Anal. Chem. 1997, 69, 2343-2348
Characterization of Immobilization of an Enzyme in a Modified Y Zeolite Matrix and Its Application to an Amperometric Glucose Biosensor Baohong Liu,† Renqi Hu,‡ and Jiaqi Deng*,†
Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China, and Quality Assurance Division, Shanghai Volkswagen Automotive Company Limited, Shanghai 201805, People’s Republic of China
A new approach to construct an amperometric biosensor is described. Without using bovine serum albuminglutaraldehyde, glucose oxidase (GOx) was immobilized on a dealuminized Y zeolite (DAY)-modified platinum electrode to construct a glucose sensor. The large specific surface area of the zeolite substrate resulted in high enzyme loading. The immobilized GOx in this manner was stable and could maintain its high activity for at least 3 months. The interactions between the zeolite and the enzyme were investigated by means of Fourier transform infrared spectra, and the pore distribution and the surface acid property of DAY were preliminarily studied. The results showed that the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme immobilization. This resulting biosensor exhibited good reproducibility and selectivity, owing to the uniform pore structure and unique ion-exchange property of the zeolite. The biosensor responded rapidly to glucose in the linear range from 2.0 × 10-6 to 3.0 × 10-3 M, with a detection limit of 0.5 µM. Improvement of the performance and long-term stability of enzyme electrodes has been one of the main focuses of biosensor research since the development of the first glucose biosensor by Clark and Lyons in 1962.1 Among the numerous advances reported in this field, the immobilization of enzymes on electrodes in the design and optimization of biosensors has attracted great interest. The conventional techniques for enzyme immobilization include covalent attachment to the electrode surfaces,2-4 entrapment by ion-exchange polymers,5,6 conducting polymers, and nonconducting polymers,7-12 and cross-linking in bovine serum albumin-glutaraldehyde13 or regenerated silk fibroin14 immobilization matrix, etc. †
Fudan University. Shanghai Volkswagen Automotive Co. Ltd. (1) Clark, L. C.; Lyons, C. Ann. N.Y. Acad. Sci. 1962, 102, 29. (2) Narasimham, K.; Wingard, L. B., Jr. Anal. Chem. 1986, 58, 2984. (3) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615. (4) Oisson, B.; Lundback, H.; Johansson, G.; Scheller, F.; Nentwig, J. Anal. Chem. 1986, 58, 2984. (5) Firtier, G.; Vaillancourt, M.; Belanger, D Electroanalysis 1992, 4, 275. (6) Mizutani, F.; Yabuki, S.; Katsura, T. Anal. Chim. Acta 1993, 274, 201. (7) Khan, G. F.; Kobatake, E.; Ikariyama, Y.; Aizawa, M. Anal. Chim. Acta 1993, 281, 527. (8) Cosnier, S.; Innocent, C. Anal. Lett. 1994, 27, 1429. (9) Shaolin, M. J. Electroanal. Chem. 1994, 370, 135. (10) Zhang, Z.; Bao, W.; Liu, C. Talanta 1994, 41, 875. (11) Pandey, P. C. J. Chem. Soc., Faraday Trans. 1988, 84, 2259. (12) Wang, J.; Wu, H. Anal. Chim. Acta 1993, 283, 683. (13) Mascini, M.; Iannello, M.; Palleschi, G. Anal. Chim. Acta 1983, 146, 135. ‡
S0003-2700(96)00930-4 CCC: $14.00
© 1997 American Chemical Society
Since 1970s, some inorganic materials, such as silica, alumina, glass, hornblende, zeolite, etc., have been proven to be promising as the immobilization matrices15-21 due to their good mechanical, thermal, and chemical stability. Zeolites are preferred since they have unique structural characteristics and are resistant to biodegradation. Meanwhile, zeolite-modified electrodes have attracted considerable interest, owing to their unique ion-exchange and electrocatalytic properties.22 Yet, the immobilization of enzymes on such zeolite-modified surfaces has not been used in connection with amperometric biosensors. The aim of the present study is to improve the analytical performance of biosensors through the immobilization of enzymes on a zeolite-modified electrode. As an enzyme immobilization matrix, zeolite can entrap enzymes only through physical or chemical actions after its pretreatment, without employing the conventional cross-linkers.23,24 It has been utilized to immobilize invertase25 and trypsin26 in industrial production. However, the zeolite pore diameter was too small, and it normally required the introduction of surface-active groups for binding