Fabrication of microfluidic devices incorporating bead-based reaction and microarray-based detection system for enzymatic assay

In this study, we developed a novel microfluidic device for enzyme-based assays that incorporates heterogeneous reaction and microarray-based detection systems. The microfluidic device had two serial chambers connected through microchannels; one for the reaction between analytes and enzymes, and the other for the quantitative detection of analytes. The reaction chamber was filled with glass microbeads covalently bound to enzymes via aminopropyltriethoxysilane (APTES). Enzyme-immobilized microbeads 70 μm in diameter were retained within the reaction chamber using a microfilter composed of micropillars with 30 μm interspaces. In the detection chamber, a poly(ethylene glycol)-based hydrogel microarray was fabricated using photolithography, which could immobilize other protein molecules or fluorescent dyes for the optical analysis of enzyme-catalyzed reaction. Different concentrations of glucose were detected within the microfluidic system where the reaction chamber was filled with glucose oxidase (GOX)-immobilizing glass microbeads, and a horseradish peroxidase (POD)-entrapping hydrogel microarray was placed in the detection chamber. A sequential bienzymatic reaction resulted in the conversion of non-fluorescent Amplex Red into fluorescent resorufin within the hydrogel microarray and glucose concentrations ranging from 1 to 10 mM were successfully detected by measuring the change of emission intensity of resorufin inside the hydrogel microarray.