24864
J. Phys. Chem. B 2006, 110, 24864-24868
Using Novel Polysaccharide-Silica Hybrid Material to Construct An Amperometric Biosensor for Hydrogen Peroxide Guan-Hai Wang and Li-Ming Zhang* Laboratory for Polymer Composite and Functional Materials, Institute of Optoelectronic and Functional Composite Materials, School of Chemistry and Chemical Engineering, Sun Yat-Sen (Zhongshan) UniVersity, Guangzhou 510275, China ReceiVed: September 2, 2006; In Final Form: October 9, 2006
A new type of sol-gel organic-inorganic hybrid material was developed and used for the fabrication of an amperometric hydrogen peroxide biosensor. This material was prepared from natural chitosan and recently introduced completely water-soluble precursor, tetrakis(2-hydroxyethyl) orthosilicates (THEOS), by the solgel process without the addition of organic solvents and catalysts. The gelation time for the sol-gel transition and dynamic rheological properties of the resultant gel matrix could be modulated by the amount of added THEOS. The structure of the hybrid gel was made up of a network and spherical particles, as confirmed by SEM observation. By electrochemical experiments, it was found that such a hybrid gel matrix could retain the native biocatalytic activity of the entrapped horseradish peroxidase and provide a fast amperometric response to hydrogen peroxide. The linear range for the determination of hydrogen peroxide was found to be from 1.0 × 10-6 to 2.5 × 10-4 mol/L with a detection limit of 4.0 × 10-7 mol/L. The apparent Michaelis-Menten constant was determined to be 2.198 mmol/L. To improve the analytical characteristics of the fabricated biosensor, the effects of applied potential and pH value on the steady-state current were studied. Under the optimized experimental conditions, the fabricated biosensor was found to have good analytical performance, reproducibility, and storage stability.
Introduction In recent years, amperometric biosensors based on horseradish peroxidase (HRP) have been proven as the most effective tools for the determination of hydrogen peroxide, which is of great importance in chemical, biological, clinical, and many other fields.1-5 Different from conventional analytical techniques such as chromatographic, colorimetric, and photometric methods, such electrochemical sensors offer an attractive route because of their simplicity, high sensitivity, and selectivity. Among various materials used to immobilize HRP for the construction of an amperometric hydrogen peroxide biosensor, silica sol-gel matrix has drawn much attention owing to its physical rigidity, chemical inertness, high photochemical biodegradation, and thermal stability.6-9 Up to now, such material is usually obtained by using tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS) as the precursor. However, these precursors suffer from poor water solubility, which is unfavorable for the gel formation. To increase the solubility, methanol or ethanol is usually added. As a result, the resultant matrix has poor biocompatibility. In this work, a new type of hybrid gel matrix was developed and used for the construction of the amperometric hydrogen peroxide biosensor. This matrix material was prepared by the sol-gelprocessoftetrakis(2-hydroxyethyl)orthosilicates(THEOS), a completely water-soluble silica precursor,10,11 in the presence of chitosan macromolecules as the nucleating centers for precipitated silica owing to the formation of hydrogen bonds between hydroxyl groups and silanols. It is known12 that chitosan is a polycationic polysaccharide derived from naturally occurring * To whom correspondence should be addressed. E-mail: ceszhlm@ mail.sysu.edu.cn.
