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A noninterference polypyrrole glucose biosensor
Biosensors and Bioelectronics 22 (2006) 639–643
A noninterference polypyrrole glucose biosensor Cheng Chen, Yan Jiang, Jinqing Kan ∗ School of Chemistry and Chemical Engineering, Yangzhou University,Yangzhou 225002, PR China Received 28 September 2005; received in revised form 24 January 2006; accepted 26 January 2006 Available online 15 March 2006
Abstract In order to eliminate the interference of impurities, such as ascorbic acid, a noninterference polypyrrole glucose biosensor was constructed with a four-electrode cell consisting of a polypyrrole film electrode, a polypyrrole-glucose oxidase electrode, a counter electrode and a reference electrode. The pure catalytic current of glucose oxidase (GOD) can be obtained from the difference between response currents of two working electrodes with and without GOD. The effects of potential, pH and temperature on analytical performance of the glucose biosensor were discussed. The optimum pH and apparent activation energy of enzyme-catalyzed reaction are 5.5 and 25 kJ mol−1 , respectively. The response current of the biosensor increases linearly with the increasing glucose concentration from 0.005 to 20.0 mmol dm−3 . The results show the glucose biosensor with under 2% of relative deviation has good ability of anti-interference. The glucose biosensor was also characterized with FT-IR and UV–vis spectra. © 2006 Elsevier B.V. All rights reserved. Keywords: Noninterference; Four-electrode cell; Glucose biosensor; Ascorbic acid; Infrared spectroscopy; Ultraviolet spectroscopy
1. Introduction Biosensors have received a great deal of interest of many researchers (Gros and Comtat, 2004; Kan et al., 2004; Singh et al., 2004; Gerard and Malhotra, 2005; Tahir et al., 2005), due to their abroad application in many fields, such as medical diagnostics, process control, pharmaceutical products, food analysis and defence applications. Until now the major obstacle for application of biosensors is the interference signal that results from electro-oxidizable species in the measured system, for example ascorbic acid. In general, there are the following approaches to eliminate the interference. One approach is to employ a permselective membrane that minimizes the access of interference substances to the electrode surface (Poyard et al., 1999; Xu et al., 2002a,b; Ward et al., 2002; Sung et al., 2004). The other approach is to lower the detection potential by using electron mediators that transport electrons between enzyme and electrode (Campuzano et al., 2002; Garcıa Armada et al., 2004; Krikstopaitis et al., 2004; Serban and El Murr, 2004). The third one is to eliminate the interference by pre-oxidation of the interference substances (Choi et al., 2002; Xu et al., 2004). The last
one is a chemical amplified method that addition of activator of immobilized enzyme improves electrochemical response of the determined substrate, so the original solution can be diluted to the concentration of interfering constituents being decreased to negligible levels (Hasebe and Ujita, 1998). Compared with previous works, we present a new noninterference polypyrrole (PPy) glucose biosensor based on fourelectrode cell containing two working electrodes, and not only can it effectively eliminate interference but also fabrication is easy and inexpensive. The effects of potential, pH and temperature on the properties of the PPy glucose biosensor are studied. FT-IR and UV–vis are used to characterize the PPy glucose biosensor. The results are offered through comparing three-electrode cell with the four-electrode cell in glucose solution with and without ascorbic acid at different potentials. 2. Experimental 2.1. Principle of determination The catalytic reaction of glucose biosensor is as follows: GOD
∗
Corresponding author. Tel.: +86 514 7975590 9415; fax: +86 514 7975590 8410. E-mail address: [email protected] (J. Kan). 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.01.023
Glucose + O2 −→Gluconic acid + H2 O2 The determination of the response current is based on the formation of H2 O2 . During the enzyme-catalyzed reaction, the
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were referred to the SCE, and the temperature was 25 ± 0.2 ◦ C unless otherwise stated. 2.3. Preparation of sensor
Fig. 1. The schema of the four-electrode system. (1) Potentiostatic system based on a potential control amplifier; (2) electrolytic cell; (3) current subtractor; W1—PPy-glucose oxidase electrode; W2—PPy film electrode; C—counter electrode; R—reference electrode; I = IW1 − IW2 .
