A NADH biosensor based on diphenylalanine peptide/carbon nanotube nanocomposite

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Abstract

The fabrication and notably improved performance of composite electrodes based on the nanocomposite of diphenylalanine peptide-covered multiwalled carbon nanotube (PP–MWCNT) is described. The synthesis of the nanocomposite of PP–MWCNT is a self-assembly process of diphenylalanine peptide (PP) along carbon nanotube (MWCNT) via aromatic stacking interaction combined with hydrogen bond of diphenylalanine peptide. PP–MWCNT modified electrode was fabricated by a simple casting method, and studied with cyclic voltammetry (CV) and chronoamperometry. PP–MWCNT modified electrode showed a high, direct and unmediated response to β-reduced coenzyme nicotinamide adenine dinucleotide (NADH) at a potential of 0.600 V (vs. SCE), which had reduced the overvoltage of NADH oxidation by 0.200 V in comparison with the bare electrode. Furthermore, the current response of NADH at PP–MWCNT modified electrode is about five times higher than that of the bare electrode. Thus, PP–MWCNT provides a new candidate for fabrication of biosensor based on β-coenzyme nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenases. Herein, an ethanol biosensor was prepared by crosslinking ethanol dehydrogenase (ADH), bovine serum albumin (BSA) and PP–MWCNT onto the electrode. The ethanol biosensor exhibited a good linearity ranged from 30 μM to 700 μM with a high sensitivity of 30.00 nA/μM cm−2 and with a low detection limit of 12 μM.

Research highlights

► An electrochemical active nanocomposite of diphenylalane dipeptide/multiwalled carbon nanotubes has been prepared on the basis of noncovalent assembly. ► The as-prepared nanocomposite can offer a remarkable decrease of the overvoltage of NADH oxidation, which makes it available in a high-sensitive ethanol biosensor.

Introduction

On the virtue of participation in more than 300 dehydrogenases as key electron mediators in living cells [1], NADH and its oxidized form nicotinamide adenine dinucleotide (NAD+) are of great importance in design and fabrication of amperometric biosensor for substrate of NAD dependent dehydrogenases, which has been widely used in food control, environmental, and clinical analysis [2], [3]. It is a challenging task of electrochemical sensing of NADH at low working potential in the micro/sub-micro molar level without any interference. Although the reversible potential of NADH/NAD+ couple is estimated to be −0.32 V (vs. NHE) at neutral pH [4], NADH oxidation is irreversible at unmodified electrodes, and take place at considerable high overpotential [5], which limits the selectivity of the determination in a real sample. Furthermore, the reaction at a high overpotential involves radical intermediates that cause electrode fouling [6], [7] and the loss of analytical sensitivity, reproducibility, and operational lifetime [8], [9].

Many efforts have been intensively devoted to decrease the overpotential of NADH oxidation. One approach is to immobilize new mediators on the electrode surface to ameliorate analytical performance of the based electrode in virtue of promotion of electron shift between electrode interfaces. The mediators are studied intensively, such as catechol derivatives [10], phenoxazine dyes [11,12], prussian blue analogue [13], organic salt [14] and conducting polymer [15]. Another method is to introduce new electrode materials to the based electrode to enhance the analytical response to NADH by decreasing overpotential for NADH oxidation, minimizing surface fouling, and improving mass-diffuse and electron-transfer kinetics. The nanomaterials are involved in peptide nanotube [16], [17], Au nanoparticle, TiO2 nanostructured film [18], and carbon materials [2], [19], [20], especially carbon nanotube [21], [22].

Diphenylalanine nanotube, reported by Reches [23], is a dipeptide nanotube of β-amyloidal diphenylalanine structural motif associated with Alzheimer disease, and forms on basis of self-assembly mechanism. The peptide nanotubes are particularly important because they can allow chemical modification [24], [25], [16], [17]. Moreover, they can be used as nanowires and nanoscaffolds in micro- and nano-electronics device [23], [26], [27], [28]. The diphenylalanine nanotube is a biocompatible organic nanotube, which can be constructed as amperometric biosensor to monitor NADH and phenol [16], [17].

Here we report a self-assembly strategy to synthesize PP–MWCNT. PP–MWCNT is special, because it combines the good biocompatibility of diphenylalanine peptide with the excellent electroconductivity of carbon nanotube. Thus, PP–MWCNT can be used as a novel biosensing platform to improve the analytical performance of the based electrode. The present work is aimed at designing and fabricating the highly sensitive amperometric enzyme biosensor based on PP–MWCNT. As a model, ADH was introduced to demonstrate the advantages of the novel biosensor, which is explored extensively in this work.

Section snippets

Chemicals

d-diphenylalanine and 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP) were obtained from Sigma–Aldrich. HNO3, H2SO4, Glutaraldehyde (GA) solution, NADH and NAD+ were obtained from ACROS. ADH from Saccharomyces cerevisiae (⩾300 unit mg−1) and BSA were obtained from Sigma. Multiwalled carbon nanotube (MWCNT, ∼5 μm length, 10–30 nm diameter, and 95% purity) was obtained from Shenzhen Nanotech Port Co., Ltd. China. All reagents were of analytical grade, and the solutions were prepared with de-ionized water

Morphological investigation of PP–MWCNT

The as-treated MWCNT can be dispersed in aqueous solution when it was oxidized and shorten by the mixed acid of HNO3 (65%) and H2SO4 (98%). The morphology of as-treated MWCNT, as shown in Fig. 1A, is clean. PP was formed on the basis of self-assembly process in the de-ionized water, as shown in Fig. 1B, which is comprised of stiff nanotubes with 5–10 μm length and 200–500 nm diameter.

PP–MWCNT was prepared by adding HFP solution of d-diphenylalanine into the dispersed solution of MWCNT. PP–MWCNT

Conclusions

PP–MWCNT was prepared, which was based on a self-assembly strategy. This novel nanocomposite can speed up the electron transfer between the electrode interfaces when it was immobilized on the surface of the based electrode. As an electron promoter, PP–MWCNT can offer a remarkable decrease of the overvoltage of NADH oxidation, and it can be used to determine NADH with a comparative high sensitivity and a comparative low detection limit. In this work, an ethanol biosensor based PP–MWCNT was

Acknowledgments

The authors are most grateful to the National Natural Science Foundation of China (Nos. 90406016 and 20903082), Department of Science and Technology of Zhejiang Province (No. 2007F70041), and Zhejiang Normal University for the financial supports.

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