enzymes.25-27 With this in mind, we regulated the zeolite’s pore size and surface properties28 in order to make it more suitable for enzyme loading before it was used in the enzyme electrode. Compared with an unmodified one, a modified zeolite which possesses mesopores is more favorable for enzyme immobilization. Biosensors constructed with such modified zeolites have many advantages over other natural or synthetic materials, such as long-term stability, high sensitivity and selectivity, fast response, and good reproduc(14) Qian, J.; Liu, Y.; Liu, H.; Yu, T.; Deng, J. J. Electroanal. Chem. 1995, 397, 157. (15) Weetall, H. H.; Havewala, N. B.; Pitcher, W. H.; Detar, C. C.; Vann, W. P.; Yaverbaum, S. Biotechnol. Bioeng. 1974, 16, 295. (16) Lee, Y. Y.; Fratzke, A. R.; Wun, K.; Tsao, G. T. Biotechnol. Bioeng. 1976, 18, 389. (17) Johnson, D. B.; Thornton, D.; Ryan, P. D. Biochem. Soc. Trans. 1974, 2, 494. (18) Thornton, D.; Byrne, M. J.; Flynn, A.; Johnson, D. B. Biochem. Soc. Trans. 1974, 2, 1360. (19) Thronton, D.; Flynn, A.; Johnson, D. B.; Ryan, P. D. Biotechnol. Bioeng. 1975, 17, 1679. (20) Flynn, A.; Johnson, D. B. Int. J. Biochem. 1977, 8, 243. (21) Flynn, A.; Johnson, D. B. Int. J. Biochem. 1977, 8, 501. (22) Rolison, D. Chem. Rev. 1990, 90, 867. (23) Doudelka-hep, M.; Strike, D. J.; Rooij, N. F. Anal. Chim. Acta 1993, 281, 466. (24) Ioanis, K.; Adam, H. Anal. Chem. 1992, 64, 1008. (25) Iyengar, L.; Prabhakara Rao, A. V. S. J. Gen. Appl. Microbiol. 1981, 27, 339. (26) Mukherjea, R. N.; Bhattacharya, P.; Ghosh, B. K. Biotechnol. Bioeng. 1977, 19, 1259. (27) Iyengar, L.; Prabhakara Rao, A. V. S. J. Gen. Appl. Microbiol. 1982, 28, 255. (28) Schwarz, J. A. J. Catal. 1988, 114, 433.
Analytical Chemistry, Vol. 69, No. 13, July 1, 1997 2343
ibility. So far, the intermolecular interactions between zeolites and enzymes are not clear, and the attempt to investigate how the structural and acidic properties of matrices affected the adsorption of enzymes is an attractive goal. In this work, a high-performance biosensor was constructed for the first time, based on the immobilization of glucose oxidase on a dealuminized Y zeolite (DAY)-modified platinum electrode according to a very simple technique. Such a porous substrate could provide not only a large specific surface area for high enzyme loading but also a desirable microenvironment to transform the enzymatically produced hydrogen peroxide more efficiently to electronic signal. The experiments indicated that both the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme adsorption. The performance of the prepared enzyme sensor and the application of this sensor to the determination of glucose in human serum were investigated. It is hoped that the attractive properties of zeolite enzyme electrodes would find various practical applications. EXPERIMENTAL SECTION Reagents. Glucose oxidase (GOx, from Aspergillus niger, EC 1.1.3.4. 150 000 units g-1) was purchased from Sigma Chemical Co. D-Glucose and poly(vinyl alcohol) (PVA, average degree of polymerization 2000 ( 100) were obtained from Shanghai Chemical Reagent Co. Glucose solutions were allowed to mutarotate overnight prior to use. All solutions were freshly prepared with doubly distilled water and analytical grade chemicals. A 0.1 M phosphate buffer solution (pH 7.0) served as the supporting electrolyte. Apparatus. Experiments were carried out using a conventional three-electrode system consisting of a saturated calomel reference electrode (SCE), a platinum wire counter electrode, and a zeolite-modified working electrode (a PVA membrane electrode was also used for comparison). Amperometric measurements were carried out with the FDH 3204 cyclic voltamperograph (Scientific Equipment Co., Fudan University, China), in connection with a Type 3086 X-Y recorder (Tokyo, Japan). A magnetic stirrer and a stirring bar provided convective mass transport. Fourier transform infrared (FT-IR) spectra were obtained in the range of 2000-400 cm-1 on a Mattson 1020 FT-IR instrument at room temperature. The X-ray diffraction (XRD) results were measured on a Rigaku D/MAX-IIA XRD diffraction meter (Japan). Preparation of Modified Y Zeolite. NaY was purchased from Fushun Chemical Engineering factory and used as received. XRD results show high purity, and the lattice parameter a0 is calculated to be 24.67 