Anal. Chem. 1988, 6 0 , 2534-2536
2534
The minimum quantity of material that we have detected by using the PL methodology is -0.1 hg, corresponding to a 1.0-wL injection of a 0.0100% n-butylamine solution in hexane. The detection limit for acetic acid is significantly poorer, -0.2 mg. This variability in response is another illustration of the chemically specific interactions taking place at the semiconductor-gas interface. In principle, the use of other emissive semiconductors, having different steric and electronic surface landscapes, can increase the versatility of this detection system. Such studies are presently in progress in our laboratories.
Q, v)
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ACKNOWLEDGMENT We thank John S. Taylor, Jim Yu, and Stanley H. Langer for helpful discussions. We thank a reviewer for helpful comments. LITERATURE CITED
Time Flgure 8. Repetition of the experiment described in Figure 3, using a multicomponent sample of the indicated species. Conditions: ambient n-CdS:Te detector; 458-nm excitation; 600-nm emission; He flow rate, 70 mL/min; oven temperature, 100 'C.
found from -0.2 to 1.5 mg; these ranges should be treated as approximate since they are detector dependent. The useful working range can be extended above these concentration limits by constructing calibration curves.
Meyer, G. J.; Lisensky, G. C.; Ellis, A. 8.Proc. flectrochem. SOC. 1987, 87-9, 438-448. Meyer, G. J.; Lisensky, G. C.: Ellis, A. 8.J. Am. Chem. Sac. 1988. 110, 4914-4918. Meyer, G. J.; Luebker. E. R. M.; Lisensky, G. C.; Ellis, A. B. I n Photochemistry on Solid Surfaces; Anpo, M., Ed.; Elsevier: Amsterdam, in press. Hollingsworth, R. E.; Sites, J. R. d . Appl. Phys. 1982, 5 3 , 5357-5358 and references therein. Ellis, A. 8.I n Chemistry and Structure at Interfaces: New Laser and Optlcal Techniques; Hall, R. E., Ellis, A. 8..Eds.; VCH Publishers: Deerfield Beach, FL, 1986; Chapter 6 and references therein. Stair, P. C. J. Am. Chem. SOC. 1982. 104, 4045-4052. Karas, B. R.; Streckert, H. H.; Schreiner. R.; Ellis, A. 8.J. Am. Chem. SOC.1981, 103. 1648-1651.
RECEIVED for review May 6, 1988. Accepted July 18, 1988. This research was supported by the Office of Naval Research and the 3M Company.
CORRESPONDENCE Electropolymerized Cobalt Tetrakis(o-aminopheny1)porphyrin Film Mediated Enzyme Electrode for Amperometric Determination of Glucose Sir: In recent years, based on the recent, remarkable developments in the field of chemically modified electrodes ( I , 2), immobilized enzyme chemically modified electrodes (IECMEs), which combine the specificity and selectivity of an enzyme for its natural substrate with the advantages of electrochemical detection, have become a research area of great interest (3-17). Enzymes are fixed onto electrode surfaces, generally by covalent attachment via intermediate linkages (e.g., using cyanuric chloride (3),glutaraldehyde (4-12,18,19), carbodiimide (4-6),and boronate (20)).More recently, they are also entrapped in a polymer matrix electrochemically prepared on electrodes (14-17). These IECMEs differ from the conventional membrane-based enzyme electrodes that have been fabricated by holding a thin layer over an electrode with some type of enzyme membrane (21,22). The IECMEs are very attractive biocatalytic (potentiometric and amperometric) sensors due to their simplicity of operation, the superior amperometric response characteristics, the capability of miniaturization, etc., compared with conventional membrane electrodes. 0003-2700/88/0360-2534$0 1.5OIO
In this paper, we report the preliminary results concerning the electrode characteristics of the IECME based on bilayer-film coating for amperometric determination of glucose. The electrode substrate is coated with two kinds of polymeric films in a bilayer state, that is, first with the cobalt tetrakis(o-aminopheny1)porphyrin polymer (abbreviated as polyCoTAPP) film, prepared by electrooxidative polymerization of the monomer (23, 24), and then with the enzyme film consisting of bovine serum albumin and glucose oxidase that were held together by cross linking with glutaraldehyde. In this sensor system for estimation of glucose concentration, O2 depletion is monitored by measuring the current for O2reduction electrocatalyzed by the poly-CoTAPP film, according to the following reaction sequence: ,&D-glucose
+ O2
glucose oxidase
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D-glUCOnO-6-laCtOne D-glucono-6-lactone + H20 0 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 22, NOVEMBER 15, 1988
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2535
+
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Where reactions 1,2, and 3 correspond to the glucose-glucose oxidase enzyme reaction, the hydrolysis reaction of Dglucono-&lactone, and the electrocatalytic reaction of O2reduction by poly-Co"TAPP (23),respectively. The present study is conceptually an outgrowth of our recent work concerning coated wire ion-selective electrodes based on bilayer film coating (25, 26).
