Use of poly(methyl methacrylate)/polyethyleneimine flow microreactors for enzyme immobilization

Abstract

Recently our group has developed a simple and practical procedure to immobilize glucose oxidase (GOD) on poly(methyl methacrylate) (PMMA) microchannels. In the present study not only was the GOD microreactor preparation optimized but also, more important, it is shown that the functionalization of PMMA microchannels with polyethyleneimine (PEI) is a methodology of wider applicability to anchor enzymes with glutaraldehyde, with a potential for the development of microanalytical devices and systems. As a demonstration, the preparation and evaluation of microreactors with immobilized ascorbate oxidase (AAO), catalase (CAT), glutamate dehydrogenase (GDH) and a dual enzymatic system (GOD and horseradish peroxidase) is reported. Direct (GOD) and indirect (AAO and CAT) chronoamperometric measurements were made in order to evaluate the reactors' activity. GDH immobilization was confirmed photometrically through a thiazolyl blue tetrazolium bromide based reaction while a Trinder's reaction based method was used to evaluate the GOD/HRP microreactor. A small volume flow cell built in the lab was connected to two optical fibers to implement spectrophotometric measurements. After 15days of use (~1000 injections), no change was observed in the efficiency of the CAT reactor (100% destruction of the analyte) while the GOD/HRP reactor that decayed to 25% of the original activity, was still useful despite the decrease in sensitivity.