Å. Modified Y zeolite (dealuminized Y-zeolite) was prepared according to the hydrothermal treatment29 of NH4Y, which was obtained by exchanging NaY with 2 M NH4NO3 four times. NH4Y was deep-bed calcinated under self-steaming conditions in a quartz reactor with i.d. 30 mm at 650 °C for 2 h; the depth of the zeolite bed was 70 mm, and the pressure of the desorbed H2O and NH3 over the zeolite was maintained at 1 kg/ cm2. H2SO4 (0.2 M) was used for the extraction of extralattice aluminum (FLA) species. The acid was added dropwise under intense stirring of the zeolite-water suspension. The optimum rate of acid addition was 1.0 mL of H2SO4 min-1 g-1 of zeolite. Then the dealuminized NH4Y was treated under self-steaming conditions at 650 °C for 2 h again, and the dealuminized Y zeolite (29) Patzelova, V.; Drahoradova, E.; Tvaruzkova, Z.; Lohse, U. Zeolites 1989, 9, 74.
2344 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
Table 1. Pore Characterization of DAYa ABET (m2/g)
Ame (m2/g)
Sme (nm)
Vme (cm3/g)
Vtotal (cm3/g)
584.47
115.90
16.0
0.2107
0.3750
aA BET, total specific surface area; Ame, mesopore area; Sme, mesopore size; Vme, mesopore volume; Vtotal, total pore volume
(DAY) was thus obtained. The XRD results show that the relative crystallinity of DAY is 74% that of NaY, and the lattice parameter a0 is 24.32 Å. Measurement of the Pore Distribution and the Point of Zero Charge of DAY. The adsorption and desorption isotherms of N2 were measured using a Digisorb 2600 instrument. The pore size distribution, relevant specific area, and pore volume were calculated according to the method of Barrett et al.30 from the adsorption branch and presented in Table 1. The point of zero charge of zeolite was determined by mass titration according to the procedure described by Subramanian et al.31 When an oxide is placed in solution, the pH of the solution changes in response to the amount of oxide added. As a consequence, the pH will decrease upon addition of oxide into the solution if the pH of the fresh solution is higher than the point of zero charge. If enough oxide is added, the pH value will reach a definite point, and this is the point of zero charge (PZC). Varying amounts of zeolite (typical values of DAY/water by weight were 0.01, 0.1, 1, 5, 10, and 20%) were added to water, and the resulting pH values were measured after 24 h of equilibration. A Model PHS-3C pH meter (the Second Analytical Instrument Factory of Shanghai), calibrated by using solutions of pH 4.00 and 10.00, was used to measure the pH. All measurements were carried out using air-saturated distilled deionized water. An open system approach was preferred because of its convenience. It has been shown that the measurement of the point of zero charge by mass titration is only slightly affected by the presence of weakly adsorbing ions like CO32- and HCO3-.31 Construction of Enzyme Biosensor. A platinum disk electrode (2.0 mm in diameter) was polished with 0.3, 0.1, and 0.05 µm alumina particles, rinsed thoroughly with deionized water between each polishing step, and then washed with 1:1 nitric acid, acetone, and doubly distilled water in an ultrasonic bath successively and dried in air before use. A pretreated zeolite gel was formed by vigorously stirring 2 mg of zeolite powder with 1 mL of H2O and 10 µL of 10% poly(vinyl alcohol) (PVA) for 2 h. With a micropipet, aliquots (10 µL) of such a zeolite gel or aliquots (6 µL) of 1% PVA colloid were deposited on a platinum electrode surface and allowed to dry under ambient conditions. In this way, the zeolite-modified electrode (DAY/Pt) or PVA membrane electrode (PVA/Pt) was obtained. The uptake of GOx was accomplished by immersing this electrode in a 0.1 M phosphate buffer solution (pH 4.5) which contained 5 wt % GOx for 24 h to complete the saturation adsorption. Then, the GOx-loaded electrode was rinsed with doubly distilled water to ostracize the non-firmly adsorbed GOx until no enzyme could be detected32 in the blank solution where the electrode was immersed. Thus, the zeolite-modified enzyme electrode was obtained. (30) Barrett, E. P.; Joyner, L. G.; Halenda, P. H. J. Am. Chem. Soc. 1951, 73, 373. (31) Subramanian, S.; Noh, J. S.; Schwarz, J. A. J. Catal. 1988, 114, 433. (32) Lowry, O. H.; Rosebrough, N. J.; Lewis, F. A.; Randall, R. J. J. Biol. Chem. 1951, 193, 265.