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I
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Flgure 1. Typical cyclic voltammograms of O2reduction obtained (A, B) at a bare BPG electrode and (C, D) at a poiyCoTAPP film (thickness, ca. 1 pm) coated Bffi electrode in a phosphate buffer solution (10 mM, pH 7.1): (A, C) under N, atmosphere: (B, D) under air atmosphere. Electrode area was 2.5 X lo3 cm2. Scan rate was 200 mV s-'.
EXPERIMENTAL SECTION Reagents. Tetrakis(o-aminopheny1)porphyrin(TAPP) was synthesized according to ref 27 and metalated with cobalt in refluxing pyridine under N2 atmosphere (28). Glucose oxidase (type 11, from Aspergillus niger, abbreviated as GOx) was obtained from Sigma Chemical Co. and used without further purification. Bovine serum albumin (fraction V, abbreviated as BSA) powder was obtained from Kodak Co. Glutaraldehyde was an aqueous 50% solution (Kanto Chemical Co.). P-D-Glucose (anhydrous, for biochemistry) was obtained from Merk Co. Basal-plane pyrolytic graphite (BPG) used as an electrode substrate was obtained from Union Carbide Corp. All other chemicals were of reagent grade. Apparatus. A standard three-electrode electrochemical cell was used for all the electrochemical experiments. The electrode assembly consisted of a bare BPG electrode or an immobilized cm2) enzyme chemically modified BPG electrode (area, 2.5 X as the working electrode, a sodium chloride saturated calomel electrode (SSCE) as the reference electrode, and a spiral platinum electrode as the counter electrode. For cyclic voltammetry, a home-made instrument was employed together with an X-Y recorder (Graphtec Co., Tokyo). All the experiments were performed at room temperature (25 i 1 "C). The potentials were measured and are quoted with respect to the SSCE. Preparation of GOx electrodes based on bilayer film coating. The BPG electrode substrates were coated with two kinds of polymer films in a bilayer state. The inner layer is an electroactive polymer film (the thickness is typically several micrometers) that was prepared by electrooxidative polymerization of cobalt tetrakis(o-aminopheny1)porphyrin(CoTAPP) in acetonitrile solution containing 2 mM CoTAPP and 0.1 M NaC104, according to the procedure recently reported by Bettelheim et al. (23,24).The poly-CoTAPP film coated BPG electrode thus prepared was further coated with the GOx film (thickness ca. several tens of micrometers) consisting of a matrix of BSA and GOx that were held together by cross linking with glutaraldehyde according to a published procedure (8-11,17,18). Two microliters of enzyme matrix solution consisting of 10-100 mg mL-' GOD and 15 wt % BSA in 50 mM phosphate buffer (pH 7.0) and 1.2 ILLof 25 wt 70 glutaraldehyde solution were mixed on the previously prepared poly-CoTAPP film-coated BPG electrode with a microsyringe and were allowed to cross link. After the crosslinking reaction was completed, the electrode was washed by immersion in distilled water and then in 10 wt % glycine solution to remove any glutaraldehyde excess from the electrode surface. In this case, it is expected that amino groups in the poly-CoTAPP film that still remain without participating in the electropolymerization (23,24) do take part in the cross-linking reaction and thus this contributes to the improvement in adherence of the poly-CoTAPP film and the enzyme film.
RESULTS AND DISCUSSION Prior to the examination of the electrode characteristics of the IECME sensor based on a bilayer film coating, we confirmed qualitatively the electrocatalysis of O2 reduction by the poly-CoTAPP film deposited on BPG electrodes in the same solution (phosphate buffer solution (pH 7.1) as that employed in the examination of the sensor characteristics. The details concerning the catalytic O2reduction by poly-CoTAPP and its analogues in 0.5 M H2S04 and 1 M NaOH solutions
2 1 Flgure 2. Typical steady-state current response of the IECME based on bilayer film coating to change in glucose concentration. The current was measured by holding the electrode potential at -0.55 V vs SSCE under the condition of air bubbling. The amount of GOx in the enzyme film was 80 mg cm-*. Steady-state currents 1, 2,3,4. and 5 correspond to glucose solutions of 0, 0.28,0.55,0.82,and 1.1 mM, respectively. The arrows indicate the injection points of glucose solution. Other experimental conditions are the same as those in Figure 1.
