HFtP molecules than the latter. Furthermore, the membrane-carrying electrodes showed worse linearity and larger scatter in sensitivity among the plates prepared. Stability of the HRP Electrode. The surface coverage of HRP on the electrode was indirectly estimated from spectrophotometric measurements on quartz plates modified with HRP through aforementioned procedures, because the absorption tail of tin oxide extending into the visible range interfered with direct measurements. The fact that tin oxide and silica exhibit nearly the same reactivity toward organosilanes (14) would ensure the validity of this method. A molar absorption coefficient of 9 X lo4 M-' cm-' for HRP (due to the heme moiety) at 403 nm (15) was used. The surface coverage of HRP for freshly prepared plates was found to be ca. 4.3 X 10l2 molecules cm-2 (or about 2300 A2 per HRP molecule); this value indicates that the enzyme molecules are immobas a roughly monomolecular layer. Figure 3 shows the temporal evolution of the surface coverage and of the sensor response to a 1.0 X lo4 M hydrogen peroxide solution. The near-parallelism seen between these two quantities suggests that the decrease in the response is mainly due to the detachment of the enzyme molecules rather than their inactivation. It is recently stated that the reaction of glutaraldehyde with an amino group is a complicated one (16,17), and that the mode of coupling depends on a support to which the enzyme is immobilized (16),and/or on the purity grade of glutaraldehyde solution (17). Use of glutaraldehyde from Sigma Chemical (grade 11) improved the stability (Figure 3). Treatment of the modified electrode with a sodium borohydride aqueous solution for the purpose of reducing (stabilizing) the Schiff base bonding resulted in a response decrease by 50% and no improvement in stability. Elucidation
2354
ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989
I0-8
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Figure 4. Simulated sensor outputs (curves)and experimental ones (symbols) for mediator concentrations of 1.0 mM (1, O),0.2 mM (2, 0),and 0.05 mM (3, A).
E,b mV re1 vs Ag/ re1 response AgCl responsec timeC
ferrocenemonocarboxylicacid +150 1.0 (std) 1.0 (std) 1.6 ferrocenedicarboxylicacid +150 0.8 ferrocenecarboxaldehyde t100 0.2 1.5 potassium ferrocyanide +lo0 0.7 1.4 Mediator concentration = 0.2 mM. Potential applied. eResrJonseto a 1.0 X lo4 M hvdroaen Deroxide solution. of the cause(s) for the enzyme detachment is of much practical significance but is beyond the scope of the present work. Other modes of coupling, such as amide bonding, may render the enzyme-immobilized electrode more stable. Kinetic Analysis. The reaction of HRP with HzOz and an electron donor as mediator (Med) can be formulated as follows (18): ferriperoxidase compound I compound I1
-
+ HzOz
+ Med(red) + Med(red)
compound I
compound I1
(1)
+ Med(ox)
ferriperoxidase
(2)
+ Med(ox) (3)
The good agreement between the time-courses of the HRP surface coverage and of the sensor response (Figure 3) suggests that the present system is not diffusion limited. Then, application of the steady-state approximation to the surface concentration of ferriperoxidase, compound I, and compound I1 leads to the following equation for the stationary catalytic current i: l / i = ks/[H2OZ] + kM/[Med(red)l
10-5
10-4
10-3
10-2
[Glucosel/ M Figure 5. Dependence of cathodic current increase on glucose concentration for the GOx/HRP bilayer-modmed electrode. Concentration of mediator is 0.2 mM. Electrode potential is 4-150 mV vs Ag/AgCI.