Figure 2. Influence of pH on immobilization of glucose oxidase on DAY-modified electrode at room temperature. Figure 1. FT-IR spectra of (a) NaY, (b) GOx-NaY, (c) DAY, and (d) GOx-DAY in the range of 2000-400 cm-1 at room temperature.
Experimental Procedure. Amperometric response of the enzyme electrodes to glucose was measured in a stirred, airsaturated cell containing 5 mL of 0.1 M phosphate buffer (pH 7.0) by applying a potential of +0.55 V (vs SCE) to the enzyme electrodes. When the electrode had reached a steady state, aliquots of stock glucose solution were added, and the response current for the oxidation of enzymatically liberated hydrogen peroxide was measured. Unless otherwise indicated, the temperature was kept at 30.0 ( 0.5 °C during the measurements. RESULTS AND DISCUSSION Intermolecular Interaction between Zeolite Matrix and Glucose Oxidase. Type Y zeolite is an alkali salt of aluminosilicate having a maximum internal pore diameter of 0.8 nm. It is normally unreactive and requires the introduction of surfaceactive groups for binding enzymes. However, a dealuminized Y zeolite (DAY) has formed mesopores (from 2.0 to 60.0 nm) which were proved by the hysteresis loop in the adsorption and desorption isotherms of N2. The pore distribution was calculated according to the method of Barrett et al.30 from the adsorption branch and presented in Table 1. The meospore volume was about 56% of the total volume, which favors the adsorption of glucose oxidase. The interactions between DAY and glucose oxidase were also studied by the infrared spectra. The Fourier transform infrared spectra of NaY, GOx-NaY, DAY, and GOx-DAY are shown in Figure 1. It can be seen that NaY had adsorption bands at 1006 and 783 cm-1, characteristics of frame symmetric (rOTOf, T ) Si, Al) and asymmetric (rOTfrO, T ) Si, Al) flexible vibrations.33 But in the IR spectrum of DAY, these bands shifted to higher frequency, 1070 and 825 cm-1, respectively, and new bands at 1712, 1421, and 1319 cm-1 were observed. This suggested that part of the aluminum ions escaped from the crystal lattice in DAY, and the mesopores formed. When GOx was immobilized on NaY, the IR spectrum (33) Anderson, M. W.; Klinowski, J. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1449.
