polypyrrole biosensor by galvanostatic method in various pH aqueous solutions

polypyrrole biosensor by galvanostatic method in various pH aqueous solutions

Biosensors and Bioelectronics 19 (2003) 141 /147 www.elsevier.com/locate/bios Fabrication of glucose oxidase/polypyrrole biosensor by galvanostatic ...

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Biosensors and Bioelectronics 19 (2003) 141 /147 www.elsevier.com/locate/bios

Fabrication of glucose oxidase/polypyrrole biosensor by galvanostatic method in various pH aqueous solutions Yuh-Ming Uang a, Tse-Chuan Chou b,* a b

Chemical Engineering Department, Wu-Feng Institute of Technology, Chia-Yi, Taiwan, ROC Chemical Engineering Department, National Cheng Kung University, Tainan 701, Taiwan, ROC

Received 15 May 2002; received in revised form 26 November 2002; accepted 16 April 2003

Abstract The pH effect of pyrrole electropolymerization in the presence of glucose oxidase (GODx) on the performance and characteristic of galvanostatically fabricated glucose oxidase/polypyrrole (Ppy) biosensor is reported. Preparing the GODx/Ppy biosensors in 0.1 M KCl saline solution with various pH containing 0.05 M pyrrole monomer and 0.5 mg/ml GODx at 382 mA/cm2 current density for 100 mC/cm2 film thickness, both the galvanostatic responses and characteristics of these resulted biosensors were obtained. The results revealed that the galvanostatic glucose biosensor fabricated at neutral pH condition exhibited much higher sensitivity than those fabricated at lower or higher pH conditions, and had a good linearity form zero to 10 mM glucose with the sensitivity of 7 nA/ mM. Finally, the long-term stability and the kinetic parameters, Michaelis constant and maximum current, of this biosensor were also reported. # 2003 Elsevier B.V. All rights reserved. Keywords: Immobilization; Conducting; Amperometric; Galvanostatic; Potentiostatic

1. Introduction The determination of glucose is one of the most popular and well-known biosensor applications. In particular, glucose is of special importance because of its involvement in human metabolic process. Diabetics do not produce enough insulin in their pancreases to control adequately the level of glucose in blood. Previously, substantial blood samples had to be taken and analyzed in a medical laboratory with consequent time delays and uncertainty about the diabetic’s condition. With the currently available glucose biosensors, the patient can be extracted only one small drop of blood and obtain a direct digital readout of the glucose concentration within few minutes. Thus, numerous efforts were devoted to develop a glucose biosensor with fast and accurate response in the past decades. The immobilization technique for localizing enzyme at the

* Corresponding author. Tel.: /886-6-275-7575x62639; fax: /8866-236-6836. E-mail address: [email protected] (T.-C. Chou). 0956-5663/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0956-5663(03)00168-4

surface of various electrodes plays a very important role in the research of glucose biosensor. Frequently, the conventional immobilizations of enzyme have combined the use of a discrete macroscopic membrane to reduce electrode fouling and to alleviate interferences caused by the electroactive species present in the sample. But, this problem can be successfully hurdled by the new enzyme immobilization technique with the conducting polymers. The popular conducting polymers for immobilizing enzyme are polypyrrole, polyaniline, polythiophene, etc. and their advantages for fabricating the amperometric biosensor have been elucidated in many literature (Bidan, 1992; Barlett and Cooper, 1993; Cosiner, 1999). Since polypyrrole is compatible to most enzymes and can be easily synthesized from pyrrole monomer in either aqueous or organic solution, the glucose oxidase/ polypyrrole (GODx/Ppy) biosensor is the most attractive topic in biosensor area. In general, the GODx/Ppy bio-film could be prepared by potentiostatic, galvanostatic or cyclic voltammetric electropolymerization method. As regards the numerous researches of the GODx/Ppy glucose biosensor since