Table I. Efficiencies of Electron Mediators in HzOe Detection
electron mediatorn
I"
(4)
where kS and kM are constants, on the assumption that i is proportional to the generation rate of Med(ox). Plots of l / i (the output signal was taken as i) vs 1/[Hz02]for a series of measurements gave averaged values of ks and k M (mean f standard error) as (1.50 f 0.08) X loF8and (1.24 f 0.07) X lo4 M cm2nA-', respectively. Figure 4 shows the calibration curves simulated with these k s and k~ values. The curves agree closely with the experimental values in the region of relatively low concentration, thus ensuring the validity of the surface process analysis according to eq 1-3. Evaluation of Mediators. The efficiencies of other electroactive species as the mediator have been evaluated and the results are summarized in Table I. Ferrocenemonocarboxylic acid is thus far the best choice with regard to both
sensitivity and response speed of the sensor. For a thorough evaluation of the efficiency of mediators, however, a difference in redox potentials between the mediator and HRP, an effect of steric hindrance, and a charge or a hydrophobicity of the mediator should be further taken into account. Application to the Flow System. The HRP-modified tin oxide plate was used as a working electrode in an electrochemical detector for a flow injection analytical system. The sensor was operated at a potential of +150 mV vs Ag/AgCl, and at a flow rate of 0.1 mL min-'. A 100-pL aliquot of a sample solution made up with a carrier buffer containing 0.2 mM ferrocenemonocarboxylicacid was injected. This method enabled determination of hydrogen peroxide down to a concentration of lo-' M, and the assay of each sample was completed within 2-4 min. Optimization of the operational conditions is currently attempted in view of improving the sensitivity and analysis repetition frequency. GOx/HRP Bienzyme Electrode as Glucose Sensor. Figure 5 depicts the performance of the bienzyme electrode as a glucose sensor. In comparison with the GOx monolayer-modified electrode described in a previous work (9),the present sensor exhibits about 10-fold higher sensitivity, 50-fold lower detection limit, and 6-fold faster response. This is in line with the acceleration of hydrogen peroxide redox processes, which are slow on a bare tin oxide electrode (Figure 2). The bienzyme electrode was then used to assay the glucose concentrationin a serum,with a nominal concentrationof 14.1 mM as determined by the supplier by means of the hexokinase and GOx-PAP methods. An aqueous solution obtained by 200-fold dilution of the original serum with the working buffer, thus possessing a nominal glucose concentration of 7.1 X M, gave a sensor response corresponding to a glucose concentration of 8.7 X M on the calibration curve (Figure 5). The sensor response, however, contained a reduction current arising from unidentified electroactive substances in the serum. Thus, the net response after subtraction of the cathodic current at a bare tin oxide electrode from the apparent response corresponded to a glucose concentration of 7.4 X M, which is in good agreement with the nominal value. In most of our previous works (9-11) hydrogen peroxide generated by glucose oxidation was directly oxidized on a tin oxide electrode. The high overpotential for direct oxidation of hydrogen peroxide necessitated sensor operation at +800 to +900 mV vs Ag/AgCl, so that electroactive substances present as impurities in samples are liable to interfere with the assay. In contrast, the coupling of HRP with GOx in the
Anal. Chem. 1989, 61, 2355-2361
present system permits sensor operation at substantially milder potentials (+lo0 to +150 mV vs Ag/AgCl, see Table I), and hence the sensor output is contaminated to a lesser extent with redox reaction of indifferent substances. Dilution of samples is not preferred for practical use. In view of this, the linear range of the sensor should be extended up to levels of concentration in sera. A work is currently under way toward this end.
ACKNOWLEDGMENT The authors are grateful to Professors S. Mizusawa and T. Ohno and Dr. H. Kobayashi, Faculty of Engineering, Chiba University, for their help in the FIA experiments. LITERATURE CITED Nagy, 0.; von Storp, L. H.; Guilbautt, 0. 0. Anal. Chim. Acta 1973, 66, 443-455. Aizawa. M.; Karube. I.: Suzuki, S. Anal. Chim. Acta 1974, 69, 43 1-437. Hahn, Y.; Olson, C. L. Anal. Chem. 1879, 51, 444-449. Kulys, J. J.; Pesliakiene, M. V.; Samalius, A. S. Bhlectrochem. Bioenerg. 1881, E , 81-88. ~ ~ lJ. yJ.;~Samelius, , A. s. Liet, TsR &ks/u Akad. Darb., ser, 1982, 3-9; Chem. Abstr. 1982, 97, 68449m.
