Sensors and Actuators B 123 (2007) 715–719

Mediatorless catalytic oxidation of NADH at a disposable electrochemical sensor K.S. Prasad a , J.-C. Chen a , C. Ay b , J.-M. Zen a,∗ a

b

Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Center for Energy Research and Sensor Technology, National Chiayi University, Chiayi City 60004, Taiwan Received 23 August 2006; received in revised form 1 October 2006; accepted 4 October 2006 Available online 14 November 2006

Abstract We report here a simple, selective and sensitive method for the determination of NADH in neutral aqueous solution by using an electrochemically preanodized screen printed carbon electrode (SPCE* ) without the addition of any redox mediator. The preanodization procedure makes the SPCE more electroactive towards NADH oxidation with a decrease of ∼300 mV in the overpotential without electrode fouling. It is proposed that surface reorientation to generate more edge plane sites during the preanodization procedure, as confirmed by a distinctive Raman band at ∼1360 cm−1 , is the main cause for the facilitated electron transfer for the oxidation of NADH. Presence of large excess of ascorbic acid does not interfere the detection of NADH and the SPCE* shows individual voltammetric peaks for ascorbic acid and NADH. Under optimized conditions, the obtained calibration plot shows a linear range up to 100 ␮M with a detection limit (S/N = 3) of 157 nM by flow injection analysis. © 2006 Elsevier B.V. All rights reserved. Keywords: NADH oxidation; Screen printed carbon electrode; Preanodization; Flow injection analysis

1. Introduction The electrochemical oxidation of nicotinamide adenine dinucleotide (NADH), a cofactor required in the enzymatic reaction of dehydrogenase, is essential in the development of dehydrogenase-based biosensors. Direct electrochemical oxidation of NADH often occurs with high overpotential leading to the interference from other easily oxidizable species in the sample of interest [1]. Electrode fouling is another concern since NADH gets oxidized to form NAD+ through a (2e− + 1H+ ) process with the cleavage of C H bond to form dimers at the electrode surface [2]. This can then attribute sluggish heterogeneous electron transfer and thereby reduces the sensitivity of the electrode [1,3]. Ceans et al [4] reported the electrocatalytic oxidation of NADH and ascorbic acid on electrochemically pretreated glassy carbon electrodes however the oxidation of NADH in presence of ascorbic acid was not demonstrated. Various chemically modified electrodes have been reported to decrease the high overpotential and to eliminate the electrode fouling effect [2,5–10]. Mean-

∗

Corresponding author. Tel.: +886 4 22840411 506; fax: +886 4 22862547. E-mail address: [email protected] (J.-M. Zen).

0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.10.012

while use of mediators with fast electron transfer to catalyze NADH has also been intensively studied [11–18]. Nevertheless the development of a rapid, cheap, disposable and sensitive electrochemical sensor for NADH determination is still a matter of interest. Recently, Banks and Compton [19] observed an interesting behavior of NADH at an edge plane pyrolytic graphite electrode and proposed that it can conveniently replace carbon nanotube modified electrode [5] for the routine sensing of NADH due to its simplicity of preparation and cost. Edge plane pyrolytic graphite electrodes are advantageous for use as electrode substrates in electroanalysis owing to the highly reactive edge plane sites which allow low detection limits, high sensitivities, improved signal to noise characteristics and low overpotentials. Similarities to the above mentioned principle, in this report another approach for the electrochemical oxidation of NADH by using a disposable preanodized screen printed carbon electrode (designated as SPCE* ) was demonstrated. Surface reorientation to generate more edge plane sites during the preanodization procedure is proposed as the main cause for the facilitated electron transfer for the oxidation of NADH. The generation of more edge plane sites during the preanodization procedure was characterized by a distinctive Raman band at ∼1360 cm−1 [20,21].

