Elsevier

Bioelectrochemistry

Volume 89, February 2013, Pages 19-25
Bioelectrochemistry

Direct electrochemistry of alcohol oxidase using multiwalled carbon nanotube as electroactive matrix for biosensor application

https://doi.org/10.1016/j.bioelechem.2012.08.007Get rights and content

Abstract

Rapid detection of alcohol is important in clinical diagnosis and fermentation industry. An octameric alcohol oxidase (AOx) (Mr 675 kDa) from Pichia pastoris, immobilized on multiwalled carbon nanotubes—Nafion® (MWCNT-Nf) matrix and encapsulated with polyethylenimine (PEI) on gold electrode (AuE), showed a redox peak at 0.21 V (vs. Ag/AgCl electrode at pH 7.5) for oxidation of alcohol. The electron transfer rate constant and surface coverage of the immobilized AOx were 1.69 ± 0.15 s 1 and 2.43 × 10 12 mol cm 2, respectively. Studies on response and kinetics of Au-MWCNT‐Nf‐AOx-PEI bioelectrodes for alcohol showed a linear response in the range of 8 μM–42 μM, response time of 55 s for steady state current, and detection limit of 5 μM. The bioelectrode retains ~ 90% of the original response even after four weeks when stored in potassium phosphate buffer pH 7.5 at 4 °C. The fabricated bioelectrode was found to exclude interference caused by the common electroactive species such as ascorbic acid, uric acid, lactic acid, glucose and urea. The bioelectrode also showed reliable response characteristics in blood serum samples. The findings of the investigation have established the direct electrochemistry of the AOx protein and its potential biosensor application for quantitative detection of alcohol in blood serum.

Highlights

► Direct electrochemistry of alcohol oxidase in carbon nanotube matrix-based bioelectrode was established. ► The bioelectrode showed reliable response characteristics in the sample. ► Nearly 90% of the original response of the bioelectrode retained even after four weeks. ► The bioelectrode excluded interference caused by the common electroactive species of body fluid.

Introduction

The detection and quantification of alcohols with high sensitivity, selectivity and accuracy are required in many different areas, such as, clinical and forensic analysis, food, beverage, and pulp industries [1]. Many analytical methods based on chemical, chromatographic, and spectroscopic principles have been developed for the determination of ethanol. Although, a few of these methods are reliable, they are complex, time consuming and require prior separation processes, expensive instrumentation and trained operators. Such disadvantages could be overcome by using biosensor devices incorporating the enzymes as bio-recognition elements [2]. Biosensors reported so far for quantitative detection of alcohol are yet to develop properly for commercial applications. Alcohol biosensors described in the literature are largely based on alcohol dehydrogenase (ADH) [3], which normally require the presence of co-factor nicotinamide adenine dinucleotide (NAD+) [4], [5], [6], [7]. The major constrain for developing ADH based alcohol biosensor is the need of supplementing the co-factor (NAD+) to the reaction medium [8]. Although there is a continuous effort to regenerate the co-factor in the reaction system to sustain the catalytic activity of ADH, efficient cofactor regenerating system for biosensor application of ADH is yet to develop properly. Alcohol oxidase (AOx) (EC 1.1.3.13) as bio-recognition element for sensing alcohol has stimulated interest since the last decade [1]. Some potential advantages of AOx based bio-recognition system are identified among which the irreversible catalytic oxidation of alcohols and the avidly bound flavin-based co-factor to the redox center of the enzyme are pertinent to the biosensor applications. Moreover, the knowledge on molecular and functional characteristics of this unexplored group of redox enzymes is rapidly growing since recent past. AOx catalyzes the oxidation of alcohols using molecular oxygen as the electron acceptor and producing corresponding carbonyl compound and hydrogen peroxide in the reaction medium. The reaction may be followed by measuring O2 or H2O2 concentration using optical or electrochemical detections [1], [8], [9], [10], [11], [12], [13], [14], [15], [16]. The oxygen-based detection, however, has practical inconveniences and limitations such as, poor response, low accuracy and reproducibility, and high background signal that may raise the minimum detection level of the substrate alcohol [17]. Nevertheless, H2O2 based sensor has the advantages of the relative ease of manufacturing and the possibility of constructing them in small sizes, the high potential (~ 600 mV) necessary to oxidize H2O2 poses a problem of electrochemical interference due to the presence of reducing compounds (e.g. ascorbic acid, uric acid, bilirubin and acetaminophen), particularly in blood serum sample. Moreover, the response of the H2O2 based sensor is slow. The second generation AOx based amperometric biosensors using electron transport mediator that shuttles electrons between the redox center of the enzyme and the electrode are also not known so far since no suitable mediator has yet been found for AOx.

