Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents

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Abstract

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.

Introduction

Immobilized enzyme based biosensors have been widely used for analyses in food and fermentation industries (Coulet and Bertrand, 1979, Scheller and Karsten, 1983, Sheper, 1992), in environmental monitoring (Schmidt and Scheller, 1989) and in clinical diagnosis (Mason and Townshend, 1984).Biosensors for glucose and sucrose have been widely used for food and fermentation sample analysis (Danielsson, 1994, Hundech et al., 1992, Scheller and Renniberg, 1983, Xu et al., 1989, Matsumoto et al., 1988). An important consideration in the practical application of biosensors is the operational life of the sensing element particularly while monitoring the food and fermentation analysis where the substrate concentrations are high. When immobilized enzymes are used for this purpose, the activity loss due to denaturation and deactivation of the enzyme diminishes the life of the sensor. Therefore techniques to enhance the storage and operational stability of the enzyme electrode are important in the application of electrochemical biosensors. Attempts to stabilize enzymes reported in literature include the use of cationic polyelectrolytes (Gavalas and Channiotakis, 2000), polyelectrolytes and sugar alcohols (Gibson et al., 1992), immobilization of enzyme and polyelectrolyte complex on CPG (Appleton et al., 1997) and immobilization of enzyme on carbon paste electrodes in the presence of various additives (Lutz et al., 1995).

One of the suitable enzyme immobilization methods for biosensor applications is crosslinking by using glutaraldehyde. Glutaraldehyde being a strong bifunctional reagent, modifies the enzyme drastically, leads to conformational changes and loss of activity (Broun, 1976). This deleterious effect can be minimized by using inert proteins like BSA, gelatin, thrombin and lysine. These proteins avoid excessive of intramolecular crosslinkages within the enzyme and enhance the intermolecular linkages between the enzyme and inert proteins (Broun et al., 1973). While it is known that inert proteins can act as enzyme stabilizing agents, its application has been restricted to BSA and gelatin. In view of the reported observations that complimentary surface protein (Chang and Mahoney, 1995) and durability of carrier protein (Gabel et al., 1970, Gabel, 1973) play an important role in stabilization of enzymes, it is quite possible that stable proteins other than BSA and gelatin showing better complimentarity with the enzyme provide a better stability of free as well as immobilized enzyme preparations. Stabilization of desired enzyme can be achieved by using certain proteins, which may be catalytically active or inactive. In this paper we refer to them as protein based stabilizing agents (PBSA). However, one should keep in mind, that if the PBSA is an enzyme, its products should not interfere with the biochemical reaction of the desired enzyme electrode. In our laboratory we have constructed a batch biosensor for glucose using immobilized GOD and for sucrose using immobilized multienzyme system (invertase, mutarotase and glucose oxidase) and tested for repeated use. In an attempt to enhance the operating stability of the biosensor, we have found that incorporation of lysozyme as PBSA can achieve this objective substantially, and the results are reported in this paper.

Section snippets

Materials

Glucose oxidase (E.C. 1.1.3.4.) from Aspergillus niger (specific activity—180 IU/mg), invertase (E.C. 3.2.1.26) from yeast (specific activity—400 IU/mg), mutarotase (E.C. 5.1.3.3.) from hog kidney (specific activity—2500 IU/mg), lysozyme, BSA, gelatin and glutaraldehyde were procured from M/s sigma, USA, the cellophane membrane molecular weight cut-off 6000–8000 from Spectra/por, USA and oxygen permeable teflon membrane from WTW, Germany. For the biosensor, the dissolved oxygen was measured by

Results and discussion

Fig. 1 demonstrates the operational stability of the enzyme electrode for glucose containing GOD immobilized with different PBSAs in 50 mM phosphate buffer, pH 6.0. Lysozyme was found to be the best for the stabilization of GOD among the PBSAs tested, followed by BSA and gelatin. Immobilization without any additive gave virtually no enzyme activity. Repeated measurements with 30 μl of 10% glucose solution were carried out. For GOD immobilized with lysozyme, it was possible to analyse 750

Acknowledgments

Financial support from Department of Science and Technology, Government Of India, is gratefully acknowledged. Director, CFTRI, Mysore, is thanked for the facilities and encouragement. M.D. Gouda, is thankful to the CSIR, India, for the award of senior research fellowship. Help from K.C. Gulla, in the preparation of the manuscript is also acknowledged.

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