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A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 356 (2006) 215–221 www.elsevier.com/locate/yabio
A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment Dan Shan a, Yuanyuan He a, Shanxia Wang a, Huaiguo Xue a
a,*
, Hao Zheng
b
School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China b Department of Chemistry, Zhejiang University, Hangzhou 310027, China Received 1 April 2006 Available online 21 June 2006
Abstract A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrileco-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (<30 s) with a linear range of 5 · 106 to 3 · 103 M and a sensitivity of 6.82 mA M1 cm2. The apparent Michaelis–Menten constant ðK app M Þ was 7.3 mM. 2006 Elsevier Inc. All rights reserved. Keywords: Glucose biosensor; Electrochemical entrapment; Poly(acrylonitrile-co-acrylic acid); Porous film; Copolymer
The development of biosensors has received great attention due to their potential application to food analysis, clinical diagnostics, and environmental monitoring [1– 6]. The research aims at designing and constructing highperformance biosensors with excellent characteristics such as accuracy of measurements, sensitivity, linear range, operational stability, lifetime for storage, and sensor-tosensor reproducibility. One important step in achieving this goal is the effective immobilization of enzyme or the other biomolecules on appropriate supports. The characteristics of immobilized enzyme are influenced drastically by the immobilization method and support materials. The main conventional approaches for enzyme immobilization on electrode surfaces include adsorption, crosslinking, covalent binding, and electropolymerization. For the method of crosslinking, glutaraldehyde generally is used as a chemical crosslinking agent that contains complicated chemical species of a documented cytotoxic
*
Corresponding author. E-mail address: [email protected] (H. Xue).
0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.06.005
nature and can cause the denaturation of the immobilized enzyme to some extent [7]. For electropolymerization, the activity of the enzyme may be strongly affected due to the coexistence with the organic monomer [8]. Adsorption contains a very simple and facile procedure, but there are some disadvantages for enzyme immobilization via adsorption such as low quantity of immobilized enzyme and easy release of enzyme from membrane surface. An alternative technique is electrochemical entrapment or doping. With this technique, sequential formation of a polymer film or enzyme layer is possible. The biomolecule can retain its native activity during the biosensor fabrication process, forming the biosensor with improved analytical performance [9–11]. On the other hand, a large surface area and high absorption ability are required for the support materials of enzyme immobilization. Porous polymer materials with the polar group can satisfy these requirements. Different porous polymeric materials have found wide application as immobilization matrix of enzyme [11–15]. In previous work, we reported a microporous polymer polyacrylonitrile with the pore size suitable for enzyme immobilization
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Porous PANAA film-based glucose biosensor / D. Shan et al. / Anal. Biochem. 356 (2006) 215–221
[11]. However, the sensitivity and stability of the obtained glucose biosensor were not very satisfactory. This may be ascribed to the hydrophobicity of the host of organic polymer that can induce denaturation processes [16]. Carboxyl (–COOH) grafting in polymer will overcome this shortcoming and improve the hydrophilicity of the polymer matrix [17,18]. The developed hydrophilicity is expected to promote the enzymatic reaction under heterogeneous conditions. It was reported that the modification of 0.1–50% of the available carboxylic functions could be achieved to meet different needs for biochemical probes or biomolecular hydrogel scaffoldings [19]. Thus, the chemically modified polymer with carboxyl group was expected to improve the affinity toward enzyme [20]. In this context, we synthesized the copolymer of acrylonitrile and acrylic acid by radical copolymerization. The copolymer was fabricated into porous film and used as a carrier for the enzyme immobilization. Glucose oxidase (GOD)1 was selected as a model enzyme to explore the first applicability of the copolymer as a matrix for enzyme immobilization in the design of amperometric glucose biosensor because GOD is well studied, inexpensive, stable, and applied practically [21–24]. Materials and methods Materials GOD (EC 1.1.3.4, type II from Aspergillus niger) was purchased from Amresco. Acrylonitrile and acrylic acid were distilled under reduced pressure before use. All other reagents were of analytical grade and were used as received without further purification. Synthesis of copolymer poly(acrylonitrile-co-acrylic acid) Copolymerization of acrylonitrile and acrylic acid was conducted by radical polymerization in dimethyl sulfoxide (DMSO) solution as follows [25]. In a reaction vessel of 250 ml capacity, 12.1 g of acrylonitrile, 1.34 g of acrylic acid, 47.3 g of DMSO, and 0.61 g of azobisisobutyronitrile were introduced. Polymerization was carried out at 60 C for 24 h under nitrogen flow. The reaction was terminated by ethanol, and the final product was washed by ethanol. The copolymer was then dried under vacuum at room temperature. The intrinsic viscosity of copolymer in N, N-dimethylformamide (DMF) was determined by Ubbelodhe-type viscometer at 30 C. Molecular weight (Mg = 48,000) of poly(acrylonitrile-co-acrylic acid) was calculated by the Mark–Houwink equation: 1 Abbreviations used: GOD, glucose oxidase; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; PANAA, poly(acrylonitrileco-acrylic acid); Pt, platinum; SCE, saturated calomel electrode; PBS, phosphate-buffered solution; SEM, scanning electron microscope; EIS, electrochemical impedance spectra; SEM, scanning electron microscope; pI, isoelectric point; RSD, relative standard deviation.