hydrogen peroxide is detected by the amperometric current method during oxidation at the biosensor (Mu et al., 1991). H2 O2 → O2 + 2H+ + 2e− The cell used to determine the response current is a fourelectrode cell with 0.1 mol dm−3 phosphate buffer with and without the substrate. The four-electrode cell consists of a PPyGOD electrode (working electrode 1, W1), a PPy film electrode (working electrode 2, W2), a platinum foil electrode (counter electrode, C) and a saturated calomel electrode (SCE, reference electrode, R). Its schema is shown in Fig. 1. Two working electrodes are controlled at the same potential by the potentiostatic system, the difference between the response currents (I) at two working electrodes is directly determined through the current subtractor. As two PPy films are same, the interferential currents caused by impurities, such as ascorbic acid, are also same at two working electrodes, and the difference between them is zero. When the four-electrode cell is placed in the buffer solution containing substrate, the response current (IW1 ) at W1 contains that of enzyme catalytic reaction and interferential currents, however, the only interferential currents (IW2 ) at W2. The difference between W1 and W2 (i.e. I = IW1 − IW2 ) is the response current of pure enzyme catalytic reaction. 2.2. Materials and apparatus Pyrrole was distilled under reduced pressure prior to use. GOD used for preparing the PPy glucose biosensor was type II Aspergillus niger (Sigma Chemical Co.). All other reagents used were of the analytical grade. Buffer solution is 0.1 mol dm−3 NaH2 PO4 + Na2 HPO4 . The solutions were prepared with double-distilled water. A CMBP-1 bipotentiostat/galvanostat was used for preparation of PPy film and immobilization of GOD. YDDH-2 precision biopotentiostat, which was accurate within ±2 nA, was used for determination of the response current. The pH values of the solutions were determined using a PXD-12 pH meter. The PPy glucose biosensor was characterized with FT-IR spectra and UV–vis spectra. FT-IR spectra were recorded on a Tensor 27 FT-IR spectrometer (Bruker). UV–vis spectra were recorded on an UV-2550 spectrometer (Shmadzu). All potentials given here
The same two platinum foils (4 mm × 3 mm) were placed in parallel in a solution containing 0.1 mol dm−3 pyrrole and 0.1 mol dm−3 NaCl at pH 2 and connected with bipotentiostat/galvanostat, the potential was set at 0.7 V for 9 min. The cohesive and uniform PPy films were formed on two platinum foils, respectively. Two PPy film electrodes were washed thoroughly with the buffer. Then GOD was immobilized into one of PPy films by two-step process to form PPy-GOD electrode (Xue and Mu, 1995). The specific process is as follows: the potential of the PPy film was swept to −0.20 V in the buffer, at which PPy film was reduced continuously for 20 min, then the reduced PPy film was moved into the buffer containing 0.75 mg cm−3 GOD (pH 5.5), the potential of the PPy film was then swept to 0.60 V, at which it was continuously oxidized for 30 min, the active GOD was immobilized into the PPy film. PPy-GOD and PPy film electrodes were act as W1 and W2, respectively. The sensor was kept at 4 ◦ C when not in use. 2.4. Measurement of the response current The four-electrode cell was set at a constant potential. The background currents of two working electrodes in the buffer without substrate was measured firstly, when they reached steady values, and the difference between them was zero, the fourelectrode cell was moved in the buffer with substrate. The maximum difference of response currents between two working electrodes was taken as the determining value in the following experiments. 3. Results and discussion 3.1. Elimination of interference The interference of impurities with the determination of the response current at different potentials, such as ascorbic acid, is shown in Fig. 2. Curve 1, curve 2 and curve 3 show the relationship between potential and response current determined by the three-electrode cell with W1 as the working electrode, which are measured in (1) 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid; (2) 0.1 mmol dm−3 ascorbic acid; and (3) 1.0 mmol dm−3 glucose, respectively. The response current in curve 4 is determined by the four-electrode cell in the phosphate buffer containing 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid. The results in Fig. 2 and following figures (Figs. 3 and 4) are average results of data from the same PPy-GOD electrode. All data are measured repeatedly at least three times and the relative deviation between them is less than 2%, the experimental data at 0.45 and 0.55 V in curve 3 are listed in Table 1. It illustrated that the PPy glucose biosensor exhibits good reproducibility. It can be seen from Fig. 2 that the difference between the response currents in curve 1 and curve 2 is almost equal to
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Table 1 Response current measured repeatedly three times at potential 0.45 V and potential 0.55 V in curve 3 Potential (V)
Fig. 2. The relationship between potential and response current in the phosphate buffer containing (1) 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid; (2) 0.1 mmol dm−3 ascorbic acid; (3) 1.0 mmol dm−3 glucose; (4) 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid; (1), (2) and (3) determined by three-electrode cell; (4) by four-electrode cell. pH 5.5.