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The stability of the treated electrode was then checked and the response of NADH was finally evaluated by cyclic voltammetry (CV) and flow injection analysis (FIA). The preanodization procedure can not only make the SPCE more electroactive towards NADH oxidation but also provide low susceptibility to electrode fouling. We believe that the SPCE* is a good alternative for the routine sensing of NADH due to its simplicity of preparation, low susceptibility to electrode fouling, low detection limit and insensitivity to interference from ascorbic acid. 2. Experimental 2.1. Chemicals and reagents ␤-Dihydronicotinamide adenine dinucleotide reduced disodium salt (MP Biomedicals) and ascorbic acid (Sigma) were used as received without further purification. All other chemicals used were of ACS certified reagent grade. Aqueous solutions were prepared with doubly distilled and deionized water. Unless otherwise mentioned, a pH 7.4, 0.1 M phosphate buffer solution (PBS) was used as supporting electrolyte in all experiments.

3. Results and discussion 3.1. Electrochemical oxidation of NADH Fig. 1 compares the cyclic voltammograms obtained for the oxidation of NADH at a bare SPCE and the SPCE* in pH 7.4 PBS. As can be seen, anodic oxidation of NADH takes place at a much higher potential (ca. 0.73 V) on a bare SPCE. However, a gradually decrease of peak current in subsequent cycles indicates the fouling of the electrode surface (data not shown). Obviously dimerization of the oxidized product NAD+ occurs easily at higher potentials and hence it gets adsorbed on the electrode surface. Compared to the CV behavior observed on a bare SPCE, the SPCE* exhibited a significant shift in peak potential to a less positive value (ca. 0.40 V, Fig. 1B). Furthermore, the peak current was unaltered in the subsequent cycles (Fig. 1C) indicating low susceptibility to electrode fouling of the SPCE* . The negative shift in the oxidation potential also reflects a faster electron transfer reaction at the preanodized electrode. It

2.2. Apparatus Electrochemical experiments were carried out with a CHI 400 electrochemical workstation (CH Instruments, Austin, TX, USA). The three electrode system consists of an SPCE* as working electrode, an Ag/AgCl reference electrode and a platinum auxiliary electrode. The disposable SPCEs (geometrical area = 0.2 cm2 ) were purchased from Zensor R&D (Taichung, Taiwan). The FIA system is consisted of a Cole-Parmer microprocessor pump drive, a Rehodyne model 7125-sample injection valve (20 ␮l loop) with interconnecting Teflon tube and a Zensor SF-100 thin-layer detecting electrochemical cell specifically designed for SPCE (Zensor R&D, Taichung, Taiwan). A carrier solution of pH 7.4 PBS was used throughout the FIA experiments. Room temperature Raman spectra were recorded with a 3D Nanometer Scale Raman PL Microscopy system by using a Nanofinder® 30 (Tokyo Instruments, INC) that used a He/Ne laser beam with an excitation wavelength of 633 nm and a CCD detector (Andor DU401-BV) with a readout speed of 1–32 ␮s/pixel at −70 ◦ C to record the Raman scattered light intensity. 2.3. Procedure For all experiments, a bare SPCE was electrochemically cleaned by cycling the potential between −0.8 and 1.2 V. The applied potential and time for preanodization was then optimized carefully to get good electrochemical behavior for NADH. In most cases, electrode was electrochemically oxidized (i.e., preanodization) by applying a potential at 2.0 V versus Ag/AgCl for 90 s in pH 7.4 PBS under stirred condition. The as-prepared electrode was further used for the voltammetric studies of NADH.

Fig. 1. Cyclic voltammetric responses of a SPCE (A) and the SPCE* (B) in 0.1 M PBS (pH 7.4) without (dotted line) and with (dark line) 1 mM NADH at v = 50 mV/s. (C) Cyclic voltammetric responses of 1 mM NADH at the SPCE* for 24 continuous cycles.

