Elsevier

Polymer

Volume 46, Issue 7, 10 March 2005, Pages 2211-2217
Polymer

Viscoelastic and swelling properties of glucose oxidase loaded polyacrylamide hydrogels and the evaluation of their properties as glucose sensors

https://doi.org/10.1016/j.polymer.2004.12.039Get rights and content

Abstract

Here we report the immobilization of glucose oxidase in polyacrylamide hydrogels carried out by aqueous crosslinking copolymerization of acrylamide and N,N′ methylene bisacrylamide in the presence of the enzyme. The swelling and viscoelastic properties of the hydrogels were evaluated as a function of the content of crosslinker of the polymer chains and enzyme concentration. Amperometric measurements were also carried out to evaluate the system as a glucose biosensor.

Introduction

Polymer gels are a unique class of macromolecular networks that contain a large fraction of solvent within their structure [1], [2], [3]. The ability of polymer gels to undergo substantial swelling and collapsing, up to 1000 times in volume [4], as a function of their environment is one of the most notable properties of these materials [5]. The phenomenon of gel volume transitions, which can be induced by temperature, pH, or ionic strength, among other stimuli, has prompted researchers to investigate gels as potential actuators, artificial muscles, sensors, controllable membranes for separations and modulators for delivery of drugs [6], [7], [8], [9], [10], [11]. Gels are particularly appropriate for biomedical applications because of their ability to simulate biological tissues [12].

Gels are usually formed by free radical polymerisation of monomers in the presence of a difunctional crosslinking agent [13], [14]. They can be made either in bulk or in nano or microparticles. The bulk gels are easy to handle and to study, but have very slow swelling rate, while the gel nanoparticles act quickly to an external stimulus, but are too small for some applications [15].

In recent years, polyacrylamide-based hydrogels have received considerable attention because of their use in many applications [16], [17], [18], [19]: specific sorbents, support carrier in biomedical engineering, aggregating agents, soil improvement agents, polymer processing or improving textiles, paper strengthening agent, adhesive, paints, oil salvaging agents, etc. The kinetics of network formation in free-radical copolymerisation of acrylamide and N,N′-methylene-bis-acrylamide in aqueous solution has been extensively studied and it has been reported that the polyacrylamide network exhibits inhomogeneous crosslink distribution [20]. Partially hydrolysed polyacrylamide gels exhibit volumetric phase transition by changing the temperature, pH, and solvent composition [21]. More studies on polyacrylamide gels have focused in the gelation conditions, swelling properties, phase transitions in swollen gels and their applications. However, there are few studies that deal with the determination of the structure of polyacrylamide gels and its derivation from viscoelastic measurements [22].

Polyacrylamide gels have been extensively used for the matrix of electrophoresis and to encapsulate drugs, enzymes and proteins for application in drug delivery systems and biosensors [23], [24]. For example, the immobilisation of enzymes into polymer gels allows their stabilisation and the retention of the enzymatic activity for long periods of time. Nevertheless, the modifications induced by the enzyme in the molecular structure and in the viscoelastic and swelling properties of the gel have been little studied [25].

In the present study, we have investigated the viscoelastic and the swelling properties of polyacrylamide gels and polyacrylamide gels with entrapped glucose oxidase (GOx) with the aim to prepare a biosensor. The principal objective is to study the effect of the introduction of GOx on the molecular structure and properties of the gels. This objective was performed using two methods: (i) swelling experiments and, (ii) viscoelastic measurements. The swelling experiments of polymer gels provides a good approach to determination of the average molecular weight of the network chains in the gel, and thus, the effect of GOx on the structure of the gel. Likewise, the viscoelastic experiments permit to determine the shear modulus, G, which can be related to the molecular weight of the network chains using the theory of rubber elasticity, and hence to study the effect of the introduction of GOx in the gel structure. Finally, amperometric measurements were carried out to evaluate the GOx loaded polyacrylamide gels as glucose sensor.

Section snippets

Materials

The acrylamide (AAm) (Panreac), the initiator ammonium persulfate (Fluka), the crosslinker N,N′ methylene bisacrylamide (Bis) (Aldrich), the accelerator N,N,N′,N′ tetramethylethylendiamine (TEMED) (Bio-Rad), the d+glucose (Merck) and the enzyme (EC 1.1.3.4.) GOx from Aspergillus niger (Sigma) were used as received.

Preparation of pure gels

PAAm gels were prepared by crosslinking copolymerization of AAm with a small amount of Bis in aqueous solution. The polymerization was initiated by the ammonium persulfate/TEMED redox

Swelling experiments

Fig. 1 depicts the evolution of the swelling degree (defined as the inverse of the polymer volume fraction, v2) with time for pure PAAm gels of different content of crosslinker. From this figure it can be observed that: (i) the freshly prepared gels (t=0 min) swell when immersed in an excess of water, at a rate which is dependent on the content of crosslinker of the samples: the time needed to achieved the equilibrium of swelling diminish as the content of crosslinker increases; (ii) the

Molecular weight between crosslinks

It is interesting to study the elastically effective crosslink densities of the PAAm gels as a function of the content of crosslinker. For this purpose the crosslinking reactions were carry out until complete conversion and the final gels were swollen in deionized water until equilibrium was reached.

From the equilibrium degrees of swelling of the PAAm hydrogels, the number average molecular weight between adjacent crosslinks, Mc, were evaluated using the Peppas and Merrill [27] swelling

Conclusion

The analysis and interpretation of the viscoelastic and swelling properties of polyacrylamide hydrogels on the basis of different theories points to the existence of an inherent inhomogeneous structure in a microscopic scale.

Glucose oxidase can be immobilised in polyacrylamide gels by aqueous crosslinking copolymerisation of acrylamide and N,N′-methylene bisacrylamide in the presence of the enzyme. The effects of the entrapment of the enzyme on the properties of polyacrylamide gels can be

Acknowledgements

The authors are grateful to the Spanish Ministerio de Educación y Ciencia (MEC) (MAT99-1179, MAT2002-2978, BMF2000-0620 and PGC2000-2246-E) for financial support.

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