Response current (A)
Average (A)
Deviation (A)
Relative deviation (%)
0.45
0.145 0.141 0.142
0.143
0.002 −0.002 −0.001
1.40 −1.40 −0.70
0.55
0.252 0.250 0.250
0.251
0.001 −0.001 −0.001
0.40 −0.40 −0.40
the response current in curve 3 at the corresponding potential, while the response current in curve 4 is nearly same as that in curve 3, and curve 4 is well coincident with curve 3, thus the interference of ascorbic acid can be eliminated effectively by the four-electrode cell. Although interference may arise with other components in the real samples, it is anticipated that the interferences of other impurities in real plasma samples can be eliminated via this four-electrode cell system. 3.2. Effect of pH The pH dependence of the PPy glucose biosensor is investigated over the range from 3.5 to 9.0 in the phosphate buffer containing 1.0 mmol dm−3 glucose (the figure is omitted). The response current increases with the increasing pH in the range from 3.5 to 5.5, and then decreases as pH increases further. The maximum response current appears at pH 5.5, which is the optimum pH for the immobilization of GOD, and it is the same as the optimum pH of free enzyme (Liu et al., 1996). This indicates that the optimum pH of GOD is hardly influenced by PPy film.
Fig. 3. The relationship between glucose concentration and the response current of the PPy glucose biosensor in the phosphate buffer, pH 5.5, 0.5 V.
Fig. 4. The relationship between temperature and the response current of the PPy glucose biosensor in the phosphate buffer containing 1.0 mmol dm−3 glucose, pH 5.5, 0.5 V.
3.3. Effect of glucose concentration The relationship between glucose concentration and the response current of the PPy glucose biosensor in the phosphate buffer is shown in Fig. 3. The response current is linear with glucose concentration in the range from 0.005 to 20.0 mmol dm−3 . The Lineweaver–Burk plot of 1/current versus 1/[S], based on the experiment data from Fig. 3, can be obtained (the figure is omitted). Since the biosensor response is kinetic, the app apparent Michaelis–Menten constant KM can be calculated for the immobilized enzyme by amperometric method as sugapp gested by Shu and Wilson (1976). KM and the maximum current response Imax are calculated from the slope and interapp cept (KM = 23.3 mmol dm−3 , Imax = 5.88 A). The value of app KM here is smaller than that (33 mmol dm−3 ) of the soluble GOD from A. niger (Almeida et al., 1993). According to the app character of Michaelis–Menten constant (KM ), the smaller the app value of KM , the stronger will be the affinity between enzyme and substrate (Segel, 1976), thus, the interaction between glucose and GOD of the PPy glucose biosensor is stronger than that of free GOD.
