Co-immobilization of cholesterol oxidase and horseradish peroxidase in a sol-gel film

Physical adsorption, physically entrapped sandwich and microencapsulation techniques have been utilized for the immobilization of cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) on tetraethyl orthosilicate (TEOS) derived sol-gel films. The enzyme activity studies indicate no significant leaching of enzyme from the microencapsulated and physically entrapped enzyme sandwich films. The ChOx activity has been assayed as a function of cholesterol concentration in these films, and spectrophotometric and electrochemical techniques have been used for the estimation of cholesterol concentration in each instance. Spectrophotometric measurements exhibit a response time of 10, 30 and >100 min for physically entrapped, physisorbed and microencapsulated films, respectively. A similar order of response times has been found for these enzyme sol-gel films using an electrochemical technique. A fast response time of 50 s has been observed amperometrically with a physically entrapped enzyme sandwich sol-gel film. The results obtained from spectrophotometric and electrochemical measurements using the three systems yield linearity for 2-10 mM cholesterol. All enzyme sol-gel films are found to be stable for about 8 weeks at 25 degrees C and 12 weeks at 4-5 degrees C. (C) 2000 Elsevier Science B.V. All rights reserved.