Immobilized biomolecules on plasma functionalized cellophane. I. Covalently attached alpha-chymotrypsin

Surface morphology changes of hydrazine-RF-plasma-exposed cellophane surfaces were monitored under 40 kHz and 13.56 MHz CW and pulsed discharge environments and the immobilization of alpha-chymotrypsin onto plasma-modified substrates was studied. It has been shown, using SEM and AFM techniques, that significantly different cellophane topographies are generated under different frequency and pulsing parameter conditions. ESCA and ATR-FTIR analyses of plasma-modified surfaces indicated the presence of primary amide and primary amine functionalities. It was found that the relative ratios of crystalline vs amorphous zones of the nascent surface layers can also be controlled by properly selected plasma parameters, including the duty cycles of pulsed plasma environments. Enzyme immobilization reactions with alpha-chymotrypsin were accomplished both from oxygen-plasma-generated carbonyl and hydrazine-plasma-created primary amine functionalities by anchoring the biomolecules either directly to the cellophane surface or by involving spacer molecules. It was found with the cellulose substrates that fairly good enzyme activity was retained without the necessity of intercalated spacer chains. It appears that the ability of the cellulose substrate to swell in the aqueous environment allows sufficient freedom of mobility for the immobilized enzyme to retain a significant part of its activity on the cellulose. However, the activities both of the free enzyme in the presence of cellophane, and that of the immobilized enzyme molecules are significantly diminished in comparison to the activity of the free enzyme, as a result of the incorporation of these molecules into the swollen network. Potential applications of immobilized enzymes from cold-plasma-functionalized surfaces are discussed.