[g] = 2.78 · 104 M0.76, where [g] is intrinsic viscosity and M is molecular weight [26]. The chemical structure of poly(acrylonitrile-co-acrylic acid) (PANAA) is shown as follows: CH2
CH COOH
CH2
CH CN
Preparation of the bioelectrode PANAA was dissolved in DMF (weight concentration 3%), coated onto the platinum (Pt) electrode (3 · 3 mm), and then fabricated into porous membrane by the phase inversion process [27]. The amount of copolymer was controlled by volume of the copolymer solution. The electrolysis cell used for bioelectrode construction consisted of a copolymer-coated Pt working electrode, a Pt counter electrode, and a saturated calomel electrode (SCE) that was carried out in 0.1 M phosphate-buffered solution (PBS, pH 6.5) containing GOD at a constant potential of 0.60 V (vs. SCE) for a given time. During the oxidation, the enzyme with negative charge moved to anode and was entrapped into PANAA film, forming the PANAA/GOD electrode. This construction procedure of the enzyme electrode here is called electrochemical entrapment. Another kind of enzyme electrode was constructed by the simple physical adsorption method. The porous PANAA film-modified Pt electrode was immersed directly into the enzyme solution for the same time to make the enzyme adsorb on the copolymer film. Then both enzyme electrodes were washed thoroughly with 0.1 M phosphate buffer solution and stored in PBS at 4 C. Measurements and apparatus Because GOD catalyzes, in the presence of molecular oxygen, the oxidation of b-D-glucose into gluconic acid and hydrogen peroxide, the amperometric detection of glucose was assayed by potentiostatting the PANAA/GOD electrode to oxidize the enzymically generated hydrogen peroxide. The cell used to determine the current response consisted of a PANAA/GOD electrode, a Pt electrode, an SCE, and 0.1 M PBS containing glucose. The method of amperometric determination was described in detail elsewhere [28]. The apparatus used for determining the current response was a YD-1 precise potentiostat. Micrographs were obtained with an XL-30E scanning electron microscope (SEM). Electrochemical impedance spectra (EIS) measurements were realized by an Autolab/PGSTAT30 (Eco Chemie) with a three-electrode system. The EIS were performed in 5 mM Fe(CN)63/4 containing 0.1 M KCl. The amplitude of the applied sine wave potential was 5 mV. The impedance measurements were recorded at a
Porous PANAA film-based glucose biosensor / D. Shan et al. / Anal. Biochem. 356 (2006) 215–221
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bias potential of 180 mV within the frequency range of 0.05–10 kHz. Results and discussion Comparison of electrochemical entrapment and simple physical adsorption Electrochemical entrapment of GOD into PANAA film was compared with simple physical adsorption. Simple physical adsorption was performed by immersing PANAA-modified Pt electrode into 0.1 M PBS (pH 6.5) containing 8 mg ml1 GOD for a defined time. Electrochemical entrapment of GOD into polymer film was conducted by applying a potential of 0.60 V in the same enzyme solution. After being washed thoroughly with PBS to remove the enzyme not firmly immobilized, both of the obtained electrodes were used for determination of amperometric current response to 1 mM glucose solution (pH 5.5) at an applied potential of 0.60 V. As can be seen in Fig. 1, both of the biosensors’ responses increase with increasing immobilization time at the beginning and then reach a steady-state value gradually. The bioelectrode constructed by the electrochemical entrapment achieves the steady-state response within only 2 min, whereas that formed by simple physical adsorption needs 10 min. This probably is ascribed to the enhancement of enzyme transfer velocity onto electrode surface under electric field condition. On the other hand, the steady-state response of the biosensor prepared by electrochemical entrapment apparently was higher than that obtained by simple physical adsorption. This result seems to be due to the fact that the stronger driving force immobilized a larger amount of enzyme during electrochemical entrapment. Because the isoelectric point (pI) of GOD was 4.3, the net charge of GOD is negative in a pH 6.5 solution. In this case, GOD with negative charge is more favorable to move to anode
Fig. 1. Relationship between biosensor response and immobilization time by the electrochemical entrapment method (curve a) and the simple physical adsorption method (curve b).