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3.4. Effect of temperature The effect of temperature on the response current of the PPy glucose biosensor in the phosphate buffer containing 1.0 mmol dm−3 glucose is shown in Fig. 4. In the temperature range of 273.15–321.15 K, the response current increases with the increasing temperature, and does not appear the optimum temperature in this range, which is similar to the electrode prepared by physical entrapment (Sun et al., 1993). The optimum temperature of free GOD is at around 30 ◦ C (Brahim et al., 2002), in this case the PPy film can provide a good microenviroment to protect active GOD. According to the Arrhenius equation: Ea ln k = ln A − RT here k is the rate constant and Ea is the apparent activation energy. Since the electrode surface areas, the quantity of the enzyme and concentration of substrate are constant, the response current is proportional to the rate constant k (Belanger et al., 1989). ln k can be replaced with ln I in the formula. The relationship between ln I and 1/T is plotted (the figure is omitted), and one straight line is obtained. The apparent activation energy can be calculated from the slope and the value of Ea in the phosphate buffer is 25 kJ mol−1 , which is less than that obtained by other experiments (Fortier et al., 1990). 3.5. UV–vis spectra Fig. 5 shows the UV–vis spectra of films electrodeposited on the platinum-sprayed quartz glasses. Curve b is the UV–vis spectrum of PPy film, there is one absorption band at 478 nm which is assigned as –* absorption according to the literature (Shen and Wan, 1998). Curve a is the UV–vis spectrum of the PPy film with GOD. There is one absorption band at 358 nm of GOD immobilized in PPy film, which is similar to that of Diazo-resins/GOD (Zhang et al., 2004).
Fig. 6. The infrared spectra of PPy films with (b) and without (a) GOD.
3.6. FT-IR spectra Fig. 6 shows the FT-IR spectra of films electrodeposited on the platinum-sprayed quartz glasses. Curve a is IR spectrum of PPy, which is in accordance with the literature (Satoh et al., 1997). Curve b is IR spectrum of PPy film with GOD. There are three absorptions in the range of 3100–2700 cm−1 in curve b, which is the characterized bands of GOD (Yang et al., 2004), this implies that GOD is effectively immobilized in PPy film and is also consistent with the result of UV–vis spectrum. 4. Conclusion In this work, a noninterference glucose biosensor based on four-electrode cell is presented. Experimental results show that it can eliminate effectively interference of ascorbic acid, which is a significant task for real world glucose sensing. The response current of the glucose biosensor is linear with glucose concentration app in the range from 0.005 to 20.0 mmol dm−3 . Imax and KM of the −3 biosensor are 5.88 A and 23.3 mmol dm , respectively. The activation energy of enzyme-catalyzed reaction is less than that of the earlier work, which is favorable for the enzyme catalytic reaction. The results of UV–vis spectra and FT-IR spectra show that GOD has been immobilized in PPy. The work to explore this noninterference PPy glucose biosensor into the selective determination of glucose in real human plasma samples is in progress. Acknowledgement This project was supported by National Science Foundation of China (No. 20273058). References
Fig. 5. The UV–vis spectra of PPy films with (a) without (b) GOD.
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C. Chen et al. / Biosensors and Bioelectronics 22 (2006) 639–643 Brahim, S., Narinesingh, D., Guiseppi-Elie, A., 2002. Polypyrrole-hydrogel composites for the construction of clinically important biosensors. Biosens. Bioelectron. 17 (1–2), 53–59. Campuzano, S., Galvez, R., Pedrero, M., Manuel de Villena, F.J., Pingarron, J.M., 2002. Preparation, characterization and application of alkanethiol selfassembled monolayers modified with tetrathiafulvalene and glucose oxidase at a gold disk electrode. J. Electroanal. Chem. 526 (1–2), 92–100. Choi, S.H., Lee, S.D., Shin, J.H., Ha, J., Nam, H., Cha, G.S., 2002. Amperometric biosensors employing an insoluble oxidant as an interference-removing agent. Anal. Chim. Acta 461 (2), 251–260. Fortier, G., Brassard, E., Belanger, D., 1990. Optimization of a polypyrrole glucose oxidase biosensor. Biosens. Bioelectron. 