Electrochemistry Communications 10 (2008) 1094–1097

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Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom

An electrochemically preanodized screen-printed carbon electrode for achieving direct electron transfer to glucose oxidase Ting-Hao Yang, Chi-Lung Hung, Jyh-Harng Ke, Jyh-Myng Zen * Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan

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Article history: Received 21 April 2008 Received in revised form 2 May 2008 Accepted 7 May 2008 Available online 21 May 2008 Keywords: Glucose oxidase Direct electron transfer Preanodization Disposable Screen-printed carbon electrode

a b s t r a c t Here we report the unique property of a preanodized screen-printed carbon electrode (SPCE*) that can allow direct electron transfer (DET) reaction of glucose oxidase (GOx). The GOx can be immobilized in the composite of oxygen functionalities and edge plane sites generated during preanodization without additional cross-linking agents. The electron transfer rate of GOx is greatly enhanced to 4.38 s 1 as a result of the conformational change of GOx in the microenvironment enabling the accessibility of active site for GOx to the electrode. The analytical versatility is further improved with the aid of Nafion film. As a consequence, the as-prepared electrode can be used as a glucose biosensor and the number of potential foreign species is then restricted by molecular size, permeation and/or (bio)chemical reaction. Most importantly, the disposable nature of the proposed electrode is expected to promote the DET-related researches. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction The direct electron transfer (DET) from the redox center (i.e., flavin adenine dinucleotide, FAD/FADH2) of glucose oxidase (GOx) to electrode is difficult to observe because the active site is deeply embedded within a protective protein shell. To improve the communication between active site and electrode, one successful approach generally referred to as ‘‘wired” enzyme electrodes has involved binding redox-active centers (mediators) and enzymes in a polymeric matrix immobilized on an electrode surface [1–6]. Nonetheless, electrode interfaces need to be designed containing a pathway that allows efficient electron transfer and the complicated procedure usually limits their broad application. The use of a suitable matrix to immobilize GOx is a more convenient way to accomplish the purpose of DET and even with higher sensitivity and better stability to act as glucose biosensors [7–15]. It was reported that carbon nanotube (CNT) can promote the DET due to the presence of the oxygen-contained groups on the surface [13,16,17]. The effect of oxygenated species at CNT was studied and the rate of electron transfer was found to be dominated by the functionalities in the CNT end, especially the carboxylate moieties [18,19]. On the other hand, Compton’s group studied the location and nature of electron transfer processes on CNT-modified electrodes and concluded that the edge plane-like defects on CNT as the key to the high electrocatalytic activity toward several biological molecules [20–24]. * Corresponding author. E-mail address: [email protected] (J.-M. Zen). 1388-2481/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2008.05.020

In our earlier studies we have reported exclusive applications of disposable preanodized screen-printed carbon electrode (designated as SPCE*) with improved electrochemical activity [25–29]. The disposable SPCE* with the introduction of edge plane carbonyl groups was found to act more or less like an edge plane graphite electrode or CNT. Based on flexibility and robustness of the SPCE*, a simple and facile alternate to allow DET reaction of GOx and the discussion on enzyme denaturation are established in this study. Note that Dong and Wang previously reported that DET reaction can take place between the adsorbed GOx and the anodized glassy carbon electrode, but the adsorbed enzyme can not catalyze its substrate due to its denaturation [30]. They suggest that the adsorbed enzyme loses its native bioactivity owing to the great change in the structure, i.e., the GOx molecule adsorbs onto the anodized electrode surface in a holoenzyme form and then extends gradually to an unfolded structure. This is, however, not the case at the SPCE* as the GOx can retain its bioactivity. Overall, the fact that GOx can be immobilized easily without additional cross-linking agents together with the disposable nature of the proposed electrode is expected to promote the DET-related researches. 2. Experimental GOx (EC 1.1.3.4 from Aspergillus niger, Sigma), Nafion (5 wt% in mixture of lower aliphatic alcohols and water, Aldrich), b-D-glucose, Na2HPO4, NaH2PO4, uric acid and ascorbic acid were of ACS certified reagent grade. A pH 7.4 phosphate buffer solution (PBS) was used in all studies. Water was obtained from a Millipore purification system. Voltammetric measurements were carried out

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with a CH Instruments (CHI 627) electrochemical workstation in a three-electrode cell assembly. A bare SPCE or SPCE* (working electrode), an Ag/AgCl, 3 M KCl (reference electrode) and a platinum wire (auxiliary electrode) were used to complete the cell setup. The SPCE with a working area of 0.196 cm2 and a conductive track radius of 2.5 mm was purchased from Zensor R&D (Taichung, Taiwan). Electrodes were then preanodized by applying a potential at 2.0 V vs. Ag/AgCl for 300 s in pH 7.4 PBS. The GOx solution was prepared by dissolving 20 mg GOx in 1 mL PBS. 3. Results and discussion Fig. 1 compares the cyclic voltammograms at SPCEs with/without preanodization and enzyme-immobilization in pH 7.4 PBS. For enzyme-immobilization, the GOx solution (5 lL) was spread evenly onto the working electrode surface with a microsyringe and let dried for 2 h. As can be seen, no peak responses were observed at SPCEs without enzyme-immobilization (curves a and c). A pair of well-defined and nearly symmetric redox peaks can only be observed with a formal potential of 0.45 V at the SPCE* with enzyme-immobilization (curve d). The controlled experiments clearly indicate that the redox peaks are derived from GOx, which is similar to the reported results [7,13,15]. The electrochemical response of GOx immobilized onto the heterogeneous surface is due to a redox reaction of FAD/FADH2 [10]. Note that FAD is known to undergo a two-electron coupled with two-proton redox reaction, i.e., GOx/FAD + 2e + 2H+ = GOx/FADH2. Thus the anodic and cathodic peak potentials of GOx immobilized on the surface of SPCE* should be pH-dependent. This is indeed the case for our system. As shown in Fig. 2, an increase of the solution pH leads to a negative shift in potential for both anodic and cathodic peaks. The slope for a linear plot of E1/2 vs. pH is –62 mV/pH, which is close to the theoretical one (–59 mV/pH) for a reversible, two-proton coupled with two-electron redox reaction process. Overall, the oxygen functionalities and edge plane-like sites formed at the SPCE* play an important role in facilitating the electron transfer between the GOx and electrode. Fig. 3A shows the cyclic voltammograms at different scan rates ranged from 10 to 150 mV/s. A good linear relationship for the peak current and scan rate (Fig. 3B) indicates a surface-controlled electrode process. An estimation of the electron transfer rate constant (ks) can be made from the peak-to-peak separation value using the model of Laviron [26,31,32]. Taking a charge transfer coefficient a