Fig. 2. Operational stability of the biosensors constructed by electrochemical entrapment (curve a) and simple physical adsorption (curve b). Immobilization time: 30 min.
and was anchored into the PANAA network, forming the more sensitive PANAA/GOD electrode. To further evaluate the effect of electrochemical entrapment on biosensor characteristics, the operational stability of each kind of bioelectrode was investigated by consecutive measurements of its response to 1 mM glucose (Fig. 2). The response of the biosensor prepared by electro-
Fig. 3. SEM photographs of PANAA film (A) and PANAA/GOD film (B).
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chemical entrapment had no apparent change within 20 successive measurements, whereas only 52% of initial activity had been retained for the biosensor prepared by simple physical adsorption. Good operational stability and higher response indicate that electrochemical entrapment is an improved procedure of enzyme immobilization. Thus, this procedure was used for construction of the PANAA/GOD electrode in the following experiments. Characteristics of PANAA and P PANAA/GOD film The surface morphologies of the membranes were examined by SEM imaging. The typical SEM micrographs of PANAA film before and after immobilized electrochemical GOD are shown in Fig. 3. PANAA film shows a porous structure with a pore diameter of 50–200 nm (Fig. 3A). This provides a large surface for enzyme loading. In general, the size of an enzyme particle is in the range of 10–100 nm. Thus, an enzyme can be immobilized into the PANAA pores. After anchoring GOD into polymer, the pores of copolymer film diminished markedly, and the enzyme (bright part) apparently was observed on the polymer film (Fig. 3B). EIS of Fe(CN)63/4 solution is an effective tool for probing the properties of surface-modified electrodes. The semicircle diameter of the Nyquist plot of impedance spectra equals the charge transfer resistance Rct [29], which controls the charge transfer kinetics of redox probe Fe(CN)63/4 at the electrode interface. Fig. 4 displays the Nyquist plots of the impedance spectroscopy of the PANAA- and PANAA/GOD-modified electrodes. There is a small semicircle domain for the PANAA electrode (Fig. 4, curve a, RCT 92 X). An apparent increase in RCT is observed when GOD was immobilized into PANAA film by electrochemical entrapment (Fig. 4, curve b, RCT 1050 X). This might be caused by the hindrance of the macromolecular structure of GOD to the electron transfer, and it also confirmed the successful immobilization of GOD.
Fig. 4. Nyquist plots of the EIS for PANAA film (curve a) and PANAA/ GOD film (curve b).
Optimization of the biosensor fabrication parameters When GOD was electrochemically immobilized into PANAA film, the response of the obtained biosensor was affected by immobilization time, enzyme concentration, and the thickness of copolymer. The relationship between biosensor response and the immobilization time is shown in Fig. 1 (curve a). The biosensor response reaches a steady-state value after it is entrapped electrochemically for 2 min. To ensure equilibrium, 10 min was selected for anchoring time during the fabrication of the PANAA/ GOD bioelectrode. The enzyme concentration might affect the amount of GOD anchored into PANAA matrix. The bioelectrode response was determined in 1 mM glucose solution (pH 5.5) after it was constructed at different concentrations of GOD (Fig. 5, curve a). The biosensor response increases linearly with GOD concentration below a concentration of 8 mg ml1 and was then observed to be saturated at enzyme concentrations higher than this value. Thus, immobilization of GOD in PANAA electrode was conducted at an enzyme concentration of 8 mg ml1 for further experiments. The film thickness can be adjusted easily by varying the volume of coating polymer solution. An initial increase in response is observed with 10 ll of PANAA loading, at which point the response decreases (Fig. 5, curve b). This phenomenon was attributed to the fact that an increasing thickness increases the amount of adsorbed GOD, although a thick film is not beneficial for electrode response because of the increase in the diffusion barrier. Therefore, the configuration with 10 ll of PANAA was chosen for further experiments. Effect of operational parameters on the biosensor response The PANAA/GOD electrode shows a fast bioelectrochemical response to glucose (response time <30 s).
Fig. 5. Influence of the enzyme concentration (curve a) and the amount of coating polymer (curve b) on the biosensor response.
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The experimental variables that can affect the amperometric determination of glucose, namely pH value of buffer solution, applied potential, and temperature, have been investigated. The pH effect on the biosensor performance was investigated by measuring the current response to 1 mM glucose (Fig. 6, curve a) in various pH solutions. In the range of pH values from 4.5 to 7.0, an optimal response current was the one found at approximately pH 5.5, which is in good agreement with the pH value of 5–6 reported for the native GOD [30,31]. This indicates that the immobilization procedure kept the native activity of GOD. The potential was increased from 0.30 to 0.70 V, and the effect of applied potential on the current response of the biosensor in 0.1 M PBS (pH 5.5) containing 1 mM glucose is shown in Fig. 6 (curve b). The biosensor response increased with increasing applied potential from 0.3 to 0.6 V and then reached a pseudoplateau for higher potentials. Taking into account the sensitivity of the biosensor and interference of easily oxidizable compounds in serum, 0.6 V was selected as the best compromise of applied potential in subsequent experiments. The thermal stability of the bioelectrode was also studied. Before the amperometric detection, the bioelectrode was immersed into the buffer solution at different temperatures to reach thermal equilibrium. The effect of temperature on the biosensor response to 1 mM glucose was evaluated between 2 and 45 C (Fig. 6, curve c). The biosensor response increases when the temperature rises from 2 to 35 C and then decreases as the temperature is further increased. The maximum response appears at approximately 35 C. At higher temperatures, the current response decreases due to the denaturation of the enzyme. To keep the stability of the biosensor, we chose 25 C as the operating temperature of the biosensor in the experiments.