5 (6), 473–490. Garcıa Armada, M.P., Losada, J., Cuadrado, I., Alonso, B., Gonzalez, B., Casado, C.M., Zhang, J., 2004. Preparation of biosensors based in a siloxane homopolymer with interacting ferrocenes for the amperometric detection of peroxides. Sens. Actuators B 101 (1–2), 143–149. Gerard, M., Malhotra, B.D., 2005. Application of polyaniline as enzyme based biosensor. Curr. Appl. Phys. 5 (2), 174–177. Gros, P., Comtat, M., 2004. A bioelectrochemical polypyrrole-containing Fe(CN)6 3− interface for the design of a NAD-dependent reagentless biosensor. Biosens. Bioelectron. 20 (2), 204–210. Hasebe, Y., Ujita, S., 1998. Highly sensitive flow detection of uric acid based on an intermediate regeneration of uricase. Analyst 123 (8), 1775–1780. Kan, J., Pan, X., Chen, C., 2004. Polyaniline-uricase biosensor prepared with template process. Biosens. Bioelectron. 19 (12), 1635–1640. Krikstopaitis, K., Kulys, J., Tetianec, L., 2004. Bioelectrocatalytical glucose oxidation with phenoxazine modified glucose oxidase. Electrochem. Commun. 6 (4), 331–336. Liu, Y., Zhang, X., Liu, H., Yu, T., Deng, J., 1996. Immobilization of glucose oxidase onto the blend membrane of poly(vinyl alcohol) and regenerated silk fibroin: morphology and application to glucose biosensor. J. Biotechnol. 46 (2), 131–138. Mu, S., Xue, H., Qian, B., 1991. Bioelectrochemical responses of the polyaniline glucose oxidase electrode. J. Electroanal. Chem. 304 (1), 7–16. Poyard, S., Martelet, C., Jaffrezic-Renault, N., Cosnier, S., Labbe, P., 1999. Association of a poly(4-vinylpyridine-co-styrene) membrane with an inorganic/organic mixed matrix for the optimization of glucose biosensors. Sens. Actuators B 58 (1–3), 380–383. Satoh, M., Ishikawa, H., Amano, K., Hasegawa, E., 1997. Structure and properties of polypyrrole synthesized under air and oxygen-free conditions. Synth. Met. 84 (1–3), 167–168. Segel, I.H., 1976. Biochemical Calculations, second ed. Wiley, New York, p. 152.
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A noninterference polypyrrole glucose biosensor Cheng Chen, Yan Jiang, Jinqing Kan ∗ School of Chemistry and Chemical Engineering, Yangzhou University,Yangzhou 225002, PR China Received 28 September 2005; received in revised form 24 January 2006; accepted 26 January 2006 Available online 15 March 2006
Abstract In order to eliminate the interference of impurities, such as ascorbic acid, a noninterference polypyrrole glucose biosensor was constructed with a four-electrode cell consisting of a polypyrrole film electrode, a polypyrrole-glucose oxidase electrode, a counter electrode and a reference electrode. The pure catalytic current of glucose oxidase (GOD) can be obtained from the difference between response currents of two working electrodes with and without GOD. The effects of potential, pH and temperature on analytical performance of the glucose biosensor were discussed. The optimum pH and apparent activation energy of enzyme-catalyzed reaction are 5.5 and 25 kJ mol−1 , respectively. The response current of the biosensor increases linearly with the increasing glucose concentration from 0.005 to 20.0 mmol dm−3 . The results show the glucose biosensor with under 2% of relative deviation has good ability of anti-interference. The glucose biosensor was also characterized with FT-IR and UV–vis spectra. © 2006 Elsevier B.V. All rights reserved. Keywords: Noninterference; Four-electrode cell; Glucose biosensor; Ascorbic acid; Infrared spectroscopy; Ultraviolet spectroscopy
1. Introduction Biosensors have received a great deal of interest of many researchers (Gros and Comtat, 2004; Kan et al., 2004; Singh et al., 2004; Gerard and Malhotra, 2005; Tahir et al., 2005), due to their abroad application in many fields, such as medical diagnostics, process control, pharmaceutical products, food analysis and defence applications. Until now the major obstacle for application of biosensors is the interference signal that results from electro-oxidizable species in the measured system, for example ascorbic acid. In general, there are the following approaches to eliminate the interference. One approach is to employ a permselective membrane that minimizes the access of interference substances to the electrode surface (Poyard et al., 1999; Xu et al., 2002a,b; Ward et al., 2002; Sung et al., 2004). The other approach is to lower the detection potential by using electron mediators that transport electrons between enzyme and electrode (Campuzano et al., 2002; Garcıa Armada et al., 2004; Krikstopaitis et al., 2004; Serban and El Murr, 2004). The third one is to eliminate the interference by pre-oxidation of the interference substances (Choi et al., 2002; Xu et al., 2004). The last