have been recently reported by Bettelheim et al(23). Figure 1 shows the typical cyclic voltammograms of O2 reduction obtained a t a poly-CoTAPP film-coated BPG electrode and at a bare BPG electrode. It can be seen that curve D in Figure 1,observed in an air-saturated solution, shows a greatly enhanced reduction current and a positive shift in the cathodic peak potential of more than 400 mV, compared to curve B in the same figure. This fact demonstrates the catalytic reduction of O2 via the mediating Co"'/"TAPP couple in poly-CoTAPP film to H202. The redox response corresponding to the Co(III/II) process of the poly-CoTAPP film was not clearly observed especially for thicker fiis (e.g., curve C in Figure l),but the formal potential for the Co(III/II) process, obtained from the cyclic voltammograms for polyCoTAPP films containing ca. 1to 10 monolayer equivalents of electroactive porphyrin, was ca. -0.2 V vs SSCE a t pH 7.1 in agreement with that reported by Bettelheim et al. (23). In Figure 2 is shown a typical steady-state current response of the bilayer-film-coated GOx electrode to change in glucose concentration in air-saturated Na2HP04-NaH2P04solutions (10 mM, pH 7.1). In this case, the current was measured by holding the electrode potential a t -0.55 V vs SSCE under the condition of air bubbling. After injection of glucose solution, the electrode showed a rapid response time reaching 95% of the steady-state current in ca. 30-40 s. This response time is approximately comparable to or more rapid than those for previously reported amperometric enzyme electrodes (4,8, 9, 29-36). It is thought that this short response time of the IECME is primarily the result of the nonconventionally thin enzyme layer. As can be expected from the principle of this sensor system (represented by reactions 1-3), the reduction current was found to decrease with increasing the concentration of glucose when other experimental conditions were held constant. The steady-state current responses for various concentrations of glucose were employed to construct response curves for glucose. The typical results are shown in Figure 3. These results are compared with the blank current responses obtained at the bilayer-film-coated electrode without GOx in the outer BSA film. With this electrode the current is almost constant irrespective of the concentration of glucose in sample solutions. On the other hand, the currents obtained a t the
2536
Anal. Chem. 1900, 60,2536-2537
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0
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Figure 3. Calibration curves of experimental steady-state current vs glucose concentration for the IECMEs based on bilayer film coating with and without GOx. The amount of GOx confined in enzyme films was (0)0, (0)13, and (0)80 mg cm-'. Other experimental condiions are the same as those in Figure 2.
IECMEs decreased gradually with increasing the glucose concentration, depending on the amount of GOx confined in the enzyme films. For the electrode with higher loading of GOx, the dynamic range where the observed current significantly changes with glucose concentration is more narrow, as expected. The initial slopes of the current vs glucose concentration curves shown in Figure 3 were 109 and 49 nA mM-' for the electrodes with GOx of ca. 80 and 13 mg cm-2, respectively, in the enzyme films. Thus, in principle, by measuring the O2reduction current obtained at the IECME based on the bilayer film coating, one can evaluate the concentration of glucose in solution. The present electrodes do not possess a dynamic range (typically ca. 0.5-15 mM) wide enough to apply them to assay of whole blood or serum samples (4,8, 9, 29-36). However, with the kinetics of the overall enzyme reaction taken into consideration, it will be possible to expand the dynamic range, for example, by controlling the GOx loading in the enzyme film (as suggested by the data in Figure 3), its thickness, and the mass transport rates of O2 and glucose into it. The sensitivity and detection limit of our sensor were almost comparable to those of the previously reported conventional GOx sensors (4,8,9,29-36). Further study concerning the electrode characteristics of the IECME sensor (e.g., linearity of calibration, selectivity, and long-term stability) as well as the kinetics of the overall enzyme reaction is currently conducted and will be reported later. Registry No. poly-CoTAPP, 107667-67-4; 02,7782-44-1; glucose, 50-99-7; glucose oxidase, 9001-37-0; glutaraldehyde, 111-30-8;graphite, 7782-42-5.
LITERATURE CITED (1) Murray, R. W. €lectroanalytical Chemistry; Bard, A. J., Ed.; Dekker: New York, 1984; Vol. 13, p 191.
