Immobilization of galactose oxidase on self‐assembled monolayers of thiols on Au and Ag surfaces

Abstract

Galactose oxidase (GalOD) was immobilized on self-assembled monolayers of thiols on silver and gold surfaces using trans-stilbene (4,4′-diisothiocyanate)-2,2′disulphonic acid (DIDS) as the bridging compound. DIDS is the symmetrical bifunctional reagent that reacted with the amine moiety of the thiol and with primary amino groups of enzyme. The Raman measurement revealed that onto cysteamine-modified silver and gold electrodes, bands corresponding to the galactose oxidase (about 694, 1076, 1274 cm—1 on Au and 762, 1058, 1274 cm–1 on Ag ) appeared and clearly demonstrated its immobilization onto Au and Ag surfaces. Simultaneously, we have also observed changes in the ratio of trans–gauche conformers of adsorbed cysteamine molecules. Layers revealing high content of trans conformer are transformed into layers composed mainly of cysteamine molecule in gauche conformation after galactose oxidase adsorption. These observations deliver a strong support for enzyme immobilization on cysteamine-modified gold and silver surfaces. The surface plasmon resonance experiment gave a surface coverage of ~8.4 × 107 g/cm2 for gold electrode modified cysteamine using DIDS chemistry and 1.1 × 107 g/cm2 for the cysteamine only modified gold substrate and demonstrated that galactose oxidase layers immobilized with DIDS coupling reagent are quite stable and cannot be easily removed from the surface by treatment with a buffer solution. The surface plasmon resonance results indicated that in this method, a multilayer of galactose oxidase have been immobilized. Our new method of covalent attachment of enzymes seems to be quite promising as a new way of manufacturing biosensors. Copyright © 2012 John Wiley & Sons, Ltd.