Anal. Chem. 1997, 69, 2682-2687
Design of Enzyme Electrodes for Extended Use and Storage Life Golam Faruque Khan*
Department of Chemical Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 Wolfgang Wernet
New Materials Research Department, International Research Laboratories, Ciba-Japan, 10-66 Miyuki-cho, Takarazuka 665, Japan
A novel design for an extended-life amperometric enzyme electrode is presented. The enzyme electrode is fabricated by arranging the three components of a biosensor (an electrode material, an enzyme, and a stabilizer) on a shapable electroconductive (SEC) film (a polyanion-doped polypyrrole film) surface by a layer-after-layer approach. First, a thin and compact Pt black layer is prepared onto the SEC film by the heat-press method. Then, an ultrathin layer of L-lactate oxidase (LOx) is cast on the platinized SEC film, and after drying, a thin gelatin layer is prepared on the LOx-cast SEC film. Finally, the dried layer assembly is cross-linked by exposing it to a diluted glutaraldehyde solution for a very short time. The performance of the developed sensor is evaluated by a FIA system at 37 °C and under continuous polarization at 0.4 V (vs Ag/ AgCl). The developed sensor shows remarkably improved performance. The sensor produces a large response current with a wide linear dynamic range. The sensor shows an extended working life of more than 2 months and a shelf life of about 2 years when it is stored dry in a freezer at -18 °C and about 1 year when it is stored at room temperature. Over the last two decades, a considerable body of literature has accumulated regarding the design of practical amperometric enzyme electrodes for the analysis of clinically important metabolites and industrial monitoring.1-3 Most biosensors reported to date have been prepared and used under laboratory conditions, demonstrating the feasibility and characteristics of the biosensor, while the problem of long-term operational and storage stability, which is crucial for commercial development, has rarely been addressed. The use life of an enzyme electrode usually depends on the retention of the biological activity of the enzyme. This may vary from days to months, depending on the stability of the enzyme used, the method of manufacture, and the operational and storage conditions.4-6 * To whom correspondence should be addressed. Present address: L&FC, R&D, P&G Far East, Inc., 17-Koyo-cho, Naka 1-Chome, Higashi, Nada-ku, Kobe 658, Japan. (1) Mizutani, F.; Asai, M. In Bioinstrumentation; Wise, D. L., Ed.; Boston, MA, 1990, p 317. (2) Scheller, F., Schubert, F., Eds. Biosensors; Elsevier: Amsterdam, 1992; p 85. (3) Schuhmann, W.; Schmidt, H. L. In Advances in Biosensors; Turner, A. P. F., Ed.; JAI Press: London, 1992; Vol. 2, p 79.
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Glucose oxidase (GOx) was used to prepare the first enzyme electrode to measure glucose.7 The reasons were twofold: first, glucose was an important analyte in medical diagnostic tests, and second, glucose oxidase was found to be a very stable enzyme. Since that time, constructing a multitude of enzyme electrodes has not been a major problem; the problem has been to obtain stable, long-lived enzyme electrodes which can stand prolonged storage and perform well after being in use for extended periods. Unfortunately, few enzymes are as stable as GOx. Indeed, even GOx presents problems. There are dozens of patents on glucose sensors and perhaps hundreds of papers, yet only a few successful commercial probes are available. One of the major problems in expanding the use of other enzymes besides GOx has been the fabrication of long-lived biosensors with a commercially acceptable use and storage life. In a previous report,8 we developed a stable glucose sensor by adsorbing GOx at the Pt black particles prepared by electrochemical platinization on a shapable electroconductive (SEC) film. In this study, a lactate sensor is prepared by the same method of adsorbing LOx at the electrochemically prepared platinized SEC (P-SEC) film. However, it is observed that the developed lactate sensor shows poor operational stability upon continuous polarization. The deactivation of the adsorbed LOx upon continuous polarization of potential is considered to be the main reason behind the poor operational stability. To overcome this problem of limited use life, a novel biosensor design is described in this paper. The sensor is fabricated by a layer-after-layer configuration on a flexible conductive film. A schematic representation of the preparation of the layered sensor in shown in Figure 1. In the first step, a very thin (3-5 µm) and compact Pt black layer is prepared on a SEC film. Then, an ultrathin enzyme layer is cast on the Pt black layer and dried. Finally, a stabilizing layer (4-6 µm at dry condition) is prepared on the enzyme layer, and the dried layer assembly is cross-linked by chemical or photochemical means. Various stabilizers can be used in the layered structure: proteinaceous stabilizers, such as bovine serum albumin (BSA) and gelatin; and polymeric stabilizers, such as poly(vinyl alcohol), poly(vinyl pyrrolidone), poly(4) Guilbault, G. G. Enzyme Microb. Technol. 