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1986 (Foulds and Lowe, 1986; Umanˇa and Waller, 1986), most of them were devoted to this study by potentiostatic method with or without mediator (Barlett and Cooper, 1993; Trojanowicz et al., 1995; Schumann, 1995; Shin and Kim, 1996a,b; Cosiner, 1999; Garjonyte and Malinaskas, 2000), and the effect of preparing conditions in the absence of mediator, such as enzyme concentration, pyrrole concentration, film thickness, electrolyte type and applied potential, on the characteristics of the potentiostatically fabricated polypyrrole had been also well studied (Kaplin and Qutubuddin, 1995; Shin and Kim, 1996a,b; Yuan et al., 1999). For galvanostatic method, in addition to the film thickness being very easy to control by the reaction time, the resulted bio-film was more porous than that fabricated by potentiostatic method (Ha¨mmerle et al., 1992). But, only fewer researches concerning the galvanostatically fabricated GODx/Ppy biosensor were presented. Ha¨mmerle et al. (1992) presented the permeability comparison of galvanostatically versus potentiostatically fabricated GODx/Ppy film at the same film thickness, and, Os et al. (1995) applied this method to fabricate the enzyme sensor using KI as the electrolyte for the measurement of glucose free from ascorbic acid interference. Since then, no further study about the effect of preparing conditions on the characteristics of galvanostatic biosensor method was reported. In view of this aspect, our previous paper (Uang and Chou, 2002) achieved the effect of some preparing conditions, such as enzyme concentration, monomer concentration, film thickness and applied current density, on the performance and characteristic of the galvanostatically fabricated GODx/Ppy glucose biosensor. In fact, besides the factors mentioned in that study, there is still another important factor affecting the property of the resulted biosensor to be considered, is the pH of the fabrication system. Although, the suitable pH for fabricating the galvanostatic polypyrrole film in the absence of enzyme was in the range of 9/11 (Unsworth et al., 1992), and, the optimal pH for the potentiostatic biosensor to sense the glucose was in slightly acidic range (Fortier et al., 1990), rare research presented the topic about the adequate pH for fabricating the galvanostatic glucose biosensor. Consequently, the effect of pH for fabricating the biosensor by galvanostatic method on the performance of glucose sensing was then carried out by this study. In the present study, we adopted the galvanostatic electropolymerization to fabricate the GODx/Ppy biosensor at various pH saline solutions and compared the sensing performances and characteristics of those biosensors. The optimal pH for preparing the glucose biosensor was determined with the sensitivity. Meanwhile, the stability of the optimized biosensor was also investigated.

2. Experimental 2.1. Reagents Glucose oxidase (GODx) (EC type VII-S (Aspergillus niger , 24 900 U/g), pyrrole, b-D-(/)-glucose and phosphate buffer saline (PBS, pH 7.4) were purchased from Sigma. Pyrrole was distilled in a vacuum system prior to use and stored under nitrogen sealing. All other chemicals are of analytical reagent grade. All solutions were prepared by de-ionized water. The glucose stock solution was allowed to mutarotate over 24 h before testing. 2.2. Fabrication of enzyme electrode Glucose oxidase was immobilized in polypyrrole films formed by galvanostatic electropolymerization of the monomer onto the platinum working electrode surface from a gently stirred, 0.1 M KCl solutions at various pH values containing 0.05 M pyrrole monomer and 0.5 mg/ ml GODx enzyme, using a single compartment cell connected to a potentiostat (EG & G 263). The pH of saline solution was adjusted by 0.1 M HCl or 0.1 M NaOH solution. The cell is consisted of the platinum rod (99.999%) as the working electrode (diameter /2 mm, surface area /0.03142 cm2), the Pt plate as counter electrode and a silver/silver chloride electrode as reference (3 M NaCl, BAS Model mw 2030). Prior to electropolymerizing, the Pt working electrode was polished with alumina slurry down to 0.05 mm until a highly shining surface was observed, and washed with de-ionized water in an ultrasonic bath for 3 min. Finally, this electrode was cleaned in 1 M H2SO4 by cycling the potential from /200 to /1450 mV versus Ag/AgCl until the stable state was attained, and then washed with de-ionized water again. 2.3. Electrochemical sensing and morphology observation The volume of sensing electrolyte was only 1 ml, and the temperature was maintained at room temperature (259/1 8C). The GODx/Ppy biosensor was maintained at /700 mV versus Ag/AgCl in the oxygen-saturated phosphate buffer solution (PBS, pH 7.4) in order to yield a stable background current. The amperometric measurement of glucose sensing, and the steady state anodic current were measured at the same potential in the PBS solution, which stirred gently with a rate of 120 rpm after spiking a glucose stock solution. All the morphologies of the GODx/Ppy films were pictured with scanning electronic microscope (ABT/60 TOPCON, Japan) and the optical microscope (NIKON ECLIPSE ME 600, Japan).

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3. Results and discussion 3.1. Effect of pH on the response of the electropolymerization of pyrrole with GODx The galvanostatic responses of the electropolymerization of 0.05 M pyrrole with 0.5 mg/ml GODx with different pH values at 382 mA/cm2 current density were shown in Fig. 1. The results revealed that potential for initializing the electropolymerization of pyrrole was about 0.95 V versus Ag/AgCl, and then, an instantaneous voltage elevation from 0.95 to about 1.65 V versus Ag/AgCl happened in neutral and alkaline conditions. The instantaneous voltage elevation phenomenon may be due to the over-oxidation of polypyrrole, electrolysis of water in working electrode and entrapment of ‘‘large and noncoductive’’ enzyme. This particular phenomenon hindered the further electropolymerization of pyrrole and resulted in a thinner bio-film. In the middle acidic pH circumstance (pH 4 or 5), the potential for initializing the electropolymerization of pyrrole was same with that in neutral condition, whereas, then, the response potential slowly elevated from 0.95 to about 1.65 V versus Ag/AgCl. But, in lower pH condition (pH 2.8), the potential for initializing the electropolymerization of pyrrole was only about 0.75 V versus Ag/AgCl and not any voltage elevation phenomenon ever took place throughout the fabrication process. Accordingly, being in the presence of the GODx, the conductivity and