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mal conditions (Fig. 7). The linear range spans the concentration of glucose from 5 lM to 3 mM, with a correlation coefficient of 0.998. A low detection limit of 0.5 lM glucose was estimated at a signal/noise ratio of 3. The biosensor sensitivity (determined from the slope of the initial linear part of the calibration) is 6.82 mA M1 cm2. The apparent Michaelis–Menten constant ðK app M Þ of the biosensor was calculated to be 7.3 mM according to the electrochemical Lineweaver–Burk form of the Michaelis–Menten equation. This value is similar to the reported value for the free enzyme [32], illustrating the nondenaturing character of the enzyme immobilizing procedure. The reproducibility of the biosensor fabrication was evaluated via a comparison of the sensitivity of different electrodes. Six different enzyme electrodes were tested independently for glucose amperometric response, providing a relative standard deviation (RSD) value of 4.9%. This indicates, in particular, an efficient and reproducible immobilization process of GOD in porous PANAA matrix. The storage stability of the biosensor was investigated by periodical measurement of its response to glucose. When the bioelectrode was stored in PBS at 4 C for 5
Amperometric response characteristics of the biosensor The steady-state current response of the PANAA/GOD bioelectrode to glucose was determined under these opti-
Fig. 7. Calibration curves of PANAA/GOD bioelectrode for glucose under the optimal conditions: 0.1 M PBS (pH 5.5) at 25 C, Eapp = 0.60 V.
Fig. 6. The effect of pH (curve a), operating potential (curve b), and temperature (curve c) on the biosensor response to 1 mM glucose.
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Table 1 Comparison of analytical performance of glucose biosensors based on polymers Configuration of biosensor
Sensitivity (mA cm2 M1)
Response time (s)
Life span
Reference
Pt/PANAA/GOD Pt/Polyacrylonitrile/GOD Pt/Polypyrrole/GOD Pt/Polypyrrole/Polyanion/Poly(ethyleneglycol)/GOD ITO/Poly(2-fluoroaniline)/GOD GCE/Poly(3,4-ethylenedioxythiophene)
6.82 4.20 0.22 0.18–0.27 — 3.00
<30 20 15 30 70 —
5 weeks 3 weeks Lost 50% activity after 14 days — Lost 43% activity after 32 days Lost 20% activity after 2 weeks
This work [11] [33] [34] [35] [36]
weeks, no significant decrease in the response to 1 mM glucose was observed. This analytical performance can be compared with that reported in the literature for the glucose biosensors based on polymers (Table 1). Compared with the polyacrylonitrile/GOD electrode, the PANAA/GOD exhibited better analytical performance. Carboxylic groups grafted in the polymer can enhance not only the hydrophilic property of matrix but also the affinity to enzyme; thus, the sensitivity and the lifetime were effectively improved. The interferences of electroactive species to the glucose response were examined in the presence of their physiological normal levels with glucose concentration at 5.6 mM. Interferences were estimated as the percentage increase in the biosensor signal recorded in the presence of interferents compared with the steady-state biosensor response to 5.6 mM glucose alone. The influences of ascorbic acid (0.1 mM), reduced glutathione (2 mM), L-cysteine (0.02 mM), and p-acetaminophenol (0.05 mM) to the glucose response were acceptable (i.e., 3–7%); only uric acid (0.5 mM) influenced the biosensor response to glucose (i.e., 41%). Conclusion Owing to the high hydrophilicity and high adsorption ability of porous PANAA, this material constitutes an available alternative substrate for the immobilization of enzyme molecules with complete retention of their native activity. The glucose biosensor constructed by electrochemical entrapment of GOD in the polymer matrix exhibited a low detection limit, a high sensitivity, good sensor-to-sensor reproducibility, and satisfactory stability. It is expected that this simple and rapid procedure of enzyme immobilization will be useful for the development of amperometric biosensors. Acknowledgments The authors are grateful for the financial support of the National Natural Science Foundation of China (20505014), the Natural Science Foundation of Yangzhou University (HK0413156), and the Foundation of Jiangsu Provincial Key Program of Physical Chemistry at Yangzhou University.
