ИСТИНА

A simple, novel, and fast strategy for physical entrapment of biomolecules into the polymeric matrix was proposed, based on unique stimuli-sensitive behavior of functional microgels. To demonstrate this, fabrication regimes and properties of microgel/enzyme thin films adsorbed onto conductive substrates were examined. The films were formed via two-steps, sequential adsorption of a both temperature- and pH-sensitive microgel poly(N-isopropylacrylamide-co-3-(N,N,-dimethyl amino)propylmethacrylamide) (poly(NIPAM-co-DMAPMA), followed by the enzyme (tyrosinase, choline oxidase and/or butyrylcholinesterase) adsorption under different pH and temperature regimes. Enhanced deposition of poly(NIPAM-co-DMAPMA) microgel was shown at elevated temperatures exceeding the volume phase transition temperature (VPTT). A considerable increase in the amount of the adsorbed enzyme was detected when the film of the pre-adsorbed microgel is first brought into a collapsed state at T>VPTT but then was allowed to electrostatically interact with the enzyme at T<VPTT. Sponge-like approach to enzyme adsorption was applied for modification of screen-printed graphite electrodes by poly(NIPAM-co-DMAPMA)/enzyme films and the resultant biosensors were tested amperometrically. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T>VPTT and (ii) following sponge-like enzyme loading at T<VPTT, we can achieve increased biosensor sensitivities for phenol (system with immobilized tyrosinase) and for choline (system with immobilized choline oxidase). Since sponge-like approach to enzyme adsorption was found to be dependent on the molecular weight (globule size) of the enzyme, the bienzymatic systems with spatially separated enzymes was prepared in one-step as was demonstrated for the system with co-immobilized choline oxidase/butyrylcholinesterase.