one is a chemical amplified method that addition of activator of immobilized enzyme improves electrochemical response of the determined substrate, so the original solution can be diluted to the concentration of interfering constituents being decreased to negligible levels (Hasebe and Ujita, 1998). Compared with previous works, we present a new noninterference polypyrrole (PPy) glucose biosensor based on fourelectrode cell containing two working electrodes, and not only can it effectively eliminate interference but also fabrication is easy and inexpensive. The effects of potential, pH and temperature on the properties of the PPy glucose biosensor are studied. FT-IR and UV–vis are used to characterize the PPy glucose biosensor. The results are offered through comparing three-electrode cell with the four-electrode cell in glucose solution with and without ascorbic acid at different potentials. 2. Experimental 2.1. Principle of determination The catalytic reaction of glucose biosensor is as follows: GOD
∗
Corresponding author. Tel.: +86 514 7975590 9415; fax: +86 514 7975590 8410. E-mail address: [email protected] (J. Kan). 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.01.023
Glucose + O2 −→Gluconic acid + H2 O2 The determination of the response current is based on the formation of H2 O2 . During the enzyme-catalyzed reaction, the
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were referred to the SCE, and the temperature was 25 ± 0.2 ◦ C unless otherwise stated. 2.3. Preparation of sensor
Fig. 1. The schema of the four-electrode system. (1) Potentiostatic system based on a potential control amplifier; (2) electrolytic cell; (3) current subtractor; W1—PPy-glucose oxidase electrode; W2—PPy film electrode; C—counter electrode; R—reference electrode; I = IW1 − IW2 .
hydrogen peroxide is detected by the amperometric current method during oxidation at the biosensor (Mu et al., 1991). H2 O2 → O2 + 2H+ + 2e− The cell used to determine the response current is a fourelectrode cell with 0.1 mol dm−3 phosphate buffer with and without the substrate. The four-electrode cell consists of a PPyGOD electrode (working electrode 1, W1), a PPy film electrode (working electrode 2, W2), a platinum foil electrode (counter electrode, C) and a saturated calomel electrode (SCE, reference electrode, R). Its schema is shown in Fig. 1. Two working electrodes are controlled at the same potential by the potentiostatic system, the difference between the response currents (I) at two working electrodes is directly determined through the current subtractor. As two PPy films are same, the interferential currents caused by impurities, such as ascorbic acid, are also same at two working electrodes, and the difference between them is zero. When the four-electrode cell is placed in the buffer solution containing substrate, the response current (IW1 ) at W1 contains that of enzyme catalytic reaction and interferential currents, however, the only interferential currents (IW2 ) at W2. The difference between W1 and W2 (i.e. I = IW1 − IW2 ) is the response current of pure enzyme catalytic reaction. 2.2. Materials and apparatus Pyrrole was distilled under reduced pressure prior to use. GOD used for preparing the PPy glucose biosensor was type II Aspergillus niger (Sigma Chemical Co.). All other reagents used were of the analytical grade. Buffer solution is 0.1 mol dm−3 NaH2 PO4 + Na2 HPO4 . The solutions were prepared with double-distilled water. A CMBP-1 bipotentiostat/galvanostat was used for preparation of PPy film and immobilization of GOD. YDDH-2 precision biopotentiostat, which was accurate within ±2 nA, was used for determination of the response current. The pH values of the solutions were determined using a PXD-12 pH meter. The PPy glucose biosensor was characterized with FT-IR spectra and UV–vis spectra. FT-IR spectra were recorded on a Tensor 27 FT-IR spectrometer (Bruker). UV–vis spectra were recorded on an UV-2550 spectrometer (Shmadzu). All potentials given here