(2) Hillman, A. R. Elechochemical Science & Technobgy 1, Unford, R. G.. Ed.; Elsevier Applied Sclence: Amsterdam, 1987; p 103. (3) Ianniello, R. M.; Yacynych, A. M. Anal. Chem. 1981, 53, 2090. (4) Bourdillon, C.: Bourgeois, J. P.; Thomas, D. J. Am. Chem. Soc. 1980, 102,4231. (5) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. 0.; Aston, W. J.; Higgins, I.J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56,661. (6) Ianniello. R. M.; Lindsay, T. J.; Yacynych, A. M. Anal. Chem. 1982. 54, 1980. (7) Ianniello, R. M.; Yacynych, A. M. Anal. Chim. Acta 1981, 131, 123. (8) Kamin, R. A.; Wilson, G. S . Anal. Chem. 1980, 52, 1198. (9) Shu, F. R.; Wilson, G. S. Anal. Chem. 1976, 48, 1679. (10) Castner, J. F.; Wlngard, L. B. Anal. Chem. 1984, 56,2891. (11) Yao. T. Anal. Chim. Acta 1983, 748. 27. (12) Yao, T.; Fujio, Y.; Wasa, T. Nippon Kagaku Kaishi 1984, 1335. (13) Ikeda, T.; Hamada, H.; Miki, K.; Senda, M. Agric. Biol. Chem. 1985, 498,541. (14) Umana, M.; Waller, J. Anal. Chem. 1986. 58,2979. (15) Foulds, N. C.; Lowe, C. R. J Chem. Soc., Faraday Trans. 1 1988, 82, 1259. (16) Bartlen, P. N.; Whitaker, R. G. J. Electroanal. Chem. 1987, 224, 37. (17) Shinohara, H.; Chiba, T.; Aizawa, M. Proc. Sensor Symp, 6th 1986, 207. (18) Liu, C. C.; Lahoda, E. J.; Gaiasco, R. T.; Wingard, R. B. Biotecnol. Bioeng. 1975, 17, 1695. (19) Stoner, G. E.; Gileadi, E.; Ludlon, J. C.; Kirwan, D. J. Biotechnol. Bioeng. 1975, 17, 455. (20) Narasimhan, K.; Wingard, L. B., Jr. Anal. Chem. 1986, 58, 2984. (21) Guilbault, G. G. Handbook of Enzymatic Methods of Analysis; Dekker: New York, 1976. (22) Carr, P. W.; Bowers, L. D. Chemical Analysis; Elving, R. J., Winefordner, J. D., Eds.; Wiley-Interscience: New York, 1980, Vol. 56, p 206, and references therein. (23) Bettelheim, A.; White, B. A.; Murray, R. W. J. €lectroanal. Chem. i9a7 .- -. , -7 1. .7 , 371 - . .. (24) Bettelheim, A.; White, B. A.; Raybuck, S. A,; Murray, R. W. Inorg. Chem. 1987, 26, 1009. (25) Oyama, N.; Hlrokawa, T.; Yamaguchi, S.; Ushizawa, N.; Shimomura, T. Anal Chem. 1987. 59. 258. (26) Oyama, N.; Ohsaka, T.; Yoshimura, F.; Mizunuma, M.; Yamaguchi, S.; Ushizawa, N.; Shimomura, T. J. Macromol. Sci., Chem., in press. (27) Collman. J. P.; Gagne, R. R.; Reed, C. A,; Halbert, T. R.; Land, G.; Robinson, W. T. J. Am. Chem. SOC. 1975, 97, 1427. (28) Buchler. J. W. I n Porphyrlns and Mefalloporphyrins; Smith, K. M.; Ed.; Elsevier: Amsterdam, Oxford, and New York, 1975; p 181. (29) Karube, I.; Mitsuda, S.; Suzuki, S. Eur. J. Appl. Microbiol. 1979, 7 , 343. (30) Guilbault, G. G.; Lubrano, G. J. Anal. Chim. Acta 1974, 69,183. (31) Lubrano, G. J.; Guilbault, G. G. Anal. Chim. Acta 1978, 97,229. (32) Thevenot, D. R.; Sternberg, R.: Coulet, P. R.; Laurent, J.; Gautheron, D. C. Anal. Chem. 1979, 51, 96. (33) Mell, L. D.; Maloy, J. T. Anal. Chem. 1976, 48, 1597. (34) Ikeda, S.;Aoyama, N.; Ito, K.: Ookura. K.; Ichihashi, H.; Kondo, T. Nippon Kagaku Kaishi 1980, 1554. (35) Lobel, E.; Rishpon, J. Anal. Chem. 1981, 53,51. (36) Gough, D. A.; Lucisano, J. Y.; Tse, P. H. S. Anal. Chem. 1985. 57, 2351.
Noboru Oyama* Takeo Ohsaka Masaya Mizunuma Department of Applied Chemistry for Resources Tokyo University of Agriculture and Technology Koganei, Tokyo 184, Japan M a r i Kobayashi Department of Chemistry Japan Women's University Bunkyo-ku, Tokyo 112, Japan RECEIVED for review May 10,1988. Accepted August 2,1988. The present work was partially supported by Grant-in-Aid for Scientific Research (No. 62217006) for N. Oyama, from the Ministry of Education, Science and Culture, Japan.
An Alternative Method for Gas Chromatographic Determination of Volatile Organic Compounds in Water Sir: The EPA purge and trap method (1)is widely accepted as the method of choice for routine analysis of volatile organic
compounds (VOC) in water. Recently, Pankow and Rosen (2)summarized the status of this method and suggested a new
0003-2700/88/0360-2536$01.50/0 0 1988 American Chemical Society