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A porous poly(acrylonitrile-co-acrylic acid) film-based glucose biosensor constructed by electrochemical entrapment Dan Shan a, Yuanyuan He a, Shanxia Wang a, Huaiguo Xue a
a,*
, Hao Zheng
b
School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China b Department of Chemistry, Zhejiang University, Hangzhou 310027, China Received 1 April 2006 Available online 21 June 2006
Abstract A novel glucose biosensor was constructed by electrochemical entrapment of glucose oxidase (GOD) into porous poly(acrylonitrileco-acrylic acid), which was synthesized via radical polymerization of acrylonitrile and acrylic acid. The obtained biosensor showed a better stability and higher sensitivity than the biosensor prepared by simple physical adsorption. Effects of some experimental variables such as immobilization time, enzyme concentration, pH, applied potential, and temperature on the amperometric response of the sensor were investigated. The biosensor exhibited a rapid response to glucose (<30 s) with a linear range of 5 · 106 to 3 · 103 M and a sensitivity of 6.82 mA M1 cm2. The apparent Michaelis–Menten constant ðK app M Þ was 7.3 mM. 2006 Elsevier Inc. All rights reserved. Keywords: Glucose biosensor; Electrochemical entrapment; Poly(acrylonitrile-co-acrylic acid); Porous film; Copolymer
The development of biosensors has received great attention due to their potential application to food analysis, clinical diagnostics, and environmental monitoring [1– 6]. The research aims at designing and constructing highperformance biosensors with excellent characteristics such as accuracy of measurements, sensitivity, linear range, operational stability, lifetime for storage, and sensor-tosensor reproducibility. One important step in achieving this goal is the effective immobilization of enzyme or the other biomolecules on appropriate supports. The characteristics of immobilized enzyme are influenced drastically by the immobilization method and support materials. The main conventional approaches for enzyme immobilization on electrode surfaces include adsorption, crosslinking, covalent binding, and electropolymerization. For the method of crosslinking, glutaraldehyde generally is used as a chemical crosslinking agent that contains complicated chemical species of a documented cytotoxic
*
Corresponding author. E-mail address: [email protected] (H. Xue).
0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.06.005
nature and can cause the denaturation of the immobilized enzyme to some extent [7]. For electropolymerization, the activity of the enzyme may be strongly affected due to the coexistence with the organic monomer [8]. Adsorption contains a very simple and facile procedure, but there are some disadvantages for enzyme immobilization via adsorption such as low quantity of immobilized enzyme and easy release of enzyme from membrane surface. An alternative technique is electrochemical entrapment or doping. With this technique, sequential formation of a polymer film or enzyme layer is possible. The biomolecule can retain its native activity during the biosensor fabrication process, forming the biosensor with improved analytical performance [9–11]. On the other hand, a large surface area and high absorption ability are required for the support materials of enzyme immobilization. Porous polymer materials with the polar group can satisfy these requirements. Different porous polymeric materials have found wide application as immobilization matrix of enzyme [11–15]. In previous work, we reported a microporous polymer polyacrylonitrile with the pore size suitable for enzyme immobilization
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[11]. However, the sensitivity and stability of the obtained glucose biosensor were not very satisfactory. This may be ascribed to the hydrophobicity of the host of organic polymer that can induce denaturation processes [16]. Carboxyl (–COOH) grafting in polymer will overcome this shortcoming and improve the hydrophilicity of the polymer matrix [17,18]. The developed hydrophilicity is expected to promote the enzymatic reaction under heterogeneous conditions. It was reported that the modification of 0.1–50% of the available carboxylic functions could be achieved to meet different needs for biochemical probes or biomolecular hydrogel scaffoldings [19]. Thus, the chemically modified polymer with carboxyl group was expected to improve the affinity toward enzyme [20]. In this context, we synthesized the copolymer of acrylonitrile and acrylic acid by radical copolymerization. The copolymer was fabricated into porous film and used as a carrier for the enzyme immobilization. Glucose oxidase (GOD)1 was selected as a model enzyme to explore the first applicability of the copolymer as a matrix for enzyme immobilization in the design of amperometric glucose biosensor because GOD is well studied, inexpensive, stable, and applied practically [21–24]. Materials and methods Materials GOD (EC 1.1.3.4, type II from Aspergillus niger) was purchased from Amresco. Acrylonitrile and acrylic acid were distilled under reduced pressure before use. All other reagents were of analytical grade and were used as received without further purification. Synthesis of copolymer poly(acrylonitrile-co-acrylic acid) Copolymerization of acrylonitrile and acrylic acid was conducted by radical polymerization in dimethyl sulfoxide (DMSO) solution as follows [25]. In a reaction vessel of 250 ml capacity, 12.1 g of acrylonitrile, 1.34 g of acrylic acid, 47.3 g of DMSO, and 0.61 g of azobisisobutyronitrile were introduced. Polymerization was carried out at 60 C for 24 h under nitrogen flow. The reaction was terminated by ethanol, and the final product was washed by ethanol. The copolymer was then dried under vacuum at room temperature. The intrinsic viscosity of copolymer in N, N-dimethylformamide (DMF) was determined by Ubbelodhe-type viscometer at 30 C. Molecular weight (Mg = 48,000) of poly(acrylonitrile-co-acrylic acid) was calculated by the Mark–Houwink equation: 1 Abbreviations used: GOD, glucose oxidase; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; PANAA, poly(acrylonitrileco-acrylic acid); Pt, platinum; SCE, saturated calomel electrode; PBS, phosphate-buffered solution; SEM, scanning electron microscope; EIS, electrochemical impedance spectra; SEM, scanning electron microscope; pI, isoelectric point; RSD, relative standard deviation.