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Fig. 2. Raman spectrum of a bare SPCE before (A) and after (B) preanodization at 2.0 V (vs. Ag/AgCl) for 90 s. Laser power at sample was 30 mW at 633 nm with 10 s detector exposure.

is well-known that preanodization of a glassy carbon electrode has a great influence on electron transfer through the generation of C O or C OH functional group on the electrode surface [22–27]. Activation of carbon electrodes can also induce the formation of edge planes as well as surface carbonyl groups on the graphite edge planes [20,21]. Note that the increase in background current observed in the cyclic voltammogram for the SPCE* (Fig. 1B) is a proof of the formation of redox active groups (mainly carbonyl functionalities). We speculate that electrocatalytic oxidation of NADH at the SPCE* may be due to the surface carbonyl functionalities on the edge planes of SPCE* formed during the preanodization procedure at an applied potential of 2.0 V (versus Ag/AgCl). This would behave like an electron transfer mediator for the oxidation of NADH as reported for glassy carbon electrodes [4,28]. The generation of more edge plane sites during the preanodization procedure was characterized by a distinctive Raman band at ∼1360 cm−1 [20,21]. As shown in Fig. 2, the Raman spectrum of a bare SPCE changes dramatically during preanodization. The E2g band at ∼1582 cm−1 (basal plane) and a relatively smaller peak intensity of D (disorder) band at ∼1360 cm−1 (edge plane) were observed in the case of a bare SPCE without preanodization. Upon preanodization at 2 V versus Ag/AgCl for 90 s, there is a substantial change in the intensity of D (disorder) band at ∼1360 cm−1 together with a broadening of the ∼1582 cm−1 band. Since the D/E2g ratio is higher in the case of SPCE* , the result would clearly indicate the introduction of edge planes after preanodization. As stated earlier, the D band at 1360 cm−1 arises from the k-vector selection rule from reduced symmetry at graphite edge planes [20,21]. Our recent X-ray photoelectron spectroscopy studies on exclusively SPCE* also confirms the production of carbonyl functionalities ( C O, C OH, C( O) OH, etc.) on the electrode surface [29]. Based on the peak sensitive parameters, the carbon to nitrogen ratio was calculated as 0.07 and 0.11 for SPCE and SPCE* , respectively. The marked increase in the ratio after the preanodization procedure proves the activation of SPCE surface. Scheme 1 sketches the electrochemical oxidation of NADH at the SPCE* with surface carbonyl functionalities on edge plane sites. Most importantly, voltammetric response of NADH oxidation at the SPCE* is in good agreement with that of an edge plane pyrolytic graphite electrode [19]. The dependence of peak

Scheme 1.

current (ip ) with scan rate (v) for the oxidation of NADH was investigated at the SPCE* . There was a gradual increase in the ip with respect to the increase in v. A linear relationship between ip and v1/2 confirms the electrochemical oxidation of NADH at SPCE* is a diffusion-controlled electron transfer process. A Tafel plot (Fig. 3) was then constructed at a quasi-steady condition (i.e., v = 10 mV/s) for 1 mM NADH in pH 7.4 PBS and a Tafel slope (ba ) of 0.17 V/decade was obtained. Assuming a 1e− transfer process in the rate-determining step (na = 1), according to the equation of ba = 2.303RT/na Fαa , the anodic transfer coef-

Fig. 3. Tafel plot for 1 mM NADH in 0.1 M PBS (pH 7.4) under different potential of charge transfer region at the SPCE* with a scan rate of 10 mV/s.

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Fig. 4. Effect of flow rate on the detection of 500 ␮M NADH at the SPCE* by FIA. Carrier solution was 0.1 M PBS (pH 7.4) and applied potential was 0.4 V.