1980, 2, 258-264. (5) Mascini, M.; Iannello, M.; Palleschi, G. Anal. Chim. Acta. 1983, 146, 135148. (6) Coulet, P. R.; Bardeletti, G.; Sechaud, F. In Bioinstrumentation and Biosensors; Wise, D. L., Ed.; Marcel Dekker Inc.: New York, 1991; Chapter 25, p 753. (7) Clark, L. C., Jr.; Lyons, C. Ann. N. Y. Acad. Sci. 1962, 105, 20-45. (8) Khan, G. F.; Wernet, W. J. Electrochem. Soc. 1996, 143, 3336-3342. S0003-2700(96)01208-5 CCC: $14.00
© 1997 American Chemical Society
To asses the benefit of the layered design, the performance of the developed lactate sensor is evaluated in a flow injection analysis (FIA) system by applying an extreme experimental condition. Sensors are polarized continuously at 0.4 V, the temperature of the system is kept constant at 37 °C, and samples are automatically measured at 30 min intervals.
Figure 1. Schematic illustration of the layered sensor on the shapable electroconductive (SEC) film.
(ethylene glycol), polyurethane, (diethylamino)ethyldextran, etc. In the present study, a proteinaceous stabilizer, gelatin, is used. The enzyme layer is sandwiched between a Pt black layer and a gelatin layer. In this assembly, the enzymatically generated H2O2 can easily be oxidized at the Pt black surface at a relatively low potential, thus producing a large response current, and it recycles the oxygen needed for the enzymatic reaction. In addition, the gelatin layer ensures both operational and storage stability of the underlying enzyme layer. The release of enzymes from the enzyme layer and the dissolution of the gelatin layer are completely overcome by the cross-linking of the enzyme and gelatin at the sandwiched condition. The cross-linking introduces a clamping effect on the enzyme which produces a strap across the surface of the protein, reducing domain mobility and degradation of the 3-D structure.9 However, uncontrolled cross-linking severely suppresses the activity of the most of enzymes. Some enzymes such as LOx are completely deactivated upon crosslinking with glutaraldehyde unless the extent of the cross-linking is controlled. In this study, this is done by performing the crosslinking in dry conditions and by controlling the time of exposure to the glutaraldehyde solution. The applicability of the layered system is, therefore, extended to almost any oxidase enzyme producing H2O2. This design is particularly useful for those enzymes which are generally deactivated by cross-linking with glutaraldehyde. In the present study, the feasibility and the characteristics of the layered sensor system have been examined with an important enzyme, L-lactate oxidase (from Pediococcus sp.), for the determination of lactate. Blood lactate concentration is an indicator of certain pathological states, such as shock, respiratory insufficiencies, and heart disease,10 and a very useful indicator for assessing the general physical condition of an athlete or a racing animal.11 Several LOx-based enzyme sensors have been described in the literature.12-17 However, only a very few enzyme-based lactate analyzers are available on the market. A limited use and shelf life and a short dynamic range of detection are reported to be the main problems for commercial development of the lactate sensor. (9) Gibson, T. D.; Woodward, J. R. In Biosensors and Chemical Sensors; Edelman, P. G., Wang, J., Ed.; ACS: Washington, DC, 1992; Chapter 5, p 40. (10) Mascini, M.; Moscone, D.; Palleschi, G. Anal. Chim. Acta. 1984, 157, 45. (11) Toffaletti, J. G. Clin. Chem. News 1989, 9, 14. (12) Hu, Y.; Zhang, Y.; Wilson, G. S. Anal. Chim. Acta. 1993, 281, 503-511. (13) Urban, G.; Jobst, G.; Aschauer, E.; Tilado, O.; Svasek, P. Varahram, M. Sens. Actuators B 1994, 18-19, 592-596. (14) Mizutani, F.; Yabuki, S.; Hirata, Y. Anal. Chim. Acta. 1995, 314, 233-239. (15) Huang, Y. L.; Khoo, S. B.; Yap, M. G. S. Anal. Chim. Acta 1993, 283, 763771. (16) Mullen, W. H.; Churchouse, S. J.; Keedy, F. H.; Vadgama, P. M. Clin. Chim. Acta 1986, 157, 191-198. (17) Shimoji, N.; Naka, K.; Uenoyama, H.; Hamamoto, K.; Yoshika, K.; Okuda, K. Clin. Chem. 1993, 39 (11), 2312-2314.