[g] = 2.78 · 104 M0.76, where [g] is intrinsic viscosity and M is molecular weight [26]. The chemical structure of poly(acrylonitrile-co-acrylic acid) (PANAA) is shown as follows: CH2
CH COOH
CH2
CH CN
Preparation of the bioelectrode PANAA was dissolved in DMF (weight concentration 3%), coated onto the platinum (Pt) electrode (3 · 3 mm), and then fabricated into porous membrane by the phase inversion process [27]. The amount of copolymer was controlled by volume of the copolymer solution. The electrolysis cell used for bioelectrode construction consisted of a copolymer-coated Pt working electrode, a Pt counter electrode, and a saturated calomel electrode (SCE) that was carried out in 0.1 M phosphate-buffered solution (PBS, pH 6.5) containing GOD at a constant potential of 0.60 V (vs. SCE) for a given time. During the oxidation, the enzyme with negative charge moved to anode and was entrapped into PANAA film, forming the PANAA/GOD electrode. This construction procedure of the enzyme electrode here is called electrochemical entrapment. Another kind of enzyme electrode was constructed by the simple physical adsorption method. The porous PANAA film-modified Pt electrode was immersed directly into the enzyme solution for the same time to make the enzyme adsorb on the copolymer film. Then both enzyme electrodes were washed thoroughly with 0.1 M phosphate buffer solution and stored in PBS at 4 C. Measurements and apparatus Because GOD catalyzes, in the presence of molecular oxygen, the oxidation of b-D-glucose into gluconic acid and hydrogen peroxide, the amperometric detection of glucose was assayed by potentiostatting the PANAA/GOD electrode to oxidize the enzymically generated hydrogen peroxide. The cell used to determine the current response consisted of a PANAA/GOD electrode, a Pt electrode, an SCE, and 0.1 M PBS containing glucose. The method of amperometric determination was described in detail elsewhere [28]. The apparatus used for determining the current response was a YD-1 precise potentiostat. Micrographs were obtained with an XL-30E scanning electron microscope (SEM). Electrochemical impedance spectra (EIS) measurements were realized by an Autolab/PGSTAT30 (Eco Chemie) with a three-electrode system. The EIS were performed in 5 mM Fe(CN)63/4 containing 0.1 M KCl. The amplitude of the applied sine wave potential was 5 mV. The impedance measurements were recorded at a
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bias potential of 180 mV within the frequency range of 0.05–10 kHz. Results and discussion Comparison of electrochemical entrapment and simple physical adsorption Electrochemical entrapment of GOD into PANAA film was compared with simple physical adsorption. Simple physical adsorption was performed by immersing PANAA-modified Pt electrode into 0.1 M PBS (pH 6.5) containing 8 mg ml1 GOD for a defined time. Electrochemical entrapment of GOD into polymer film was conducted by applying a potential of 0.60 V in the same enzyme solution. After being washed thoroughly with PBS to remove the enzyme not firmly immobilized, both of the obtained electrodes were used for determination of amperometric current response to 1 mM glucose solution (pH 5.5) at an applied potential of 0.60 V. As can be seen in Fig. 1, both of the biosensors’ responses increase with increasing immobilization time at the beginning and then reach a steady-state value gradually. The bioelectrode constructed by the electrochemical entrapment achieves the steady-state response within only 2 min, whereas that formed by simple physical adsorption needs 10 min. This probably is ascribed to the enhancement of enzyme transfer velocity onto electrode surface under electric field condition. On the other hand, the steady-state response of the biosensor prepared by electrochemical entrapment apparently was higher than that obtained by simple physical adsorption. This result seems to be due to the fact that the stronger driving force immobilized a larger amount of enzyme during electrochemical entrapment. Because the isoelectric point (pI) of GOD was 4.3, the net charge of GOD is negative in a pH 6.5 solution. In this case, GOD with negative charge is more favorable to move to anode
Fig. 1. Relationship between biosensor response and immobilization time by the electrochemical entrapment method (curve a) and the simple physical adsorption method (curve b).
