Electrochemical Biosensors with Cross-Linked Hydrogels

Abstract

Leland C. Clark Jr. and Champ Lyons introduced in 1962 the principle of the first electrochemical biosensor with immobilized glucose oxidase (GOx) [1]. This device was specifically designed and used for fast glucose analysis in blood samples from diabetics. Since then, the biosensor field has experienced important growth. In last few decades, this field has been widely explored in many different environments, such as clinical, biotechnological, food and pharmaceutical. This type of device is object of attention because it allows analysis with high specificity, sensitivity, selectivity and low response time.A key parameter for the proper functioning of electrochemical biosensors is the enzyme immobilization onto the electrochemical interface [2]. Immobilization must be performed in a way that the selected enzyme maintains either structure and spatial orientation. In addition, substrate and product have to be able to diffuse to/from the active site, where the biological reaction takes place. One of the simplest and cost effective immobilization method is the polymer entrapment using natural origin polysaccharides. In that frame, chitosan is currently receiving a great deal of interest because of its intrinsic properties: biocompatible, biodegradable, high abundance, low cost of production and it easily forms hydrogels. However, the poor stability of the hydrogel is of major concern [3] when used as an immobilization strategy in the construction of electrochemical biosensors, since it affects the biosensor lifespan.In the present work, amperometric glucose biosensors based on glucose oxidase (GOx) immobilization onto highly ordered titanium dioxide nanotube arrays (TiO2NTAs) was used to determine the long term stability of the constructed probes. TiO2NTAs were grown by electrochemical anodization of a titanium metal disc in ethylene glycol containing ammonium fluoride (0.3%w/w) and water (2%w/w). The electrochemical synthesis was performed at 30 V for 17 hours. This anodization time yields the maximum sensitivity of the sensor. Higher times result in the nanotube walls collapse. Finally, a thermal annealing was performed at 500 ⁰C for 3 h in air to crystallize TiO2 nanotubes from amorphous to anatase phase.In the sensor architecture chitosan was used as a base material for enzyme immobilization in the hydrogel matrix: Ti/TiO2NTAs/GOx–Chitosan. In order to increase the stability of the immobilization matrix, different crosslinking agents were evaluated onto the chitosan matrix: glutaraldehyde, hypochlorous acid and poly(ethylene glycol) diacrylate. Non-modified chitosan provided a biosensor lifespan around 30 days. In contrast, the used of modified chitosan either increases the long-term stability even the sensitivity of the biosensor.