Evaluation of an alcohol oxidase - based enzyme biosensor immobilized in PVA-AWP polymer

Abstract

Biosensor technology is a powerful alternative to conventional analytical techniques, harnessing the specificity and sensitivity of biological systems in small, low cost devices. One of the factors that affect the performance of a biosensor is the immobilization of the biological sensing element, or bioreceptor, on the transducer surface. In this study we determined the effect of polymer concentration, ultraviolet (UV) light exposure, and film thickness on the activity and stability of alcohol oxidase (AOX) when immobilized in a photocrosslinkable and water-soluble polymer with a poly(vinyl alcohol) azide-unit water pendant (PVA-AWP). Compared to other gel immobilization techniques, PVA hydrogels offer several advantages, such as better elasticity, low toxicity, biocompatibility with enzymes and yeast cells, mechanical and long-term stability, and biodegradability. The polymerization of PVA hydrogels are also spatially and temporally controllable by the irradiation of UV light. However, the effects of UV exposure time and film thickness of PVA hydrogels on biosensor sensitivity are not yet fully characterized. Immobilization was carried out through photocrosslinking a mixture of AOX and PVA-AWP dropcoated on a screen printed carbon electrode. The immobilized AOX was tested with varying amounts of ethanol solution. The electrical current produced during the oxidation of hydrogen peroxide by cobalt phthalocyanine was measured chronoamperometrically. The magnitude of the current output was dependent on thickness of the film on the electrode. There was no significant difference observed in the rate of current output with change in ethanol concentration between AOX immobilized in PVA-AWP and non-immobilized or free AOX, but it was significantly higher for AOX immobilized in glutaraldehyde. Enzyme activity decreased by 40%, 36% and 7% when stored for 24 hours at 23°C, 3°C and -17°C respectively. When AOX was immobilized in approximately 0.09 mm thick PVA-AWP polymer film, results showed that the lower limit of ethanol detection was 171.3 i­M.</p >