chitin by alkaline deacetylation (Figure 1) and has the advantages such as biodegradability, nontoxicity, biocompatibility, and good film-forming ability. With its attractive properties, chitosan has received much attention as a promising matrix for the immobilization of enzyme13 and as a material for the design of modified electrodes.14 Different from the previous sol-gel process of TMOS or TEOS as the silica precursor, the sol-gel process developed in this study could be easily carried out without the addition of any organic solvents. Moreover, the gelation time for the sol-gel transition and dynamic rheological properties of the resultant gel matrix could be modulated by the amount of added THEOS. Here we report the gelation characteristics for the chitosan/THEOS system and the cyclic voltammetric behavior of the resulting hydrogen peroxide biosensor as well as the optimization for its fabrication. Experimental Section Materials. The precursor THEOS was prepared by using tetraethoxysilane (Shanghai Chemical Co., China) according to the method reported by Mehrotra and Narain.15 Chitosan (Mr 190000-310000, 85-90% deacetylation) was purchased from Aldrich. HRP (EC1.11.1.7, RZ > 3.0, 250U/mg) was obtained from Boao Biotechnology Co. Ltd (Shanghai, China). The other chemicals were of analytical grade and used without further purification. Hydrogen peroxide solutions were purchased from Chemical Reagent Company (Guangzhou, China). Preparation and Characterization of Hybrid Material. The chitosan solution (1.0 wt %) was prepared first by dissolving 1.0 g chitosan in 100 mL of 0.05 mol/L acetic acid. The viscous chitosan solution was stirred overnight at room temperature. Then, the required amount of the precursor THEOS was added
10.1021/jp0657078 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/16/2006
An Amperometric Biosensor for Hydrogen Peroxide
J. Phys. Chem. B, Vol. 110, No. 49, 2006 24865
Figure 1. Structure of chitosan derived from naturally occurring chitin by alkaline deacetylation.
to carry out the sol-gel process, resulting in a chitosan-silica hybrid gel. The gelation process was monitored by rheological measurements, and the viscoelastic properties of the resulting gel were also measured at room temperature by means of an advanced rheometric extended system (ARES, TA Co.) in oscillatory mode with a cone-and-plate geometry (cone diameter, 25 mm; angle, 1°). SEM micrographs were taken by a JSM6330F field emission scanning electron microscope. Before the SEM observation, the gel sample was fixed on aluminum stubs and coated with gold. Preparation of Hydrogen Peroxide Biosensor and Its Property Measurements. The glass carbon electrode (GCE) was used as the base electrode for the biosensor construction. For the purpose of purification, GCE was polished using alumna powder and then washed by the mixed nitric acid/acetone solvents (1:1, v/v) and double distilled water. For the preparation of the amperometric hydrogen peroxide (H2O2) biosensor, a homogeneous stock solution of HRP/chitosan mixture was first formed by blending the above-mentioned 1.0 wt % chitosan solution with 5 mg/mL HRP solution at a volume ratio 2:1 (v/ v). After that, a required amount of THEOS was added. The resulting gel was rapidly deposited onto a purified GCE surface and was dried for approximately 10 min at room temperature. Then the enzyme electrode was immersed in a phosphate buffer (pH 7.4) and kept at 4 °C in a refrigerator in order to remove the excess HRP from the electrode surface. For the property measurements of the hydrogen peroxide biosensor, amperometric and cyclic voltammetric experiments were performed under various conditions with a CHI 630 electrochemical workstation (Shanghai Chenghua, China). All experiments were carried out with a conventional three-electrode system with the enzyme electrode as the working electrode and a platinum wire as the auxiliary electrode. Results and Discussion When the silica precursor THEOS was mixed with aqueous chitosan solution, no phase separation or precipitation was observed, showing a good compatibility. Moreover, we found that such mixing was sufficient to promote the synthesis of silica hybrid material. The sol-gel process could proceed at ambient temperature without the addition of any catalysts. In the absence of chitosan, however, the sol-gel transition did not occur even at reduced temperature. It seems that chitosan has a catalytic effect on the sol-gel process of THEOS. Figure 2 shows the elastic modulus (G′) and viscous modulus (G′′) as a function of time for chitosan/THEOS systems with various feed compositions. At the initial stage, the dynamic moduli are almost unchanged. With the increase of time, there is a sudden sharp increase in the rheological parameters, which represents a gel development stage. The gelation time, at which the transition into a gel state happens, follows from a crossover of storage and loss moduli curves.16 Beyond the crossover point, the G′ value becomes larger that the G′′ value. With the further
Figure 2. The elastic modulus (G′) and viscous modulus (G′′) as a function of time for chitosan/THEOS systems with various feed compositions: (a) 10 wt % chitosan + 10 wt % THEOS; (b) 10 wt % chitosan + 20 wt % THEOS; (c) 10 wt % chitosan + 40 wt % THEOS. Time corresponding to the crossover point between G′ and G′′ curves is taken as the gelation time. The experiments were carried out at 1 Hz, 0.01% strain, and 25 °C.