EXPERIMENTAL SECTION Materials. L-Lactate oxidase (LOx, 40 units/mg, 40% protein) from Pediococcus sp. was purchased from Asahi Chemical Co. (Tokyo, Japan). Gelatin from porcine skin (type A, 300 bloom) was purchased from Sigma. Hexachloroplatinic acid was purchased from Aldrich. All other chemicals were reagent grade and were used without further purification. Milli-Q water was used in all aqueous solutions. The buffer solution was prepared by mixing 0.1 M K2HPO4 and 0.1 M KH2PO4 (PBS), and pH was adjusted to 7.0. Preparation of the SEC Film. The SEC film was synthesized on rotating, stainless steel electrodes in a propylenecarbonate solution containing 5% (v/v) pyrrole, 50 mM poly(β-hydroxy ethers) (sulfation ratio, 0.25), and 3% water under galvanostatic conditions at a current density of 2 mA/cm2 and passing a charge of 5 C/cm2. The film was peeled off, washed with propylenecarbonate for several hours and then overnight with ethanol, and dried under vacuum at 50 °C for 12 h. The synthesized film was 130-150 µm thick and had a conductivity of 15-20 S/cm. Details of the synthesis and properties are reported elsewhere.18-20 The solution side of the SEC film was used for platinization. Preparation of the Pt Black Layer. (1) Electrochemical Platinization Method. Platinization of SEC films was carried out galvanostatically in an electrolyte solution containing 10 mg/ mL of hexachloroplatinic acid and 0.2 mg/mL of lead acetate. The electrolyte solution also contained 0.1 M HCl. SEC films were cut into 10 cm × 10 cm squares and pasted on a stainless steel plate with the help of Teflon adhesive tapes and carbon paste. This SEC film electrode was placed in parallel with another stainless steel plate counter electrode (larger in size) and a Ag/ AgCl reference electrode in the electrolyte solution. The galvanostatic deposition was carried out at a fixed current of 2 mA/ cm2 for 10 min (Pt black loading, about 600 µg/cm2). After platinization, the platinized SEC films were washed overnight with plenty of 0.1 M phosphate buffer solution (PBS) of pH 7 to neutralize the acid and dried under vacuum. (2) Heat-Press Method. For the preparation of fine Pt black particles, diluted hexachloroplatinic acid solution was chemically reduced by NaBH4. A diluted suspension of these particles was made in 2-methoxyethanol. This suspension was uniformly spread over a SEC film to a Pt black loading of about 3 mg/cm2 and was dried under vacuum at 60 °C. Then, the Pt black-loaded SEC film was pressed by passing through two very slowly rotating hot steel rollers. The temperature of the rollers was kept at around 150 °C. The SEC film became very soft at this temperature; thus, the compressed Pt black particles partially penetrate into the film and make a very uniformly compact and stable Pt black layer. Preparation of the Enzyme Layer. (1) Adsorption of LOx. The electrochemically prepared P-SEC film was cut into the desired pieces. The electrode side (nonplatinized side) of the (18) Wernet, W.; Stoffer, J. Eur. Pat. Appl. EP 358188, 1990. (19) Wernet, W. Synth. Met. 1991, 41, 843. (20) Yamato, H.; Wernet, W.; Ohwa, M.; Rotzinger, B. Synth. Met. 1993, 5557, 3550.