The same two platinum foils (4 mm × 3 mm) were placed in parallel in a solution containing 0.1 mol dm−3 pyrrole and 0.1 mol dm−3 NaCl at pH 2 and connected with bipotentiostat/galvanostat, the potential was set at 0.7 V for 9 min. The cohesive and uniform PPy films were formed on two platinum foils, respectively. Two PPy film electrodes were washed thoroughly with the buffer. Then GOD was immobilized into one of PPy films by two-step process to form PPy-GOD electrode (Xue and Mu, 1995). The specific process is as follows: the potential of the PPy film was swept to −0.20 V in the buffer, at which PPy film was reduced continuously for 20 min, then the reduced PPy film was moved into the buffer containing 0.75 mg cm−3 GOD (pH 5.5), the potential of the PPy film was then swept to 0.60 V, at which it was continuously oxidized for 30 min, the active GOD was immobilized into the PPy film. PPy-GOD and PPy film electrodes were act as W1 and W2, respectively. The sensor was kept at 4 ◦ C when not in use. 2.4. Measurement of the response current The four-electrode cell was set at a constant potential. The background currents of two working electrodes in the buffer without substrate was measured firstly, when they reached steady values, and the difference between them was zero, the fourelectrode cell was moved in the buffer with substrate. The maximum difference of response currents between two working electrodes was taken as the determining value in the following experiments. 3. Results and discussion 3.1. Elimination of interference The interference of impurities with the determination of the response current at different potentials, such as ascorbic acid, is shown in Fig. 2. Curve 1, curve 2 and curve 3 show the relationship between potential and response current determined by the three-electrode cell with W1 as the working electrode, which are measured in (1) 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid; (2) 0.1 mmol dm−3 ascorbic acid; and (3) 1.0 mmol dm−3 glucose, respectively. The response current in curve 4 is determined by the four-electrode cell in the phosphate buffer containing 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid. The results in Fig. 2 and following figures (Figs. 3 and 4) are average results of data from the same PPy-GOD electrode. All data are measured repeatedly at least three times and the relative deviation between them is less than 2%, the experimental data at 0.45 and 0.55 V in curve 3 are listed in Table 1. It illustrated that the PPy glucose biosensor exhibits good reproducibility. It can be seen from Fig. 2 that the difference between the response currents in curve 1 and curve 2 is almost equal to
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Table 1 Response current measured repeatedly three times at potential 0.45 V and potential 0.55 V in curve 3 Potential (V)
Fig. 2. The relationship between potential and response current in the phosphate buffer containing (1) 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid; (2) 0.1 mmol dm−3 ascorbic acid; (3) 1.0 mmol dm−3 glucose; (4) 1.0 mmol dm−3 glucose and 0.1 mmol dm−3 ascorbic acid; (1), (2) and (3) determined by three-electrode cell; (4) by four-electrode cell. pH 5.5.
Response current (A)
Average (A)
Deviation (A)
Relative deviation (%)
0.45
0.145 0.141 0.142
0.143
0.002 −0.002 −0.001
1.40 −1.40 −0.70
0.55
0.252 0.250 0.250
0.251
0.001 −0.001 −0.001
0.40 −0.40 −0.40
the response current in curve 3 at the corresponding potential, while the response current in curve 4 is nearly same as that in curve 3, and curve 4 is well coincident with curve 3, thus the interference of ascorbic acid can be eliminated effectively by the four-electrode cell. Although interference may arise with other components in the real samples, it is anticipated that the interferences of other impurities in real plasma samples can be eliminated via this four-electrode cell system. 3.2. Effect of pH The pH dependence of the PPy glucose biosensor is investigated over the range from 3.5 to 9.0 in the phosphate buffer containing 1.0 mmol dm−3 glucose (the figure is omitted). The response current increases with the increasing pH in the range from 3.5 to 5.5, and then decreases as pH increases further. The maximum response current appears at pH 5.5, which is the optimum pH for the immobilization of GOD, and it is the same as the optimum pH of free enzyme (Liu et al., 1996). This indicates that the optimum pH of GOD is hardly influenced by PPy film.
Fig. 3. The relationship between glucose concentration and the response current of the PPy glucose biosensor in the phosphate buffer, pH 5.5, 0.5 V.
Fig. 4. The relationship between temperature and the response current of the PPy glucose biosensor in the phosphate buffer containing 1.0 mmol dm−3 glucose, pH 5.5, 0.5 V.