of GOx-NaY was similar to that of NaY, which indicated that little enzyme was adsorbed on NaY. However, there were distinct differences between the IR spectra of DAY and GOx-DAY. The new adsorption bands at 1712, 1421, and 1369 cm-1 had nearly disappeared, and the other at 1194 cm-1 shifted to 1176 cm-1 in the IR spectrum of GOx-DAY. This might result from the interactions between GOx and some specific sites of DAY. Since the crystal lattice perfection of NaY was better than that of DAY, its internal surface was negatively charged. The NH3+ groups of enzyme could act with Al or Si groups in NaY, and the adsorbed water vibration band at 1637 cm-1 shifted to higher frequency, 1641 cm-1. However, due to the crystal imperfection of DAY, the -COO- groups of enzyme could also act with OH groups in DAY, and this interaction was stronger than that between NH3+ and Al or Si groups. So, the adsorbed water vibration band at 1637 cm-1 shifted to lower frequency, 1631 cm-1, after GOx was adsorbed on DAY. These results showed that there were some intermolecular interactions between GOx and DAY, and apparently DAY was a good immobilization matrix for enzymes. Immobilization Conditions. The various experimental parameters in the glucose oxidase immobilization, including the pH of the adsorption solution, enzyme concentration, pretreatment of the zeolite, and adsorption temperatures, were examined for optimum analytical performance. The influence of pH on enzyme immobilization (presented in Figure 2) is the most important factor among the variables. It has been generally accepted that a solid oxide particle in suspension in an aqueous solution tends to be mainly occupied by OH groups34 and to be electrically charged. Due to the different sites of OH groups in the particle surface, they may exhibit different acidic or basic property. Some of them can deprotonate, and the others protonate. At the point of zero charge, the amount of protonated OH groups is equal to that of deprotonated OH groups. When an oxide particle is dipped in a solution at a pH lower than its PZC, it tends to polarize positively and to adsorb compensating anions. The adsorption sites for anions and cations are charged surface groups resulting from the protonation-deprotonation equilibria of the surface hydroxyl (34) Brunelle, J. P. Pure Appl. Chem. 1978, 50, 1211.
Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
2345
Figure 3. Current output for catalytic oxidation of glucose with DAYGOx (a) and NaY-GOx (b). Successive additions of 0.1 mM glucose; operating potential, +0.55 V.
group of the solid.34-36 The point of zero charge of DAY was measured by mass titration to be 4.9. The isoelectric point of GOx was 4.2. So, at the pH range from 4.2 to 4.9, the surface of DAY could become negatively charged by adsorbing OH- ions, while GOx was positively charged by H+ ions. This may be a function of the effect of pH on the immobilization. Analytical Characteristics of the Glucose Sensor. The enzyme, glucose oxidase, catalyzes the following reaction:
β-D-glucose + O2 f δ-gluconolactone + H2O2 H2O2 f H2O + O2 + 2e
According to this reaction, hydrogen peroxide generated can be monitored with a glucose sensor based on H2O2 detection, and current variation is correlated to the concentration of glucose in the stock sample. The steady-state response of the sensor to glucose is measured at an applied potential of +0.55 V. Figure 3 shows the current output for catalytic oxidation of glucose. The current density represented the values of output current for immobilized GOx on NaY and DAY. Some blank current response for glucose was observed on the zeolite-modified sensor without GOx. However, the ratio of the blank current response without GOx to that with GOx was less than 5%. So the observed current in Figure 3 was related to the catalytic oxidation of glucose on GOx. The time required to arrive at 95% of the steady state was less than 10 s after addition of the glucose sample. Owing to the porous structure of the zeolite modified substrate, the glucose added was transferred quickly through the layer so as to be oxidized by the immobilized glucose oxidase. Hydrogen peroxide generated in the vicinity of the surface of a platinum electrode was immediately oxidized, resulting in an anodic current. The combination of quick processes of substrate transfer with (35) Vordonis, L.; Koutsoukas, P. G.; Lycourghiotis, A. J. Chem. Soc., Chem. Commun. 1984, 1309. (36) Vordonis, L.; Koutsoukas, P. G.; Lycourghiotis, A. J. Catal. 1986, 98, 296.
2346 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
Figure 4. Typical calibration plots of the DAY-GOx response to glucose with freshly prepared electrodes (b) and those after 6 months of storage (O); operating potential, +0.55 V.