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P-SEC film was covered by adhesive Teflon tape. This P-SEC film was incubated in a LOx solution (10 mg/mL in PBS of pH 7.0) for the adsorption of LOx. After 3 h, the film was taken out of the enzyme solution, and the remaining enzyme solution was completely wiped out from the film and dried at room temperature. (2) Casting of LOx. The heat-pressed P-SEC film was cut into the desired pieces. A LOx solution (20 mg/mL in PBS of pH 7.0) was uniformly spread over the Pt black layer by successive addition and dried at room temperature for 1-2 h. Preparation of the Stabilizer Layer. Here, 10 µL/cm2 of 5% gelatin solution (prepared in water and incubated 30 min at 37 °C before use) was uniformly spread over the enzyme-loaded P-SEC film and dried at room temperature for 1-2 h. This film was then dipped into a glutaraldehyde solution (5% in water) for a short time, washed immediately with a copious amount of water, and dried at room temperature for several hours. Dipping in the glutaraldehyde solution was not done when BSA was cast on the enzyme-loaded film instead of gelatin. Then, 5 µL/cm2 of glutaraldehyde solution was spread over the BSA-cast film, and after drying, the film was thoroughly washed with water and dried at room temperature. The sensor film was then stored in a freezer at -18 °C until further experimentation. Measurement of the Sensor Performance. The performance of the developed sensors was characterized by two types of measurement: (1) batch analysis and (2) flow injection analysis. (1) Batch Analysis. Sensor films were cut into small pieces, and electrodes were made with the help of aluminum sheets, carbon paste, and Teflon tape. The sensor response was determined by conventional hydrodynamic amperometry. This was done by immersing the sensor electrode with an Ag/AgCl reference electrode and Pt counter electrode in a magnetically stirred PBS solution of pH 7.0. Then, an oxidative potential of 0.4 V was applied to the working electrode. When the background current became flat, an appropriate amount of concentrated lactate solution was introduced into the cell for a preselected concentration. The faradaic current increased as a function of time and eventually reached a flat value within 20-30 s. The increased current was taken as the sensor response for the corresponding substrate concentration. A concentrated substrate was successively added to the same batch for the calibration purpose. (2) Flow Injection Analysis (FIA). The biosensor film was punched to a size of 3.5 mm diameter, fixed at an opening of 2.0 mm diameter of a specially designed flow-through cell (purchased from Bioanalytical Systems, West Lafayette, IN), and pressed with a screw-driven brass rod. The flow cell consisted of a working electrodes, a Ag/AgCl reference electrode, and a stainless steel cell body as a counter electrode. The flow cell was fixed in an oven, and the temperature of the oven was controlled at 37 °C. The carrier stream was 0.1 M PBS of pH 7.0. An uniform flow of 0.2 mL/min was controlled by a pump. A continuous polarization potential of 0.4 V (vs Ag/AgCl) was applied to the working electrode. The samples (usually 100 µL) were injected automatically by an autoinjector at 30 min intervals. A total of 48 samples were measured in a day: 35 samples of 5 mM lactate, two lots of 5-6 samples of different concentrations of lactate for the daily calibration, 2-3 samples of 2 mM H2O2, and one sample of 200 mM of lactate for the maximum response current (Imax). To assess the working stability of the sensor, the mean response for the 5 mM lactate over a day was compared with the first day response. 2684 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
Figure 2. Effect of cross-linking time on the response current of lactate sensors prepared by adsorption of LOx at the electrochemically platinized SEC films. Batch measurement at room temperature. (9) Direct cross-linking; (O, b, 0) indirect cross-linking. Exposure time (s): (O) 15, (b) 30, (0) 60.