formation rate of polypyrrole increased with the decrease of pH in fabrication. In other words, a thicker film would be formed in lower pH condition. Based upon the above results, the film thickness, being a significant factor for the sensitivity of the galvanostatically fabricated biosensor, depended not only on the input charge but also on the pH of preparing condition. This is a very important message for investigating the performance of the resulted biosensor. 3.2. Sensing performance and characteristic of the galvanostatic GODx/Ppy biosensor The sensitivity of the glucose biosensor is dependent of the activity of the GODx entrapped in the electrodeposited polypyrrole film. Yet, it is difficult to measure the activity of entrapped GODx directly. Fortunately, the activity of the immobilized enzyme can be easily evaluated by the electrochemical analysis. In other words, the amperometric current of the biosensor is proportional to the concentration of H2O2 catalytically produced by the GODx on the anode. Thus, the sensing current can determine the relative activity of immobilized GODx. The sensing performances of the galvanostatic GODx/ Ppy glucose biosensors fabricated at different from pH from 2 to 12 were listed in Table 1. The results revealed that, these biosensors performed different sensing currents from zero to 35 nA, with different corresponding

Fig. 1. Galvanostatic response curves of the electropolymerization of 0.05 M pyrrole with 0.5 mg/ml GODx in 0.1 M KCl solution with various pH values under 382 mA/cm2 applied current density for100 mC/cm2 film thickness.

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Table 1 Sensing performances of the galvanostatic GODx/Ppy biosensors fabricated in 0.1 M KCl solution containing 0.05 M pyrrole with 0.5 mg/ml GODx at various pH values under 382 mA/cm2 applied current density for100 mC/cm2 film thickness Sensor number

pH of fabrication

Sensing current (nA)


1 2 3 4 5 6 7 8

2.89/0.1 3.49/0.1 5.39/0.1 6.29/0.1 7.09/0.1 8.59/0.1 10.09/0.1 11.59/0.1

0 2.09/0.1 12.59/0.7 20.09/1.5 35.09/2.2 2.09/0.3 1.59/0.1 1.59/0.1

0 5.0 5.6 7.5 6.3 15 6.7 6.7

The sensing currents were measured for the final glucose concentration of 5 mM by fresh-made biosensors (n/5) at /700 mV vs. Ag/ AgCl after spiking glucose into the PBS solution (pH 7.4).

coefficients of variation (C.V.), after spiking the glucose into PBS solution (pH 7.4). Obviously, both the biosensors made in slightly acidic and neutral condition showed better sensitivity than that made in other conditions, and the reproducibility for making these biosensor was higher than 92%. In addition to the SEM shown as Fig. 2(A /C), the morphologies of the typical biosensors made at various pH conditions could also be clearly observed by the convenient optical microscopy shown as Fig. 3(A /C). For strong acidic condition, the poor sensitivity was mainly ascribed to the lower amount of positive charge GODx (isoelectric point, pH 4.2) entrapped onto Ppy film and the serious glucose and H2O2 diffusion barrier caused by thicker polypyrrole film shown as Fig. 2(A) and Fig. 3(A). For alkaline condition, a nonconductive, thin and uniform bio-film with no apparent macroscopic immobilized GODx (shown as Fig. 2(B) and Fig. 3(B)) would be formed due to the electrostatic attachment of strong nucleophilic reagent, OH , that resulted in the GODx being hardly immobilized onto the Ppy layer. Thus, although the thinner film could be fabricated at higher pH condition, the sensitivity performance was still very poor. Yet, for neutral fabrication condition, a thin film, but enough to effectively entrap the enzyme as shown in Fig. 2(C) and Fig. 3(C), resulted in a sensitive glucose sensing. In medium acidic case (pH 4/6.5), the enzyme were effectively immobilized and well distributed in the sensing layer similar to the neutral fabricating case, and the resulted biosensor also perform considerable sensitivity to glucose. Particularly, in medium basic condition (pH 8.5), at fist, the characteristic of the resulted biosensor was expected to be similar to that made in neutral condition. But, after the examination, the sensing performance of biosensor made in pH 8.5 was poorer than that made in neutral condition. With optical microscopic observation, the black dentritic scallops as shown in Fig. 4 gave the evidence that the

Fig. 2. Typical SEM morphologies (3000/) of the galvanostatic GODx/Ppy biosensor fabricated at various pH in the same condition stated in Fig. 1. (A) pH 2.8, (B) pH 11.5, (C) pH 7.0.