increase of time, one can observe a gradual decrease in the rate of dynamic moduli increase, and then the dynamic modulus curves level off. For the system containing 10 wt % chitosan and 10 wt % THEOS, the gelation time and the value of G′/G′′ crossover were determined from Figure 2a to be 85.6 min and 1.41 Pa, respectively. For the system containing 10 wt % chitosan and 20 wt % THEOS, the gelation time and value of G′/G′′ crossover were determined from Figure 2b to be 69.7 min and 3.23 Pa, respectively. For the system containing 10 wt % chitosan and 40 wt % THEOS, the gelation time and value of G′/G′′ crossover were determined from Figure 2c to be 28.6 min and 10.12 Pa, respectively. These results demonstrate that
24866 J. Phys. Chem. B, Vol. 110, No. 49, 2006
Wang and Zhang
Figure 4. The cyclic voltammograms of the fabricated biosensor in 0.02 mol/L PBS (pH 7.0) containing 1.0 mmol/L K4Fe(CN)6 at a scan rate of 50 mV/s. The gel matrix used was derived from the system containing 10 wt % chitosan and 10 wt % THEOS.
Figure 3. SEM micrographs of the hybrid gel derived from the system containing 10 wt % chitosan and 10 wt % THEOS: (A) dried at room temperature; (B) frozen by the liquid nitrogen.
the gelation time for the sol-gel transition and the dynamic rheological properties of the resultant gel matrix could be modulated by the amount of added THEOS. For the hybrid gel prepared in the case of 10 wt % THEOS, its morphology was examined by SEM observation. Figure 3 gives the SEM micrographs, which were taken with smaller and larger magnifications. It was found from Figure 3A that the hybrid gel consisted mostly of the spherical particles with the diameter of about 100-200 nm. In the presence of chitosan, these particles form a three-dimensional network with a highly porous structure, as shown in Figure 3B. These micrographs look very similar to the SEM pictures from the SiO2 gels reported by other research groups.10,17 To construct the amperometric biosensor for hydrogen peroxide, the gel matrix derived from the system containing 10 wt % chitosan and 10 wt % THEOS was used, and the electrocatalytic behavior of HRP entrapped in this hybrid material was evaluated by cyclic voltammetry. Figure 4 shows the cyclic voltammograms of the modified electrode in pH 7.0 phosphate buffer saline (PBS) containing 1.0 mmol/L K4Fe(CN)6 as the mediator at a scan rate of 50 mV/s. In the absence of H2O2, the enzyme electrode gives no response, and only a small background current is observed. When 0.10 mmol/L H2O2 was introduced, an electrocatalytic characteristic was observed with the increase of oxidation current and the decrease of the reduction current. This redox wave resulted from the electrochemical oxidation of H2O2 liberated from the enzymatic reaction catalyzed by the HRP on the enzyme electrode. From the mechanistic point of view, the response process of the biosensor may be summarized as follows:18
First, the immobilized HRP on the glass carbon electrode reduced hydrogen peroxide to water. Then, the HRP was regenerated using K4Fe(CN)6 as the mediator through two separate one-electron steps. At last, the mediator was recycled at the electrode, leading to an increase of its reduction current. The results presented in Figure 4 illustrate that HRP could maintain its biocatalytic activity within the used hybrid gel matrix. For the fabricated biosensor, the cyclic voltammograms corresponding to various scan rates were also investigated. As shown in Figure 5a, well-characterized redox peaks could be observed at a potential range from 10 to 300 mV/s. Moreover, it was found from Figure 5b that the peak currents were proportional to the square roots of the scan rates, which showed a typical diffusion-controlled electrochemical behavior.19 To optimize the fabrication of the biosensor, the effects of applied potential and pH on the steady-state current of the biosensor were studied in the presence of 0.10 mol/L H2O2. From Figure 6, it was found that the electrode response to H2O2 increased steadily with the change of applied potential from -0.4 to 0 V. The highest sensitivity was obtained at -0.2 V. A further increase of the negative