A new glucose sensor based on encapsulated glucose oxidase within organically modified sol–gel glass

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Abstract

A non-mediated glucose biosensor is reported based on encapsulated glucose oxidase (GOD) within the composite sol–gel glass, which is prepared using optimum concentrations of 3-aminopropyltriethoxy silane, 2-(3, 4-epoxycyclohexyl)-ethyltrimethoxy silane, GOD dissolved in double distilled water and HCl. A white, smooth film of sol–gel glass with controlled thickness is also prepared at the surface of a Pt disk electrode without GOD to study the electrochemistry of ferrocene monocarboxylic acid at the surface of the modified electrode. The electrochemistry of ferrocene monocarboxylic acid at composite sol–gel glass electrode with varying thickness is reported. The GOD-immobilized film over the Pt disk surface shows a yellow colour. The new sol–gel glass in the absence and the presence of GOD is characterized by scanning electron microscopy (SEM). The enzyme-immobilized film of different thickness is made using varying concentrations of soluble sol–gel components applied to the well of the Pt disk electrode. The enzyme is cross-lined with the 3-aminopropyltriethoxysilane, one of the composite component of sol–gel glass using glyoxal at 4°C for 4 h. The response of non-mediated enzyme sensor is studied based on cyclic voltammetry and amperometric measurements. A typical amperometric response of the enzyme sensor having varying thickness of the modified sol–gel glass film is reported. The variation of the response time as a function of the film thickness is reported. The stability of cross-linked GOD to sol–gel glass is found to be more than a month without loss of enzymatic activity when the enzyme sensor is stored at 4°C.

Introduction

The recent reports on the synthesis of sol–gel glasses 1, 2, 3, 4, 5, 6have received widespread attentions because of its application in various directions. One of the potential application of such materials is in the development of sensors particularly for attaching the sensing material to the surface of physico-chemical transducers. A number of publications are available in the literature on the applications of sol–gel glass for the development of optical and electrochemical sensors. The development of electrochemical biosensor involves the coupling of biological components with the polarizable or non-polarizable electrodes. The use of sol–gel glass for the development of electrochemical biosensors have received great attention because of its possible applications in commercialization. The development of such biosensors based on sol–gel glass is currently restricted mainly due to two major problems: (1) the requirement of controlled gelation of soluble sol–gel components at ambient conditions, (2) preparation of sol–gel glass of smooth surface, controlled thickness and porosity. Additionally, the stability of biological element within the sol–gel network is another need to develop such sensors at commercial scale.

Apparently, the synthesis of suitable bio-compatible sol–gel glass of desired thickness and porosity is of considerable interest. The soluble materials leading to the formation of sol–gel glasses are the derivatives of alkoxysilane. These alkoxysilanes in acidic and some time in basic medium generate a solid network whose physical structure can be comparable to conventional glass. However, research is needed to synthesize such sol–gel glasses suitable for the better performance as sensors and reactors of practical significance. The application of these glasses in sensors requires controlled synthesis of solid-state network with desired porosity and thickness. Additionally, the availability of a suitable group within the solid-state network provides an advantage for the cross-linking of the sensing element to the solid-state network.

Recently, we reported [3]a glucose biosensor based on sol–gel glass using trimethoxysilane and 3-glycidoxypropyltrimethoxysilane with and without glucose oxidase (GOD). The sol–gel glass without protein shows a nice network with symmetrical distribution of the pores whereas the introduction of GOD results irregular distribution of the protein throughout the gel network together with the least porosity. The present investigation is attributed to the fabrication of such sol–gel glass using a mixture of two silanes, 3-aminopropyltriethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane. These silanes under optimum conditions generate the solid-state network of desired configuration for the construction of biosensors and is reported in the present research work. The availability of the amino-group in one of the composite sol–gel glass components provides an extra event for cross-linking with protein/enzyme using bifunctional reagent (Glyoxal).

Two types of the composite sol–gel glass have been developed in the present investigation: (1) sol–gel glass of controlled thickness and porosity without enzyme (GOD), (2) sol–gel glass of controlled thickness and porosity with enzyme GOD. The electrochemistry of ferrocene monocarboxylic acid is studied on system 1. The performance of system 2 as a glucose biosensor is studied. The performance of non-mediated glucose sensors based on cyclic voltammetry and the typical amperometric responses of the biosensors having varying thickness and porosity are reported.

Section snippets

Materials and methods

3-Aminopropyltriethoxy silane was obtained from Aldrich; 2-(3,4-Epoxycyclohexyl)-ethyltrimethoxy silane was obtained from United Chemical Technologies, Petrarch™ Silanes and Silicones, Bristol, PA, USA; GOD was obtained from Sigma. All other chemical employed were of analytical grade.

Preparation of organically modified sol–gel glass electrodes

The electrode body used for the preparation of composite sol–gel glass modified electrodes was similar as described in an earlier publication [7]made from Teflon containing platinum base with a recessed depth of 2

Physical characterization of organically modified sol–gel glass electrode

The physical characteristics of the sol–gel glass with and has been examined using various composition of 3-aminopropyltriethoxy silane, 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane, enzyme dissolved in water and HCl. The content of 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane significantly affects the physical characteristics of the sol–gel matrix. The colour of the enzyme-immobilized sol–gel film is yellow whereas the colour is white in the absence of enzyme. The smooth sol–gel film

Conclusion

We report the fabrication of a new composite sol–gel glass using 3-aminopropyltriethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane, in the presence of distilled water and HCl. The resulting silane provides a very smooth surface with a rigid porous structure. The enzyme-modified sol–gel glass is reported to construct an amperometric biosensor for glucose. Under optimum composition of the sol–gel glass ingredients, a very smooth and thin stably adhered to the Pt surface was obtained

Acknowledgements

The authors are thankful to UGC, New Delhi for financial assistance.

Prem Chandra Pandey received his MSc degree in Chemistry in 1980 and PhD in 1986, both from Gorakhpur University. Since 1988, he is a lecturer in the Analytical Chemistry division at Banaras Hindu University and subsequently worked as Reader since 1996 in the same division. He is a member of the Bioelectrochemical society and the Indian Chemical society. He has worked as a visiting scientist at Ecole des Mines, France and the National Institute of Standards and Technology, Gaithersburg, USA.

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Prem Chandra Pandey received his MSc degree in Chemistry in 1980 and PhD in 1986, both from Gorakhpur University. Since 1988, he is a lecturer in the Analytical Chemistry division at Banaras Hindu University and subsequently worked as Reader since 1996 in the same division. He is a member of the Bioelectrochemical society and the Indian Chemical society. He has worked as a visiting scientist at Ecole des Mines, France and the National Institute of Standards and Technology, Gaithersburg, USA. His current interests include mediated, non-mediated and electrocatalytic biosensors, optical biosensors, ion-selective electrodes, chemically modified electrodes, electropolymerization, re-chargeable batteries, dynamics of membrane processes both in linear and non-linear regime, flow injection analysis and photo-chemistry.Sanjay Upadhyay obtained his PhD degree in 1993 from Banaras Hindu University on “liquid membrane transport”. Since 1994, he has been working as Research Associate at the Department of Chemistry. He is working on electrochemical biosensors in the group of Dr. P.C. Pandey.Harish C. Pathak received his MSc in Chemistry from Poorvanchal University in 1994. Since 1996, he has been a student of Banaras Hindu University and has been working for his PhD degree in the group of Dr. P.C. Pandey. His current interests include electrochemical biosensors and photophysical properties of bacteriorhodopsin molecule.

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