We report here an amperometric biosensor using direct electrochemistry of the large multimeric AOx from Pichia pastoris immobilized in a nano-composite matrix on the surface of gold (Au) electrode. Multiwalled carbon nanotubes (MWCNT), which have been used for fabricating bioelectrodes of many small enzymes [18], [19] due to its good electrical conductivity, high mechanical strength, and antifouling property [20], were dispersed on the surface of the electrode using hydrophobic polymer chain of Nafion (Nf) [21]. Polyethylenimine (PEI), a polycationic polymer, was used to stabilize the AOx immobilized on the nano-matrix coated on an Au electrode [22]. The fabricated AOx bioelectrode has been characterized and detailed account on the response of the bioelectrode for the substrate alcohol is presented here.

Section snippets

Chemicals and reagents

AOx from P. pastoris (21 U/mg protein), multiwalled carbon nanotube (MWCNT, size OD 10–15 nm, ID 2–6 nm, length 0.1–10 μm), Nafion117 (Nf) (5% w/v in isopropanol), polyethylenimine (PEI) (50% w/v in water solution), ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], and horseradish peroxidase (HRP) were bought from Sigma-Aldrich (USA). Hydrogen peroxide (H2O2) 50% and Ethanol 99.9% was purchased from Merck. All other chemicals were of analytical grade and used as received without

Morphological characterization of the bioelectrode

The surface morphology of bare Au, Au-MWCNT-Nf, Au-MWCNT-Nf-AOx and Au-MWCNT‐Nf‐AOx-PEI bioelectrodes was investigated using FESEM. As shown in Fig. 1, AuE shows homogenous surface (Fig. 1A), and the Au-MWCNT-Nf electrode shows uniform distribution of MWCNTs on the electrode surface (Fig. 1B) with porous morphology clearly visible from the image taken at higher magnification (inset Fig. 1B). When AOx was added on this MWCNT-Nf film, the thread like structure of MWCNT and porosity of the film

Conclusion

The direct electrochemistry of a multimeric alcohol oxidase (AOx) physically immobilized by encapsulating with PEI in a MWCNT-Nf matrix on the gold electrode surface was established for the first time. Direct electron transfer facilitated by the MWCNT between the AOx protein and the electrode was confirmed. The entrapped AOx possesses good bioactivity and electrocatalytic activity at room temperature and physiological pH. The constructed bioelectrode showed linear response at the oxidation

Acknowledgments

We acknowledge the financial assistance from DBT, India to carry out this work. The FESEM facility provided by CIF, IITG is duly acknowledged.

Madhuri Das received M.Sc. degree in Biotechnology from Gauhati University, India in 2007 and M.Tech. in Bioelectronics from Tezpur University in 2009. She is a PhD student at the Centre for Energy, IIT Guwahati, India. Her research interest is the development of novel enzyme based bioelectrode for biosensors and biofuel cell applications.

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    Functionalized CNTs have been used as electrochemical sensors and many review papers concerning CNTs-electrochemical sensors have been highlighted (Dai, 2007; Daniel et al., 2007; Gao, Guo, Liua, & Huang, 2012; Husain, 2018a; Jacobs, Peairs, & Venton, 2010; Yang et al., 2015). Das and Goswami (2013) immobilized an octameric alcohol oxidase (AOx) (Mr 675 kDa) obtained from Pichia pastoris on MWCNT-nafion (MWCNT-Nf) matrix. The complex encapsulated with polyethylenimine (PEI) on gold electrode, showed a redox peak at 0.21 V (vs Ag/AgCl electrode at pH 7.5) for oxidation of alcohol.

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Madhuri Das received M.Sc. degree in Biotechnology from Gauhati University, India in 2007 and M.Tech. in Bioelectronics from Tezpur University in 2009. She is a PhD student at the Centre for Energy, IIT Guwahati, India. Her research interest is the development of novel enzyme based bioelectrode for biosensors and biofuel cell applications.

Prof. Pranab Goswami received M.Sc. degree in Chemistry from Gauhati University, India in 1986, MS degree in Science & Technology, from BITS Pilani, Rajasthan and PhD degree in Biotechnology from Gauhati University in 1992. From 1991 to 2002, he was a scientist (Scientist B to scientist E1) at NEIST, Jorhat India. Prof. Goswami was a BOYSCAST fellow of DST, India at University of Massachusetts Boston, USA from 1996 to 1997. In 2002, he joined IIT Guwahati as faculty and currently working as a Professor at the same institute. His research interest comprises the development of enzymatic biosensors and biofuel cells for clinical and biomedical applications.

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