Fig. 2. Operational stability of the biosensors constructed by electrochemical entrapment (curve a) and simple physical adsorption (curve b). Immobilization time: 30 min.
and was anchored into the PANAA network, forming the more sensitive PANAA/GOD electrode. To further evaluate the effect of electrochemical entrapment on biosensor characteristics, the operational stability of each kind of bioelectrode was investigated by consecutive measurements of its response to 1 mM glucose (Fig. 2). The response of the biosensor prepared by electro-
Fig. 3. SEM photographs of PANAA film (A) and PANAA/GOD film (B).
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chemical entrapment had no apparent change within 20 successive measurements, whereas only 52% of initial activity had been retained for the biosensor prepared by simple physical adsorption. Good operational stability and higher response indicate that electrochemical entrapment is an improved procedure of enzyme immobilization. Thus, this procedure was used for construction of the PANAA/GOD electrode in the following experiments. Characteristics of PANAA and P PANAA/GOD film The surface morphologies of the membranes were examined by SEM imaging. The typical SEM micrographs of PANAA film before and after immobilized electrochemical GOD are shown in Fig. 3. PANAA film shows a porous structure with a pore diameter of 50–200 nm (Fig. 3A). This provides a large surface for enzyme loading. In general, the size of an enzyme particle is in the range of 10–100 nm. Thus, an enzyme can be immobilized into the PANAA pores. After anchoring GOD into polymer, the pores of copolymer film diminished markedly, and the enzyme (bright part) apparently was observed on the polymer film (Fig. 3B). EIS of Fe(CN)63/4 solution is an effective tool for probing the properties of surface-modified electrodes. The semicircle diameter of the Nyquist plot of impedance spectra equals the charge transfer resistance Rct [29], which controls the charge transfer kinetics of redox probe Fe(CN)63/4 at the electrode interface. Fig. 4 displays the Nyquist plots of the impedance spectroscopy of the PANAA- and PANAA/GOD-modified electrodes. There is a small semicircle domain for the PANAA electrode (Fig. 4, curve a, RCT 92 X). An apparent increase in RCT is observed when GOD was immobilized into PANAA film by electrochemical entrapment (Fig. 4, curve b, RCT 1050 X). This might be caused by the hindrance of the macromolecular structure of GOD to the electron transfer, and it also confirmed the successful immobilization of GOD.
Fig. 4. Nyquist plots of the EIS for PANAA film (curve a) and PANAA/ GOD film (curve b).
Optimization of the biosensor fabrication parameters When GOD was electrochemically immobilized into PANAA film, the response of the obtained biosensor was affected by immobilization time, enzyme concentration, and the thickness of copolymer. The relationship between biosensor response and the immobilization time is shown in Fig. 1 (curve a). The biosensor response reaches a steady-state value after it is entrapped electrochemically for 2 min. To ensure equilibrium, 10 min was selected for anchoring time during the fabrication of the PANAA/ GOD bioelectrode. The enzyme concentration might affect the amount of GOD anchored into PANAA matrix. The bioelectrode response was determined in 1 mM glucose solution (pH 5.5) after it was constructed at different concentrations of GOD (Fig. 5, curve a). The biosensor response increases linearly with GOD concentration below a concentration of 8 mg ml1 and was then observed to be saturated at enzyme concentrations higher than this value. Thus, immobilization of GOD in PANAA electrode was conducted at an enzyme concentration of 8 mg ml1 for further experiments. The film thickness can be adjusted easily by varying the volume of coating polymer solution. An initial increase in response is observed with 10 ll of PANAA loading, at which point the response decreases (Fig. 5, curve b). This phenomenon was attributed to the fact that an increasing thickness increases the amount of adsorbed GOD, although a thick film is not beneficial for electrode response because of the increase in the diffusion barrier. Therefore, the configuration with 10 ll of PANAA was chosen for further experiments. Effect of operational parameters on the biosensor response The PANAA/GOD electrode shows a fast bioelectrochemical response to glucose (response time <30 s).
Fig. 5. Influence of the enzyme concentration (curve a) and the amount of coating polymer (curve b) on the biosensor response.
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The experimental variables that can affect the amperometric determination of glucose, namely pH value of buffer solution, applied potential, and temperature, have been investigated. The pH effect on the biosensor performance was investigated by measuring the current response to 1 mM glucose (Fig. 6, curve a) in various pH solutions. In the range of pH values from 4.5 to 7.0, an optimal response current was the one found at approximately pH 5.5, which is in good agreement with the pH value of 5–6 reported for the native GOD [30,31]. This indicates that the immobilization procedure kept the native activity of GOD. The potential was increased from 0.30 to 0.70 V, and the effect of applied potential on the current response of the biosensor in 0.1 M PBS (pH 5.5) containing 1 mM glucose is shown in Fig. 6 (curve b). The biosensor response increased with increasing applied potential from 0.3 to 0.6 V and then reached a pseudoplateau for higher potentials. Taking into account the sensitivity of the biosensor and interference of easily oxidizable compounds in serum, 0.6 V was selected as the best compromise of applied potential in subsequent experiments. The thermal stability of the bioelectrode was also studied. Before the amperometric detection, the bioelectrode was immersed into the buffer solution at different temperatures to reach thermal equilibrium. The effect of temperature on the biosensor response to 1 mM glucose was evaluated between 2 and 45 C (Fig. 6, curve c). The biosensor response increases when the temperature rises from 2 to 35 C and then decreases as the temperature is further increased. The maximum response appears at approximately 35 C. At higher temperatures, the current response decreases due to the denaturation of the enzyme. To keep the stability of the biosensor, we chose 25 C as the operating temperature of the biosensor in the experiments.