3.3. Effect of glucose concentration The relationship between glucose concentration and the response current of the PPy glucose biosensor in the phosphate buffer is shown in Fig. 3. The response current is linear with glucose concentration in the range from 0.005 to 20.0 mmol dm−3 . The Lineweaver–Burk plot of 1/current versus 1/[S], based on the experiment data from Fig. 3, can be obtained (the figure is omitted). Since the biosensor response is kinetic, the app apparent Michaelis–Menten constant KM can be calculated for the immobilized enzyme by amperometric method as sugapp gested by Shu and Wilson (1976). KM and the maximum current response Imax are calculated from the slope and interapp cept (KM = 23.3 mmol dm−3 , Imax = 5.88 A). The value of app KM here is smaller than that (33 mmol dm−3 ) of the soluble GOD from A. niger (Almeida et al., 1993). According to the app character of Michaelis–Menten constant (KM ), the smaller the app value of KM , the stronger will be the affinity between enzyme and substrate (Segel, 1976), thus, the interaction between glucose and GOD of the PPy glucose biosensor is stronger than that of free GOD.
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3.4. Effect of temperature The effect of temperature on the response current of the PPy glucose biosensor in the phosphate buffer containing 1.0 mmol dm−3 glucose is shown in Fig. 4. In the temperature range of 273.15–321.15 K, the response current increases with the increasing temperature, and does not appear the optimum temperature in this range, which is similar to the electrode prepared by physical entrapment (Sun et al., 1993). The optimum temperature of free GOD is at around 30 ◦ C (Brahim et al., 2002), in this case the PPy film can provide a good microenviroment to protect active GOD. According to the Arrhenius equation: Ea ln k = ln A − RT here k is the rate constant and Ea is the apparent activation energy. Since the electrode surface areas, the quantity of the enzyme and concentration of substrate are constant, the response current is proportional to the rate constant k (Belanger et al., 1989). ln k can be replaced with ln I in the formula. The relationship between ln I and 1/T is plotted (the figure is omitted), and one straight line is obtained. The apparent activation energy can be calculated from the slope and the value of Ea in the phosphate buffer is 25 kJ mol−1 , which is less than that obtained by other experiments (Fortier et al., 1990). 3.5. UV–vis spectra Fig. 5 shows the UV–vis spectra of films electrodeposited on the platinum-sprayed quartz glasses. Curve b is the UV–vis spectrum of PPy film, there is one absorption band at 478 nm which is assigned as –* absorption according to the literature (Shen and Wan, 1998). Curve a is the UV–vis spectrum of the PPy film with GOD. There is one absorption band at 358 nm of GOD immobilized in PPy film, which is similar to that of Diazo-resins/GOD (Zhang et al., 2004).
Fig. 6. The infrared spectra of PPy films with (b) and without (a) GOD.
3.6. FT-IR spectra Fig. 6 shows the FT-IR spectra of films electrodeposited on the platinum-sprayed quartz glasses. Curve a is IR spectrum of PPy, which is in accordance with the literature (Satoh et al., 1997). Curve b is IR spectrum of PPy film with GOD. There are three absorptions in the range of 3100–2700 cm−1 in curve b, which is the characterized bands of GOD (Yang et al., 2004), this implies that GOD is effectively immobilized in PPy film and is also consistent with the result of UV–vis spectrum. 4. Conclusion In this work, a noninterference glucose biosensor based on four-electrode cell is presented. Experimental results show that it can eliminate effectively interference of ascorbic acid, which is a significant task for real world glucose sensing. The response current of the glucose biosensor is linear with glucose concentration app in the range from 0.005 to 20.0 mmol dm−3 . Imax and KM of the −3 biosensor are 5.88 A and 23.3 mmol dm , respectively. The activation energy of enzyme-catalyzed reaction is less than that of the earlier work, which is favorable for the enzyme catalytic reaction. The results of UV–vis spectra and FT-IR spectra show that GOD has been immobilized in PPy. The work to explore this noninterference PPy glucose biosensor into the selective determination of glucose in real human plasma samples is in progress. Acknowledgement This project was supported by National Science Foundation of China (No. 20273058). References
Fig. 5. The UV–vis spectra of PPy films with (a) without (b) GOD.
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