Enzyme Immobilization by Polydimethylsiloxane Stamping for Glucose Sensor Fabrication
Bo Wang, Bonhye Koo, Harold G. Monbouquette*
Chemical and Biomolecular Engineering Department, University of California, Los Angeles, CA 90095, USA
Most glucose sensors are based on electrochemical detection of hydrogen peroxide (H2O2) generated from the glucose oxidase (GOx)-catalyzed oxidation of glucose, and immobilization of the enzyme is a critical step in biosensor fabrication. Here, a new and effective method for GOx immobilization is presented. The enzyme is successfully transferred from a polydimethylsiloxane (PDMS) stamp surface onto a chitosan-coated electrode. Compared to manual enzyme immobilization methods, the enzyme layer generated by PDMS stamping is thinner, which leads to fast sensor response and high sensitivity. The sensor construction procedure begins with the coating of polypyrrole and Nafion on a platinum (Pt) electrode surface sequentially as permselective polymers to exclude anionic and cationic interferents (e.g., ascorbic acid and dopamine). A chitosan film is electrochemically deposited on top of the Nafion to serve as a suitable adherent layer for enzyme immobilization. GOx and bovine serum albumin (BSA) are transferred together onto the chitosan surface using a PDMS stamp. Then, the stamped layer is exposed to glutaraldehyde vapor for cross-linking. The layering process of biosensor fabrication is monitored by electrochemical impedance spectroscopy (EIS) and the chitosan surface before and after enzyme stamping is characterized by scanning electron microscopy (SEM). In testing with varying concentrations of glucose and interferents, the sensors show high sensitivity, low detection limit, good selectivity, and fast response time. This enzyme immobilization method can be used in principle to immobilize various enzymes for creation of biosensors for applications both in vitro and in vivo.
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