Unless otherwise mentioned, all the response current was based on a geometric area of 1 cm2. RESULTS AND DISCUSSION Effect of Cross-Linking. It is well known that enzymes lose activity when they are cross-linked with glutaraldehyde. To overcome this problem, a promising method of indirect crosslinking of enzymes was developed.8,21,22 The enzyme is crosslinked under dry conditions through a dried gelatin layer by exposing it to a diluted glutaraldehyde solution for a very short time. Thus, the extent of the cross-linking can be substantially controlled by the exposure time. Figure 2 shows the effect of exposure time on the response current of the lactate sensor prepared by the adsorption of LOx at the electrochemically platinized SEC films. The response current decreased with the increase of exposure time. A negligible response current was observed when the enzyme was cross-linked directly in the presence of BSA. These results suggest that LOx severely deactivates upon cross-linking with glutaraldehyde, and the extent of deactivation can be reduced by indirect cross-linking with a short exposure to the glutaraldehyde solution. For subsequent studies, lactate sensors were prepared by a 30 s exposure to the glutaraldehyde solution. Linear Range of Detection. The linear range of the calibration graphs in Figure 2 was limited up to about 2 mM without a cover membrane. The linear range can be extended by covering the enzyme electrode with a semipermeable membrane, which restricts the mass transport of lactate into the catalytic enzyme layer. Several commercially available membranes were tested. Hydrophilic polycarbonate (PC) membrane from Nucleopore (thickness, 5-10 µm; pore size, 0.015 µm) produced the necessary extended linear range for the measurement of normal and abnormal clinical lactate levels (0-16 mM). The linear range of the lactate sensor covered with a polycarbonate membrane was significantly extended up to the 30 mM level. The minimum detectable lactate concentration was about 5 µM, and the slope of the calibration graph was about 5 µA/mM. (21) Khan, G. F.; Ohwa, M.; Wernet, W. Anal. Chem. 1996, 68, 2939-2945. (22) Khan, G. F.; Ohwa, M.; Wernet, W. Proceeding of the East Asian Conference on Chemical Sensors, X’ian, China, 1995; pp 287-291.
Figure 3. Operational stability of the lactate sensor prepared by adsorption of LOx at the electrochemically prepared SEC film under continuous polarization at 0.4 V in a FIA system. (O) 37 °C; (b) 25 °C. The 5 mM lactate responses were compared. PC membrane was used.
Use Life of the Lactate Sensor Prepared by the Adsorption of LOx. The operational stability of the lactate sensor at 25 and 37 °C under continuous polarization at 0.4 V is shown in Figure 3. The sensor response continuously increased to about 120% in the first week of operation at both temperatures. The initial increase of the response current was related to the change of the diffusional properties of the gelatin matrix and the wetting of the Pt black layer. The gelatin matrix might swell to some extent in the first few days of operation, resulting in an easier excess of the analyte to the enzyme layer. This increase of the initial response current is a common phenomenon in the case of gelatincovered biosensors.8,21 The H2O2 oxidation activity of the P-SEC was found to increase slightly in the first few days of operation and then became relatively stable for several weeks (not shown here).8 The lactate response current decreased very sharply at the end of the second week. Thereafter, a slow decrease was found to continue for another 2 weeks. The shapes of the stability curves were similar at both temperatures; however, the sensor operated at 25 °C showed about 15% higher retention of the initial response current at the end of the fourth week. The stability of this lactate sensor was quite limited as compared to that observed for a glucose sensor prepared similarly with an electrochemically prepared SEC film.8 The deactivation of LOx was the main reason behind this poor use life. The elevated temperature, the enzymatic generation of H2O2, the adsorption of enzymes onto the surface of the Pt black particles, and the continuous polarization potential are supposed to be the major causes for the LOx deactivation under the present experimental conditions. It is well known that the temperature stability of the enzyme significantly improves when the enzyme is immobilized by covalent binding in a friendly environment, especially by cross-linking in a proteinaceous matrix.23 As LOx was cross-linked with glutaraldehyde in a gelatin matrix, it is expected that the elevated temperature would not be a major obstacle for the extension of its use life. Therefore, only about a 15% increase of stability was observed when the operating temperature was dropped from 37 to 25 °C. Although the elevated temperature is a common enzyme deactivating factor, it is not solely responsible for the poor stability of the lactate response. (23) Moussy, F.; Jakeway, S.; Harrison, D. J.; Rajotto, R. V. Anal. Chem. 1994, 66, 3882-3888.
