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Title:Evaluation of immobilizing horseradish peroxidase and alcohol oxidase in PVA-AWP polymer
Author(s):Beesabathuni, Shilpa N.
Advisor(s):Danao, Mary-Grace C.
Department / Program:Engineering Administration
Discipline:Agricultural & Biological Engr
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):Horseradish Peroxidase
Alcohol Oxidase
Poly (vinyl alcohol) azide-unit water pendant (PVA-AWP)
immobilization
Biosensor
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. The objectives of this study were to determine the effects of polymer concentration, ultraviolet (UV) light exposure, and film thickness on the activity and stability of horseradish peroxidase (HRP) and alcohol oxidase (AOX) when immobilized in a photo-crosslinkable and water-soluble polymer, poly (vinyl alcohol) azide-unit water pendant (PVA-AWP). The immobilized enzyme films were to be used in biosensing applications and their stability and activity were determined colorimetrically and electrochemically. 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. To determine the activity and stability of immobilized HRP, UV–Vis spectroscopy was used to analyze changes in HRP structure since the position of the Soret absorption band at 402- 403 nm of the enzyme’s heme prosthetic group can provide information on protein conformation. Position of Soret band peaks occurred between 402-403 nm for HRP immobilized in agarose and PVA-AWP (3.8 and 5% w/v). Peak absorbance of the Soret band in AWP 5% (w/v) was found to be 22%, 30%, 25% higher than those using AWP 3.8% (w/v) for UV exposure times 5, 10 and 15 minutes, respectively. From the above results, it can be concluded that UV exposure did not affect the conformation of HRP immobilized in PVA-AWP and the activity was higher in AWP (5% w/v). There was no difference (p < 0.05) observed in the enzyme activity of HRP immobilized in PVA-AWP, for UV exposure times of 5, 10 and 15 minutes, and PVA-AWP concentrations of 3.8% (w/v) and 5% (w/v). However, enzyme activity was lower (p < 0.05) when HRP was immobilized in glutaraldehyde. There was also no difference (p < 0.05) in enzyme leaching for UV 5, 10 and 15 minute exposure when immobilized in PVA-AWP. However, enzyme leaching was higher (p < 0.05) for HRP immobilized in glutaraldehyde. It can be concluded that HRP immobilized in PVA-AWP had higher activity and mechanical stability when compared to HRP immobilized in glutaraldehyde. Results from the preceding tests were applied to immobilizing AOX in PVA-AWP. An AOX-PVA-AWP mixture containing 0.5 units of AOX activity was drop-coated on a screen printed carbon electrode. The immobilized AOX was tested with varying concentrations of ethanol solution ranging from 17.1 μM to 1.71 mM. 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 difference (p < 0.05) 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 higher (p < 0.05) 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 μM.
Issue Date:2010-08-20
URI:http://hdl.handle.net/2142/16921
Rights Information:Copyright 2010 Shilpa Naidu Beesabathuni
Date Available in IDEALS:2010-08-20
2014-08-31
Date Deposited:2010-08


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