rapid electrochemical reaction yielded a fast response on the glucose sensor. Moreover, the trace also clearly demonstrated that the sensor was highly sensitive to glucose. The response current of glucose on a DAY-modified electrode was about 20 times higher than that on a NaY-modified electrode. NaY has only micropores, and the adsorbed enzymes were dispersed on its outer surfaces. The enhanced sensitivity apparently resulted from the existence of mesopores in DAY. Therefore, it was chosen for immobilizing glucose oxidase. A typical calibration plot of the glucose sensor is shown in Figure 4 (b). The linear response of the sensor to glucose was from 2.0 × 10-6 to 3.0 × 10-3 M, and the detection limit was 5.0 × 10-7 M at a signal-to-noise ratio of 3. According to the Lineweaver-Burke form of the Michaelis-Menten equation,37 the apparent Michaelis constant (Kmapp) for the immobilized glucose oxidase was calculated to be 17.52 mM, based on the data in the calibration curve. Effect of pH and Temperature on the Glucose Sensor. The effect of pH on the sensor response was evaluated (Figure 5). The optimum pH for the immobilized glucose oxidase was 6.5, while for the soluble GOx it was 5.6. This pH shift can be attributed to the effective reduction of positive charge (resulting from -amino lysine groups) due to interaction with electron-rich groups in the matrix. It is obvious that the stability of GOx against pH was improved by the zeolite immobilization. The effect of temperature has been reported to be very important on the functioning of enzyme (Figure 6). The sensitivity increased with increasing temperatures from 15 to 45 °C, but the rectilinear upper part became lower when the temperature reached 35 °C. Taking both the lifetime and response characteristics into consideration, 30 °C was selected for this work. The activation energy was calculated to be 26.76 and 28.69 kJ mol-1 for immobilized and native GOx, respectively. Additionally, the effect of temperature on enzyme reactions was generally reflected through the temperature coefficient (Q10), which was calculated to be 1.42 and 1.47, respectively. In addition, thermal treatment of the enzyme was carried out by keeping it for 30 min in 0.1 M phosphate buffer (pH 7.0) at a (37) Gortier, G.; Beliveau, R.; Leliveau, E.; Belanger, D. Anal. Lett. 1990, 23, 1607.
Table 2. Recovery of Glucose Sensor glucose concn (mM)
a
added
founda
recovery (%)
0.010 0.150 0.750 1.00 2.50
0.011 0.154 0.746 0.982 2.42
110.0 102.7 99.5 98.2 96.8
Average of three measurements in diluted blood.
Figure 5. pH profile of glucose biosensor response based on DAYGOx (b) and soluble GOx (O) at a glucose concentration of 1.0 mM at 30 °C; operating potential, +0.55 V.
Figure 7. Stability of the glucose biosensor kept in a desiccator filled with saturated sodium chloride (a), in pH 7.0 phosphate buffer solution (b), and in air (c), at 4 °C between measurements. Each plot depicts biosensor response to 1.0 mM glucose at 30 °C; operating potential, +0.55 V.
Figure 6. Relationship between temperature and the relative response activity of glucose sensor at pH 7.0 phosphate buffer solution in the presence of 0.5 mM glucose: DAY-GOx (b) and soluble GOx (O).
given temperature. The immobilized GOx retained 75% of the initial activity at 60 °C, while the native enzyme kept only 20% of the original activity. It is evident that the thermal stability of GOx was improved by the immobilization, presumably due to the unchangeability of the microenvironments and the enzyme conformation upon adsorption in the zeolite. These results indicated that the DAY-modified glucose sensor could be handled over a wider range of temperatures. Properties and Long-Term Stability of the Glucose Sensor. The recoveries of five standard glucose samples with concentrations of 1.0 × 10-5-2.5 × 10-3 M in diluted blood were determined with the calibration curve method (shown in Table 2). The average recovery was 101.4%. Moreover, the experiments showed that the resulting glucose sensor based on zeolite immobilization displayed a good reproducibility. The relative standard deviation of the concentration of a standard sample measured with six glucose sensors, which were prepared under the same conditions, was 4.8%. This good reproducibility was attributed to the uniform pore and cage structure of the zeolite substrate.