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Kulys, J. J.; Samalius, A. S. Elektrokhimiya 1984, 20, 637-641. Frew, J. E.; Harmer, M. A.; Hill, H. A. 0.; Libor, S. I. J. Electroanel. Chem. 1988, 201, 1-10, Armstrong, F. A.; Lannon, A. M. J. Am. Chem. SOC. 1987. 109, 721 1-7212. Watanabe, T.; Okawa, Y.; Tsuzuki, H.; Yoshda, S.; Nfhei, Y. Chem. Lett. 1988. 1183-1186. Okawa, Y.; Tsuzuki, H.; Yoshida, S.; Watanabe, T. Anal. Sci., in press. Tsuzukl, H.; Watanabe, T.; Okawa, Y.; Yoshida, S.; Yano. S.; Koumoto, K.; Komiyama, M.; Nihei, Y. Chem. Lett. 1988, 1265-1268. Strojek, J. W.; Kuwana, T. J. Electroanal. Chem. 1968, 16,471-483. Armstrona, N. R.: Lin, A. W. C.; Fuiihira. M.; Kuwana. T. Anal. Chem. 1876, 48; 741-750. Moses, P. R.; Wier, L.; Murray, R. W. Anal. Chem. 1975, 47, 1882-1 086. Yamazaki, I.; Yokota, K. Mol. Cell. Biochem. 1973, 2 . 39-52. Maklno, K.; Maruo, S.; Morita, Y.; Takeuchi. T. Biotechnol. Bioeng. 1988, 31, 617-619. (17) Kirkeby, S . ; Jakobsen, P.; Moe. D. Anal. Lett. 1987, 20, 303-315. (18) Yamada, H.; Yamazaki, I. Arch. Biochem. Biophys. 1974, 165, 728-738.
RECEIVED for review June 8,1989. Accepted August 10,1989. This work was supported in part by the Asahi Glass Faundation and Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (No. 63108001 and 63604517).
Interdigitated Gate Electrode Field Effect Transistor for the Selective Detection of Nitrogen Dioxide and Diisopropyl Methylphosphonate Edward S. Kolesar, Jr.,* and John M. Wiseman Air Force Institute of Technology, Department of Electrical and Computer Engineering, Wright-Patterson Air Force Base, Dayton, Ohio 45433-6583
An interdigitated gate electrode field effect translstor (IGEFET) coupled to an electron beam evaporated copper phthalocyanine thin flim was used to selectlveiy detect partper-biiiion concentration levels of nitrogen dloxlde (NO,) and dbopropyl methy@hoqhonate (DIMP). The sensor is exdted with a voltage pulse, and the time- and frequency-domain responses are measured. The envelopes of the magnitude of the normalized dlfference frequency spectrums reveal features that unambiguously distinguish NO, and DIMP exposures.
INTRODUCTION Two critical groups of environmentally sensitive contaminants are the oxides of nitrogen and the organophosphorus pesticides and their structurally affiliated compounds. It is now recognized that even trace amounts of these noxious pollutants may have an adverse effect on certain ecological systems. This recognition motivates the development of sensitive and selective personal monitoring instrumentation to detect subthreshold levels of these toxic compounds. The oxides of nitrogen, particularly nitrogen dioxide (NO2), are unintentionally emitted into the atmosphere from a number of industrial stacks and the exhaust of automobiles. In addition, NOz is known to evolve from the detonator chemical matrix in certain munitions as they age and cause corrosion of the electrical firing mechanism ( 1 ) .
In contrast to the NO2 pollutant, the organophosphorus pesticides and structurally related compounds are synthesized and valued for their deleterious effect and persistence, and their distribution is essential for their efficacy. A significant portion of the organophosphoruscontaminants contain either the phosphoryl or thiophosphoryl group. Since diisopropyl methylphosphonate (DIMP) is a phosphoryl-containingcompound, has low toxicity, and has been used in prior detector development investigations, it was selected as a model compound in this research (2-8). In recent years, coated bulk-wave piezoelectric quartz crystal microbalances (2-6,9, IO) and surface acoustic wave devices ( 11-15) have been investigated as candidate detector technologies for NO2 and the organophosphorus compounds. However, significant limitations associated with these technologies include the lack of selectivity, sensitivity to moisture, and overall response reproducibility. This paper reports the electronic properties that are modified when an interdigitated gate electrode field effect transistor (IGEFET) is coupled to an electron-beam evaporated copper phthalocyanine (CuPc) thin film which is used to selectively detect parts-per-billion (ppb) concentration levels of NO2 and DIMP. The IGEFET’s operation is based on the sensitivity of the field effect transistor’s output current to changes in the molecular structure or chemical composition of the thin film which covers the interdigitated gate electrode. These molecular and compositional changes are manifested as a correspondingchange in the gas-sensitive film’s dielectric relaxation function or, more succinctly, as a change in the
This article not subject to U.S. Copyright. Published 1989 by the American Chemical Society
