Silica sol–gel immobilized amperometric biosensor for the determination of phenolic compounds

https://doi.org/10.1016/S0003-2670(98)00064-6Get rights and content

Abstract

An amperometric enzyme electrode for phenolic compounds was developed via an easy and effective immobilization method using the sol–gel technique. The enzyme electrode comprises tyrosinase immobilized by the thin silica sol–gel layer on a carbon-paste electrode. The tyrosinase retains its bioactivity when being immobilized by the sol–gel film. Phenolic compounds were determined by the direct reduction of biocatalytically liberated quinone species at 0 mV vs. Ag/AgCl (sat. KCl). The process parameters for the fabrication of the enzyme electrode were optimised. The influence of various experimental variables was explored for optimum analytical performance of the enzyme electrode. The effect of oxygen on the response of the enzyme electrode was evaluated. The sensitivities of the enzyme electrode for catechol, phenol, p-cresol, m-cresol, o-cresol and 2-chlorophenol were 1.53, 1.28, 1.05, 0.687, 0 and 0 A M−1, respectively. The enzyme electrode retained ca. 50% of its activity after 15 days of storage in a phosphate buffer solution at 4°C.

Introduction

The determination of phenolic compounds is of great importance owing to their applicability in a broad range of chemical manufacturing processes and their inherent toxicity. Colorimetric- and ultraviolet-spectrophotometric analyses are now commonly used for the determination of phenols as standard methods 1, 2. However, these schemes may suffer from the time-consuming complicated sample pre-treatment, lacking sensitivity and may not be suitable for in situ sensing application. To address this problem, a simple, effective and fast alternative method for the determination of phenolic compounds is desirable. Amperometric biosensors for phenolic compounds based on tyrosinase have proven to be promising for this purpose 3, 4, 5, 6, 7, 8, 9.

Tyrosinase, also known as polyphenol oxidase, is a copper-containing mono-oxygenase that can catalyse the conversion of phenolic compounds to the corresponding quinones in the presence of oxygen. The liberated quinone species can be electrochemically reduced to phenolic substances at low potential without any mediator. Numerous immobilization methods have been developed to stabilise the tyrosinase in enzyme electrodes. Besides native fixing behind a dialysis membrane [10] and mixing in carbon paste [9] or graphite–epoxy [4], tyrosinase has been immobilized by entrapment within polyacrylamide gel [11], polypyrrole using amphiphilic pyrrole [12], poly(carbamoyl sulfonate) hydrogel [7] and by glutaraldehyde-mediated cross-linking either on bovine serum albumin [13] or polyamide mesh [14]. Nevertheless, the search for a simple and reliable method to immobilize and stabilise tyrosinase is still of interest.

This article describes the immobilization of tyrosinase on a carbon-paste electrode (CPE) for the determination of phenolic compounds by the sol–gel technique. The sol–gel process is a low temperature, high purity processing technology for the production of ceramic materials by hydrolysis and polycondensation of alkoxides. This technology has provided an alternative route to the analytical chemists to tailor supporting inorganic matrices with the versatility and flexibility that has been traditionally attributed to organic polymers. The porous and chemically inert sol–gel inorganic host support has been proven to be an effective and attractive matrix for the immobilization of active enzymes due to the significant advantages offered 15, 16, 17, 18, 19, 20. From our previous investigations 21, 22, we found that a thin layer sol–gel matrix is more advantageous than a monolithic sol–gel matrix for enzyme immobilization. This is due to shorter diffusion path for the substrate and less denaturation of immobilized enzyme in the thin layer matrix. In the present investigation, we report on the fabrication and characterisation of a tyrosinase enzyme electrode immobilized by the thin sol–gel film derived from tetramethoxysilane.

Section snippets

Reagents

Tetramethoxysilane (TMOS, 99%) was obtained from Janssen Chimica. Tyrosinase (EC 1.14.18.1, 4400 U mg−1) from mushroom, catechol, p-cresol, m-cresol and o-cresol were obtained from Sigma. 2-Chlorophenol and phenol were obtained from Fluka. Cetyltrimethylammonium bromide (CTAB) was obtained from Aldrich and prepared as 3.8% (w/v) solution in methanol. All other chemicals were of analytical grade and used as received without further purification. All aqueous solution were prepared in distilled

Cyclic voltammetry

Fig. 1 shows the cyclic voltammograms of tyrosinase enzyme electrode in an unstirred 0.02 M air-saturated phosphate buffer (pH 6.5) without catechol (- - -) and with 5 μM catechol (———). It was observed that the reduction current increased after catechol was added to the phosphate buffer solution on the enzyme immobilized electrode. Such an increase in the reduction current is due to the reduction of quinone species liberated from the enzymatic reaction catalysed by the tyrosinase on the enzyme

Conclusions

In this work, we report that tyrosinase has been successfully immobilized by the simple sol–gel technique on a CPE which functions as a selective and sensitive phenolic compounds biosensor. The enzyme electrode exhibits a remarkable electrochemical response in terms of sensitivity and reproducibility, since the sol–gel immobilized tyrosinase CPE not only increases the sensitivity for phenolic compounds detection but also improves the selectivity of the unmodified CPE. It has been observed that

Acknowledgements

We are grateful to Nanyang Technological University (Singapore) for the RP17/96 research grant. One of us, J. Li, is grateful for the postgraduate research scholarship awarded by NTU.

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