In addition to enhanced sensitivity and good reproducibility, the zeolite modification matrix imparted a good long-term stability to the glucose biosensor. Such stabilization action was tested in the presence of 1.0 mM glucose in diluted blood (1:10) over a period of 8 h. The sensor activity lost only 12.5% of its initial activity after more than 100 successive measurements. To improve the sensor’s lifetime, the storage stabilities of the glucose sensors under different conditions at 4 °C were examined by checking their relative response currents periodically (Figure 7). These relative activities corresponded to the ratio of the response currents in solutions containing 2.0 mM glucose with their initial values. It is evident that the sensor in a desiccator filled with saturated sodium chloride exhibited a good storage characteristic, and its activity hardly changed for 3 months. However, when the glucose sensor was stored in phosphate buffer solution, it showed a 37% loss of activity, while there was 20% loss when the sensor was kept in air at 4 °C. Desorption and dehydration of the immobilized enzymes were observed when the electrodes were kept in solutions or in air for a long time, which resulted in reduced activity. Thus, it is obvious that the lifetime could be greatly improved when the sensor was kept in such a desiccator. Up to now, this glucose sensor had been intermittently used and stored at 4 °C for more than 6 months, and it maintained 70% of its original activity and still displayed an excellent response to glucose (Figure 4, O). Study of the Interference and Determination of Glucose in Serum. In this sensor, DAY matrix possessed a unique cationAnalytical Chemistry, Vol. 69, No. 13, July 1, 1997
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Table 3. Selectivity of the Zeolite-Modified Glucose Biosensor response current (µA)a substrate glucose (1.0 mM) hydrogen peroxide ascorbic acid uric acid L-glutamate, lactate, galactose, L-cysine, L-lysine, L-aspartic acid, L-glutamic acid
DAY/PVA electrode
Table 4. Analysis Results of the Blood Serum Samples with Zeolite-Modified Glucose Biosensor and Spectrophotometric Method with GOx Peroxidase glucose concn (mM)
PVA/Pt electrode
5.04 0.27 4.77 12.28 0.11 9.16 0.07 8.37 no response
a
Response at 0.2 mM level; at +0.55 V in phosphate buffer solution (pH 7.0)
exchange property which could both prevent anionic electroactive interferents from reaching the electrode surface and keep the biosensor from fouling. Table 3 exhibits the response of glucose and some electroactive species, such as ascorbic acid and uric acid, which are generally encountered in the determination of physiological samples, on a DAY-modified electrode and a PVA membrane electrode. The results showed that the interference was significantly reduced by the use of DAY modification, and the zeolite matrix could reject the access of uric acid and ascorbic acid to the platinum electrode, while it allowed about 40% of H2O2 to penetrate it. The presence of 0.2 mM ascorbic acid and uric acid in the buffer containing 1.0 mM glucose did not affect the response current, suggesting that, especially at low glucose, there was no interference by ascorbic acid, etc., which might lead to false glucose measurements. To test the precision of the new glucose sensor, several assays were made on serum blood samples. The glucose concentration was determined by the calibration curve and presented in Table 4. Corresponding experiments were carried out with a glucose oxidase-peroxidase-4-aminoantipyrine spectrophotometric method by a local hospital. As shown in Table 4, the results displayed a good correlation between the two methods. The result of sample 5 was an average value of 16 measurements, with a relative standard deviation of 2.7%. However, the method described here did not require expensive equipment and any other pretreatment of the samples. Therefore, it is possible to obtain a reliable biosensor at very low cost, and the method is useful for application in real samples with good precision and accuracy. In summary, we have illustrated that the immobilization of enzymes on a dealuminized Y zeolite (DAY)-modified platinum
2348 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
sample
glucose biosensora
spectrophotometric method
relative error (%)
1 2 3 4 5
4.60 5.42 6.73 7.21 5.14b
4.71 5.34 6.87 7.52 5.00
-2.17 +1.44 -2.04 -4.12 +2.80
a Average of three measurements. b Relative standard deviation of 2.7% for 16 repetitive assays.
electrode enhanced the performance of first-generation glucose biosensors, including high sensitivity, long-term stability, good reproducibility, and significantly reduced interference. Such an attractive performance was attributed to the uniform porous structure of the hydrophilic zeolite immobilization matrix, which provides a favorable microenvironment for enzyme loading. It is possible to obtain a new, reliable method for immobilizing enzymes on a zeolite-modified electrode without using BASglutaraldehyde as usual. The operation of other enzyme electrodes should benefit from the unique structural features of the zeolite substrate. The zeolite-modified first- or second-generation biosensors are currently being explored. Such successful immobilization of enzyme for the construction of the titled glucose sensor in this novel and efficient strategy opens up a new approach to develop biosensors in the actual applications. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China and the Electroanalytical Chemistry Open Laboratory of Changchun Institute of Applied Chemistry of Chinese Academia Sinica.
Received for review September 12, 1996. Accepted March 21, 1997.X AC960930U X
Abstract published in Advance ACS Abstracts, May 1, 1997.
