A new and effective method for enzyme immobilization, which is used for biosensor fabrication, has been developed. Glucose oxidase served as the model enzyme. 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. In this study, the enzyme is successfully transferred at high concentration from a polydimethylsiloxane (PDMS) stamp surface onto a chitosan-coated electrode. The sensor construction procedure begins with the sequential coating of polypyrrole and Nafion on a platinum (Pt) disk electrode surface as permselective polymers to exclude anionic and cationic interferents (e.g., ascorbic acid and dopamine).[1] A chitosan film is electrochemically deposited on top of the Nafion to serve as a suitable adherent layer for enzyme immobilization.[2] GOx and bovine serum albumin (BSA) are transferred together onto the chitosan surface using a PDMS stamp. The GOx and BSA subsequently are crosslinked using glutaraldehyde vapor. There is a necessity for the development of improved, reproducible, non-manual methods for depositing enzymes selectively on microelectrodes, especially for microelectrodes less than 100 μm apart which is difficult to be modified by manual enzyme deposition.[1] The mechanical properties of the elastomeric PDMS stamps provide sufficient mechanical stability for the printing down to a feature size of 500 nm.[3, 4] In the future, this enzyme transfer method by PDMS can be used in principle to immobilize various enzymes at microscale for creation of biosensors for applications both in vitro and in vivo. Reference 1. Tseng, T.T.C. and H.G. Monbouquette, Implantable microprobe with arrayed microsensors for combined amperometric monitoring of the neurotransmitters, glutamate and dopamine. Journal of Electroanalytical Chemistry, 2012. 682: p. 141-146. 2. Tseng, T.T.C., J. Yao, and W.C. Chan, Selective enzyme immobilization on arrayed microelectrodes for the application of sensing neurotransmitters. Biochemical Engineering Journal, 2013. 78: p. 146-153. 3. Bernard, A., et al., Microcontact printing of proteins. Advanced Materials, 2000. 12(14): p. 1067-1070. 4. Qin, D., Y.N. Xia, and G.M. Whitesides, Soft lithography for micro- and nanoscale patterning. Nature Protocols, 2010. 5(3): p. 491-502.

Enzyme immobilization by polydimethylsiloxane stamping for biosensor fabrication

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.