Multiplexed enzyme-based bioassay within microfluidic devices using shape-coded hydrogel microparticles

This paper describes a multiplexed enzyme-based assay within a microfluidic device using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles. A microfluidic device was constructed by serially connecting two patterning chambers and a microfilter-integrated detection chamber through a Y-shaped microchannel. Various shapes and sizes of hydrogel microparticles entrapping different enzymes were fabricated in the patterning chamber by photolithography and collected in the detection chamber by pressure-driven flow. Due to the action of the microfilter, hydrogel microparticles were perfectly retained inside the detection chamber without passing through it. The sequential bienzymatic reactions of glucose oxidase (GOX) and peroxidase (POD) or alcohol oxidase (AOX) and POD were successfully investigated using Amplex Red within the hydrogel microparticles. Furthermore, by entrapping GOX and AOX into different shapes of hydrogel microparticles, each enzyme-catalyzed reaction was easily identified, and therefore, simultaneous detection of glucose and ethanol was possible in the concentration range from 1.0 to 10.0 mM without cross-talk by using same fluorescence indicator in a single detection chamber.