Fig. 1. Cyclic voltammetric responses of a bare SPCE (a), SPCE immobilized with GOx (b), SPCE* (c) and SPCE* immobilized with GOx (d) in 0.1 M, pH 7.4 PBS. Scan rate = 50 mV/s.

Fig. 2. Cyclic voltammetric responses of the SPCE* immobilized with GOx in different pH (a–e: 6.5, 7.0, 7.5, 8.0 and 8.5) phosphate solutions. Inset shows the plot of Ep vs. pH. Scan rate = 50 mV/s.

Fig. 3. (A) Cyclic voltammetric responses of the SPCE* immobilized with GOx in 0.1 M, pH 7.4 PBS with different scan rates (a–h: 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 and 0.15 V/s). (B) Plots of cathodic (a) and anodic (b) peak current with scan rate.

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of 0.42, the electron transfer rate constant was measured as 4.38 s 1. It is much higher than those of CNT-related papers (1.5– 1.7 s 1) [7,13]. These further suggest that the matrix greatly facilitates active site of GOx to approach the electrode. The analytical versatility is further improved with the aid of Nafion film. Nafion has been widely used in biosensor making as a protective and selective coating material and as a support for enzyme immobilization. In addition, Nafion film has negative charge, so foreign species such as ascorbic acid, uric acid, paracetamol, etc. are readily repelled [15–17]. The number of potential foreign species is then restricted by molecular size, permeation and/or (bio)chemical reaction. A 5 lL coating solution of Nafion/ GOx = 2:3 (v/v) was dropped onto the surface of a SPCE* with a microsyringe and allowed to dry at ambient temperature. Since the electrode with five layers of coating (5 lL each run) showing the best performance in glucose detection, it was used in subsequent study. The DET of GOx at the SPCE* protected with a Nafion film can then be applied in electroanalysis. The characteristics of the biosensor are further investigated by chronoamperometric experiment. Fig. 4A shows the calibration curve with a linear range spans from 0 to 900 lM glucose and deviates from linearity at higher concentration representing a typical characteristic of Michaelis–Menten kinetics. The Michaelis–Menten constant (Km) is evaluated to be 1.07 mM derived from Lineweaver–Burk equation [33]. The value is much lower than the reported 22 ± 2 mM [34,35] and 33 mM in solution phase [36]. These results show that the biosensor possesses higher biological affinity to glucose. It is thus expected that the GOx molecule does not extend gradually to an unfolded structure as reported in

the case of anodized glassy carbon electrode [30]. Of course, surface characterization of the SPCE* is needed in order to confirm the expectation. In the linear range, the electrode has a high sensitivity of 50.88 lA/(mM cm2). Fig. 4B shows the typical current– time responses for successive adding 100 lM of glucose and possible interference species as ascorbic acid and uric acid. As can be seen, both ascorbic acid and uric acid does not give any observed interference for the biosensor. The current response of the enzyme electrode increased along with glucose concentration and reached 95% of the steady-state current within 20 s. The storage stability of the electrode towards the catalytic oxidation of glucose has been examined as well. After being stored at 4 °C for 2 weeks, signals decrease by less than 5%, indicating good stability of this enzyme electrode. The reproducibility of the electrode was examined at a glucose concentration of 100 lM. The relative standard deviation is 2.7% for nine successive assays. From the above results, it can be deduced that the enzyme electrode is effective to act as an amperometric biosensor in the determination of glucose. 4. Conclusion In this paper, we demonstrate the DET of GOx at a disposable SPCE* with a high electron transfer rate of 4.38 s 1. The experimental results further confirm that the immobilized GOx remains its electrocatalytic activity for the oxidation of glucose with optimum biocompatibility and good film-forming. In addition, the electrode possesses high sensitivity and good chemical/mechanical stability to act as an amperometric biosensor for the determination of glucose. The avenue is promising for the development of other DETrelated bioelectrochemical devices. Acknowledgements The authors gratefully acknowledge financial support from the National Science Council of Taiwan. This work is supported in part by the Ministry of Education, Taiwan under the ATU plan. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

Fig. 4. (A) Calibration curve of glucose detected by the proposed electrode. (B) The obtained i–t curve with the injection of uric acid, ascorbic acid and glucose. Detection potential = 0.5 V vs. Ag/AgCl.

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