potential resulted in very little change in current response as the limiting potential had been reached. From Figure 7, it was found that the current response increased from pH 6.0 to 7.0 and decreased from pH 7.0 to 8.0, which was in agreement with that reported for soluble HRP.20 It seems that the hybrid gel matrix did not change the optimal pH value for the bioelectrocatalytic reaction of the immobilized HRP to H2O2. Therefore, the optimal potential and pH value were determined to be -0.2 V and 7.0, respectively. Figure 8a shows a typical current-time response on the successive addition of H2O2 under the optimized experimental conditions. It was observed that the biosensor responded so rapidly to the substrate that it could obtain about 95% of the steady-state current within 5 s. Such a fast response may be
An Amperometric Biosensor for Hydrogen Peroxide
J. Phys. Chem. B, Vol. 110, No. 49, 2006 24867
Figure 6. Effect of the applied potential on the peak current of the electrocatalytic H2O2 oxidation at the modified electrode in the presence of 0.10 mmol/L H2O2 in 0.02 mol/L PBS solution. The gel matrix used was derived from the system containing 10 wt % chitosan and 10 wt % THEOS.
Figure 5. (a) Typical cyclic voltammograms of the fabricated biosensor in 0.02 mol/L PBS (pH 7.0) containing 1.0 mmol/L K4Fe(CN)6 under different scan rates from 10 to 300 mV/s. (b) Plots of peak current versus the square root of the scan rate. The gel matrix used was derived from the system containing 10 wt % chitosan and 10 wt % THEOS.
ascribed to the highly porous structure of the hybrid gel matrix, which was shown in Figure 3B. Figure 8b gives the calibration curve of the biosensor for the measurement of H2O2. The biosensor has a linear range of H2O2 concentration from 1.0 × 10-6 to 2.5 × 10-4 mol/L, with a slope of 2.28 × 10-2. The detection limit of 4.0 × 10-7 mol/L was estimated at a signalto-noise ratio of 3. The apparent Michaelis-Menten constant Kapp M , which gives an indication of the enzyme-substrate kinetics, could be calculated from the electrochemical version of the Lineweaver-Burk equation:21
1/Iss ) 1/Imax + Kapp m /ImaxC where Iss is the steady-state current after the addition of substrate, C is the bulk concentration of the substrate, and Imax is the maximum current measured under saturated substrate condition. The Kapp M was determined by analyzing the slope and intercept for the plot of the reciprocals of the current versus H2O2 concentration and found to be 2.198 mmol/L. Li et al.22 developed an amperometric enzyme electrode for hydrogen peroxide by using the silica sol-gel matrix resulting from 600 µL of methanol, 50 µL of tetramethoxysilane (TMOS), 10 µL of 3.8% cetyltrimethylammonium bromide (CTAB), 10 µL of
Figure 7. Effect of pH value on the peak current of the electrocatalytic H2O2 oxidation at the modified electrode in the presence of 0.10 mmol/L H2O2 in 0.02 mol/L PBS solution. The gel matrix used was derived from the system containing 10 wt % chitosan and 10 wt % THEOS.
5 mmol/L NaOH, and 60 µL of H2O in a small test tube at room temperature. Linear calibration for hydrogen peroxide was obtained in the range from 2.0 × 10-5 to 2.6 × 10-3 under the optimized conditions. The apparent Michaelis-Menten constant of the enzyme electrode was found to be 4.8 mmol/L. In contrast, the biosensor developed in this study has a wider linear calibration range for hydrogen peroxide and a lower Kapp M value. The smaller Kapp M value means higher enzymatic activity.21 In addition, the reproducibility of the fabricated biosensor and its storage stability in a drying state at 4 °C were investigated. We found that the current response to 0.10 mmol/L H2O2 was not lowered after the biosensor was tested continuously for 20 times and the biosensor could retain about 90% of its original response after 30 days. Li et al.22 examined the storage stability of the enzyme electrode developed by using the silica sol-gel matrix resulting from TMOS and found that the enzyme electrode retained about 60% of its activity after 35 days of storage at 4 °C. In contrast, the biosensor constructed in this study has a better storage stability.