GODx was ever entrapped temporarily in Ppy film during fabrication, but it finally leached out from the thinner Ppy film due to the enzyme were not immobilized effectively. According to the results, the sensitivity of the glucose biosensor depended strongly on both parameters, the entrapped GODx amount and film thickness. With the

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Fig. 3. Typical optical microscopic morphologies of the same biosensors of Fig. 2. (A) pH 2.8, (B) pH 11.5, (C) pH 7.0. Scale bar/20 mm.

best sensitivity, the optimal pH for fabricating the galvanostatic GODx/Ppy biosensor was determined at the neutral range. As for the interference problem accompanied in glucose sensing, Ppy is known to possess of the sizeexclusion effect to eliminate the interference arouse by electroactive species (e.g. ascorbic acid, uric acid) and it has been proved (Uang and Chou, 2002; Ha¨mmerle et al., 1992) that the ascorbic acid interference decreased with the increase of film thickness and was successfully suppressed by galvanostatically fabricated Ppy while the film thickness was higher than 50 mC/cm2. 3.3. Calibration curve and kinetic data of the galvanostatic GODx/Ppy biosensor Fig. 4. Typical optical microscopic morphology of the galvanostatic fabricated GODx/Ppy biosensor fabricated at pH 8.5 in the same condition stated in Fig. 1. Scale bar/20 mm.

The sensing calibration curve of the biosensor prepared at neutral condition was shown in Fig. 5. The


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a superposition of diffusional and kinetic limitation. Besides, The response time (t95%) of this biosensor was very fast, about 15 seconds. 3.4. Long-term stability of the neutral pH fabricated glucose biosensor

Fig. 5. Glucose sensing calibration curve of the biosensor fabricated at neutral condition. The steady current is the average of triple replicates (n/3).

results revealed that this glucose biosensor exhibited a very good linearity for sensing the glucose from zero to 10 mM with a sensitivity of 7 nA/mM. The kinetic parameters, Michaelis constant (Km) and maximum response current (Imax), could be readily derived from the Lineweaver /Burke plot as shown in Fig. 6. The maximum sensing current was 300 nA and the apparent Michaelis constant was 37.6 mM. This apparent Km value was higher than that (33 mM) of the soluble GODx from A. niger (Almeida et al., 1993). The higher Km value of the immobilized GODx gave the evidence of

Fig. 6. Lineweaver /Burke type plot of Fig. 4. Determination of the apparent Michaelis /Menten constant (Km) and Maximum current (Imax) for the biosensor fabricated at neutral condition.

In addition to sensitivity and morphology, the longterm stability of this pH optimized glucose biosensor was also examined. A series of measurements of 5 mM glucose per test were executed for 20 days. The experimental data were sketched in Fig. 7. The results revealed that the signal (current) increased from 36 to 43 nA corresponding to the first two tests, that was due to the Ppy swelling and the re-conformation of GODx entrapped on bio-film. Subsequently, increasing the storage time from 3 to 13 days, the signal decreased from 43 to 19 nA. Furthermore, the signal was then gradually down to 16 nA corresponding to 20 storage days. Results revealed that this biosensor still remained about 50% of the initial sensitivity till 14 days. The reasons for decreasing the activity of entrapped GODx might be due to the enzyme being oxidized by H2O2 and leaching out from the bio-film during glucose sensing.

4. Conclusions The spatial distribution of the immobilized GODx on the electropolymerized polypyrrole film can be observed

Fig. 7. Long-term stability of the galvanostatic GODx/Ppy biosensor fabricated at the neutral condition. The amperometric measurement of 5 mM glucose in the PBS solution (pH 7.4) was executed at the potential of /700 mV vs. Ag/AgCl. The biosensor was stored in PBS solution (pH 7.4) under 4 8C while being not tested.

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not only by SEM but also by the convenient optical microscopy. The sensitivity of the galvanostatic glucose biosensor strongly depended on both the film thickness and the immobilized GODx amount, and which were influenced significantly by pH of the fabricating condition. The optimal pH for fabricating the sensitive galvanostatic GODx/Ppy glucose biosensor was in the neutral condition, and the resulted biosensor performed good reproducibility and about 2-weeks long-term stability. The results may be referred and applied to fabricate the other kinds of biosensors.

Acknowledgements The supports of the Ministry of Education for Republic of China (Program for promoting university academic excellence, Ex-91-E-FA09-5-4), the National Cheng Kung University and Wu-Feng Institute of Technology are acknowledged.

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