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mal conditions (Fig. 7). The linear range spans the concentration of glucose from 5 lM to 3 mM, with a correlation coefficient of 0.998. A low detection limit of 0.5 lM glucose was estimated at a signal/noise ratio of 3. The biosensor sensitivity (determined from the slope of the initial linear part of the calibration) is 6.82 mA M1 cm2. The apparent Michaelis–Menten constant ðK app M Þ of the biosensor was calculated to be 7.3 mM according to the electrochemical Lineweaver–Burk form of the Michaelis–Menten equation. This value is similar to the reported value for the free enzyme [32], illustrating the nondenaturing character of the enzyme immobilizing procedure. The reproducibility of the biosensor fabrication was evaluated via a comparison of the sensitivity of different electrodes. Six different enzyme electrodes were tested independently for glucose amperometric response, providing a relative standard deviation (RSD) value of 4.9%. This indicates, in particular, an efficient and reproducible immobilization process of GOD in porous PANAA matrix. The storage stability of the biosensor was investigated by periodical measurement of its response to glucose. When the bioelectrode was stored in PBS at 4 C for 5
Amperometric response characteristics of the biosensor The steady-state current response of the PANAA/GOD bioelectrode to glucose was determined under these opti-
Fig. 7. Calibration curves of PANAA/GOD bioelectrode for glucose under the optimal conditions: 0.1 M PBS (pH 5.5) at 25 C, Eapp = 0.60 V.
Fig. 6. The effect of pH (curve a), operating potential (curve b), and temperature (curve c) on the biosensor response to 1 mM glucose.
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Table 1 Comparison of analytical performance of glucose biosensors based on polymers Configuration of biosensor
Sensitivity (mA cm2 M1)
Response time (s)
Life span
Reference
Pt/PANAA/GOD Pt/Polyacrylonitrile/GOD Pt/Polypyrrole/GOD Pt/Polypyrrole/Polyanion/Poly(ethyleneglycol)/GOD ITO/Poly(2-fluoroaniline)/GOD GCE/Poly(3,4-ethylenedioxythiophene)
6.82 4.20 0.22 0.18–0.27 — 3.00
<30 20 15 30 70 —
5 weeks 3 weeks Lost 50% activity after 14 days — Lost 43% activity after 32 days Lost 20% activity after 2 weeks
This work [11] [33] [34] [35] [36]
weeks, no significant decrease in the response to 1 mM glucose was observed. This analytical performance can be compared with that reported in the literature for the glucose biosensors based on polymers (Table 1). Compared with the polyacrylonitrile/GOD electrode, the PANAA/GOD exhibited better analytical performance. Carboxylic groups grafted in the polymer can enhance not only the hydrophilic property of matrix but also the affinity to enzyme; thus, the sensitivity and the lifetime were effectively improved. The interferences of electroactive species to the glucose response were examined in the presence of their physiological normal levels with glucose concentration at 5.6 mM. Interferences were estimated as the percentage increase in the biosensor signal recorded in the presence of interferents compared with the steady-state biosensor response to 5.6 mM glucose alone. The influences of ascorbic acid (0.1 mM), reduced glutathione (2 mM), L-cysteine (0.02 mM), and p-acetaminophenol (0.05 mM) to the glucose response were acceptable (i.e., 3–7%); only uric acid (0.5 mM) influenced the biosensor response to glucose (i.e., 41%). Conclusion Owing to the high hydrophilicity and high adsorption ability of porous PANAA, this material constitutes an available alternative substrate for the immobilization of enzyme molecules with complete retention of their native activity. The glucose biosensor constructed by electrochemical entrapment of GOD in the polymer matrix exhibited a low detection limit, a high sensitivity, good sensor-to-sensor reproducibility, and satisfactory stability. It is expected that this simple and rapid procedure of enzyme immobilization will be useful for the development of amperometric biosensors. Acknowledgments The authors are grateful for the financial support of the National Natural Science Foundation of China (20505014), the Natural Science Foundation of Yangzhou University (HK0413156), and the Foundation of Jiangsu Provincial Key Program of Physical Chemistry at Yangzhou University.
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