The immobilization of glucose oxidase onto radio-frequency plasma-modified poly(etherurethaneurea)

Abstract

Glucose oxidase was covalently immobilized onto a radio-frequency plasma-modified poly(etherurethaneurea). Thin (90-100 nm) plasma-polymerized N-vinyl-2-pyrrolidone films were deposited onto poly(etherurethaneurea) films. Active sites for the immobilization were obtained via reduction with aqueous sodium borohydride and activation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. Modified poly(etherurethaneurea) films were assayed for binding and activity of the immobilized glucose oxidase layer. The results of a modified radioimmunoassay and an 'immunochemical stain' indicated that washing in 900 ml of continuously stirred 2% sodium dodecyl sulfate, 2% Triton X-100, and 20 mM sodium phosphate, pH 7.0, for 24 h each at 4°C was necessary to remove physically adsorbed glucose oxidase from the solid supports. An amperometric activity determination in 9 ml of well-stirred 20 mM sodium phosphate-0.1 M sodium chloride, pH 7.4, gave a qualitative demonstration of the activity of the immobilized enzyme on 18.75 cm² of modified poly(etherurethaneurea) film. A colorometric activity determination using the coupled reaction with o-dianisidine and peroxidase indicated that glucose oxidase covalently immobilized on approximately 2.4 cm² of modified poly(etherurethaneurea) film had an activity approximately equal to that of 13.4 nM glucose oxidase in 50 mM sodium acetate, pH 5.1, with a specific activity of approximately 32.0 U/mg at pH 5.1 and room temperature.

Keywords:

• Immobilization
• plasma modification
• polyurethane