Co-immobilization of cholesterol oxidase and horseradish peroxidase in a sol–gel film Original Research Article

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°C and 12 weeks at 4–5°C.