24868 J. Phys. Chem. B, Vol. 110, No. 49, 2006
Wang and Zhang from natural chitosan and recently introduced completely watersoluble precursor, tetrakis(2-hydroxyethyl) orthosilicates (THEOS), by the sol-gel process without the addition of organic solvents and catalysts. Its gelation time and dynamic rheological properties could be modulated by the amount of added THEOS. In particular, such a hybrid gel matrix could retain the native biocatalytic activity of the entrapped horseradish peroxidase and provide a fast amperometric response to hydrogen peroxide. The fabricated hydrogen peroxide biosensor has the characteristics of good analytical performance, reproducibility, and storage stability under the optimized experimental conditions. Acknowledgment. This work was supported by NSFC (Grants 20273086; 30470476; 20676155), NSFG (Grants 039184; 6023103), Department of Science and Technology of Guangdong Province (Grant 2004B33101003), and the NCET Program (Grant NCET-04-0810) in Universities of China. References and Notes
Figure 8. (a) Amperometric response of the fabricated biosensor to the successive addition of different concentrations of H2O2 in 0.02 mol/L PBS solution (pH 7.0) at the applied potential of -0.2 V. (b) Calibration plot between the current and the concentration of H2O2. The gel matrix used was derived from the system containing 10 wt % chitosan and 10 wt % THEOS.
Conclusions Novel polysaccharide-silica hybrid gel material with good biocompatibility and porous network structure could be prepared
(1) Mulchandani, A.; Pan, S. Anal. Biochem. 1999, 267, 141. (2) Liu, S. Q.; Ju, H. X. Anal. Biochem. 2002, 307, 110. (3) Yu, J. H.; Ju, H. X. Anal. Chem. 2002, 74, 3579. (4) Delvaux, M.; Walcarius, A.; Demoustier-Champagne, S. Anal. Chim. Acta 2004, 525, 221. (5) Tripathi, V. S.; Kandimalla, V. B.; Ju, H. Biosens. Bioelectron. 2006, 21, 1529. (6) Wang, K. M.; Li, J.; Yang, X. H.; Shen, F. L.; Wang, X. Sens. Actuators B 2000, 65, 239. (7) Navas Dı´az, A.; Ramos Peinado, M. C.; Torijas Minguez, M. C. Anal. Chim. Acta 1998, 363, 221. (8) Li, J.; Tan, S. N.; Oh, J. T. J. Electroanal. Chem. 1998, 448, 69. (9) Li, J.; Tan, S. N.; Ge, H. Anal. Chim. Acta 1996, 335,137. (10) Meyer, M.; Fischer, A.; Hoffmann, H. J. Phys. Chem. B 2002, 106, 1528. (11) Shchipunov, Yu. A. J. Colloid Interface Sci. 2003, 268, 68. (12) Sinha, V. R.; Kumria, R. Int. J. Pharm. 2001, 224, 19. (13) Ng, L. T.; Zhao, H. Electroanalysis 1998, 16, 1119. (14) Cruz, J.; Kawasaki, M.; Gorski, W. Anal. Chem. 2000, 72, 680. (15) Mehrotra, R. C.; Narain, R. P. Indian J. Chem. 1967, 5, 444. (16) Winter, H. H.; Chambon, F. J. Rheol. 1986, 30, 367. (17) Bourret, A. H. Europhys. Lett. 1988, 6, 731. (18) Aoki, K.; Kaneko, H. J. Electroanal. Chem. 1988, 247, 17. (19) Chi, Y.; Duan, J.; Zhao, Z. Electroanalysis 2003, 15, 208. (20) Harbury, H. A. J. Biol. Chem. 1997, 432, 71. (21) Cass, A. E. G., Ed. Biosensors a Practical Approach; IRL Press: Oxford, U.K., 1990; p 218. (22) Li, J.; Tan, S. N.; Ge, H. L. Anal. Chim. Acta 1996, 335, 137.
