Silica sol–gel immobilized amperometric biosensor for the determination of phenolic compounds
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.
References (31)
- et al.
Tr. Anal. Chem.
(1996) - et al.
Anal. Chim. Acta
(1995) - et al.
J. Bioelectrochem. Bioenerg.
(1993) - et al.
Biosens. Bioelectron.
(1991) - et al.
Mater. Lett.
(1990) - et al.
Sensors Acutators B
(1997) - et al.
Anal. Chim. Acta
(1996) - A. Townshend (Ed.), Encyclopedia of Analytical Science, vol. 8, Academic Press, London, 1995, p....
- A.I. Vogel, Textbook of Quantitative Chemical Analysis, Longman, UK, 1989, p....
- et al.
Analyst
(1994)
Anal. Lett.
Electroanal.
Anal. Chem.
Electroanal.
Anal. Lett.
Cited by (122)
Acetylcholinesterase based biosensor for monitoring of malathion and acephate in food samples: A voltammetric study
2014, Food ChemistryCitation Excerpt :Among these sol–gel immobilisation can be preferred since the other immobilisation methods are tedious, result in poor stability, and require expensive reagents, enzyme leaking and loss of enzyme activity. So many sol–gel derived enzyme biosensors have been developed to monitor glucose, lactate, cholesterol, dopamine, H2O2, phenols and urea (Pandey, Upadhyay, Tiwari, Singh, & Tripathi, 2001; Park, Lwuoha, Smyth, Freoney, & Mc Shane, 1997; Sampath & Lev, 1996; Yao & Takashima, 1998; Park, 1999; Li, Chia, Goh, & Tan, 1998; Pandey & Singh, 2001). In this work, we demonstrated that acetylthiocholine chloride (ASChCl) used as a substrate for the enzyme catalysed reaction.
Amperometric biosensor based on polypyrrole and tyrosinase for the detection of tyramine in food samples
2013, Sensors and Actuators, B: ChemicalA comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes
2012, BioelectrochemistryCitation Excerpt :Whilst using small volumes of electrolyte helps to prevent the components from leaking from the cell, drying of the electrolyte can contribute to power loss. As the anodes are coupled to an enzymatic printed cathode the eventual loss in cell power can neither be attributed solely to anode instability with, for example issues with stability of ABTS as a laccase mediator previously highlighted [21,30]. None of the other mediators tested shows promise in the printed ALDH anode for this application/threshold setting.