Fig. 5. FIA responses of the SPCE* with different concentration of NADH (10–500 ␮M) in 0.1 M PBS (pH 7.4). Eapp = 0.4 V vs. Ag/AgCl; Hf = 0.3 ml/min.

ficient (αa ) was calculated as 0.35. This result indicates that, in the overall 2e− oxidation of NADH, the first electron transfer is the rate-determining step. 3.2. Analytical application Analytical performance of the SPCE* towards NADH detection was explored by FIA coupled with wall-jet electrode system to further increase the sensitivity. This approach provides the feasibility for the sensitive determination of NADH. Hydrodynamic parameter of flow rate (Hf ) was optimized by using a detection potential of 0.4 V as selected from earlier CV studies. As shown in Fig. 4, considering enhanced peak current and R.S.D. value in analytical application, an Hf of 0.3 ml/min was thus uniformly chosen for further FIA experiments. The effect of applied potential (Eapp ) was then examined by fixing Hf = 0.3 ml/min and an optimized response was obtained upon repeated injection of NADH at Eapp = 0.4 V with 0.1 M, pH 7.4 PBS as carrier solution. The optimized conditions of Hf = 0.3 ml/min and Eapp = 0.4 V were thus used in subsequent FIA studies. Fig. 5 shows the typical FIA responses observed for 10–500 ␮M NADH under the optimized conditions. As can be seen, a linear calibration plot up to 100 ␮M with slope and regression coefficient of 0.0915 ␮A/␮M and 0.990, respectively, were obtained. Eleven continuous injection of 10 ␮M NADH resulting in an R.S.D. of 4.2% indicates a detection limit (DL , S/N = 3) of 157 nM. Note that the detection limit is better than that of a polythionine-modified SPCE [30] and also comparable with most reported results [5,6,16,17,19,31]. Overall the reproducibility of the system is very good and NADH also has a favorable interaction at the SPCE* . Selective detection of NADH is very important in the development of a NADH sensor. It is well known that ascorbic acid is a common interferent during the electrochemical detection of NADH in biological samples. Furthermore, in order to eliminate the interference of ascorbic acid, electrodes can either modify with anionic polymers or immobilize with ascorbate oxidase [32,33]. However, modification with anionic polymers can also

Fig. 6. Cyclic voltammetric responses of the SPCE* (dark line) and a bare SPCE (dotted line) in 0.1 M PBS (pH 7.4) with 1 mM NADH in the presence of 1 mM ascorbic acid at v = 50 mV/s.

exclude NADH as it is negatively charged in neutral pH. Here, we examined the detection of NADH in the presence of ascorbic acid at the SPCE* . Fig. 6 shows the CV responses for equimolar concentration of ascorbic acid and NADH in pH 7.4 PBS. In the case of SPCE, there is a well defined oxidation peak at ∼0.61 V with an ill-defined peak at 0.37 V, which is not stable in the subsequent scans. The SPCE could not separate the voltammetric signal of NADH from the voltammetric signal of ascorbic acid, showing the strong interference of ascorbic acid with NADH. As to the SPCE* , we can see two individual peaks at 0.13 and 0.42 V for ascorbic acid and NADH, respectively, with a peak separation of ∼285 mV. It is clear that the SPCE* can be used to detect NADH in the presence of ascorbic acid without interference. 4. Conclusions The present investigation reveals that the SPCE* shows excellent electrocatalytic activity towards NADH with a marked

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decrease in overpotential and enhanced stability compared to the performance observed at a bare SPCE. Analytical characterization of the SPCE* shows an excellent detection limit of 157 nM. In addition to this, we demonstrate the selective detection of NADH in the presence of ascorbic acid. Raman spectroscopy study provides a solid support to the formation of edge plane sites after the preanodization process. In other words, the disposable SPCE* can act more or less like an edge plane graphite electrode in the electrochemical oxidation of NADH. Most importantly, these facts offer a great promise for the design of amperometric biosensors with the immobilization of suitable dehydrogenase enzymes. Acknowledgements This work was supported by the National Science Council of Taiwan. K.S.P. acknowledges foreign student scholarship from NCHU. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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Biography K. Sudhakara Prasad received his MS degree in chemistry from Gandhigram Rural Institute, Deemed University, India, in 2003. At present he is pursuing his doctoral studies under the guidance of prof. Jyh-Myng Zen. His research interests include electroanalytical chemistry, chemical sensors and material science.