It is generally suspected that H2O2 causes the inactivation of enzymes,24,25 especially the reduced form of GOx, which is irreversibly inactivated by H2O2 in a concentration-dependent fashion, independent of whether the enzyme is immobilized or not.26,27 Recently, von Woedtke et al.28 reported that, for the amperometric glucose sensor, the generated H2O2 does not cause any considerable inactivation of GOx. Sarcosine oxidase (from Corynebecterium sp.) is reported to be safe in the presence of H2O2.29 H2O2 is also suspected to cause the inactivation of LOx; however, no report has clearly demonstrated the effect of H2O2 on LOx, especially the effect of enzymatically generated H2O2 on the stability of the biosensor response. If the effect is similar to that for GOx, then it would be negligible under the present experimental conditions as the enzyme always remained in the oxidized state. The drop in pH upon the oxidation of H2O2 may also have a harmful effect on the enzyme. However, as the lactate sample was in contact with the sensor film for only 30 s/measurement, and the sensor film was continuously washed with a strong phosphate buffer solution (0.1 M), there would not be a big change in pH that might cause significant deactivation of the enzyme. Therefore, under the present experimental conditions, the elevated temperature and presence of H2O2 might be responsible for a limited deactivation of the enzyme. It is quite likely that there was a monolayer coverage of enzymes on the surface, as LOx was freely adsorbed onto the Pt black particles surface. The interaction of the monolayer-adsorbed enzyme with the solid surface has a direct effect on the activity and the stability of the adsorbed enzyme.30 When the enzyme electrode is continuously polarized, the electrostatic interaction between the surface and the adjacent amino acid residues of the protein may change in a way that may cause the enzyme to be deactivated with time. No adequate explanation exists for the combined effect of the potential on the adsorbed enzymes; however, one can speculate that some critical amino acid residues may oxidize (or reduce, depending on the applied potential) or/ and the structural conformation of the enzyme may change in such a way that cofactors are released. The effect of the potential should be limited in the close proximity of the electrode surface. Consequently, enzymes close to the surface may be affected by the potential. Therefore, in the layered assembly, the enzyme is cast on a compact Pt black layer in such a way that there is a multilayer coverage of enzymes on the surface. In the course of continuous polarization, the enzymes very close to the surface may deactivate, but the rest of the enzyme remains active. This is the concept behind the layer-after-layer design of the biosensor. Preparation of a Compact Pt Black Layer. The electrochemical platinization produced a highly porous Pt black matrix, as shown in Figure 4A. The thickness of a 600 µg/cm2 loaded Pt black layer was estimated from the SEM observation to be about 6-8 µm. LOx readily penetrates into the pores and adsorbs when the LOx solution is spread over the electrochemically prepared (24) Scheller, F. W.; Pfeiffer, D.; Hintsche, R.; Dransfeld, I; Nentwig, J. Biomed. Biochim. Acta 1989, 48, 891-896. (25) Gough, D. A. Horm. Metab. Res., Suppl. Ser. 1988, 20, 30-33. (26) Greenfield, P. F.; Kittrell, J. R.; Rawrence, R. L. Anal. Biochem. 1975, 65, 109-124. (27) Kleppe, K. Biochemistry 1966, 5, 139-143. (28) von Woedtke, T.; Fischer, U.; Abel, P. Biosens. Bioelectron. 1995, 9, 6571. (29) Sakslund, H.; Hammerich, O. Anal. Chim. Acta 1993, 268, 331-345. (30) Gibson, T. D.; Woodward, J. R. Anal. Proc. 1986, 23, 360-362.
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Table 1. Effects of the LOx Loading on the Response Characteristics of the Layered Sensors LOx loading (µg/cm2)
sensitivitya (µA mM-1 cm-2)
linear range of detectionb (mM)
Imaxc (µA/cm2)
8 40 160 320
4.0 5.5 6.5 4.2
0-15 0-20 0-30 0-30
68.0 230.0 256.0 210.0
a The slope of the linear calibration graphs. b The linearity deviation was
