Microfluidic bioassay system based on microarrays of hydrogel sensing elements entrapping quantum dot–enzyme conjugates

► Microfluidic biosensor able to detect substrates of oxidase enzymes. ► Enzyme–quantum dot (QD) conjugates are entrapped within hydrogel microarrays. ► Hydrogel microarrays are fabricated within microchannels by photopatterning. ► The reaction between oxidase and substrate produce H2O2, which quench the QDs. ► Glucose and alcohol are detected using glucose oxidase and alcohol oxidase. This paper presents a simple method to fabricate a microfluidic biosensor that is able to detect substrates for H2O2-generating oxidase. The biosensor consists of three components (quantum dot–enzyme conjugates, hydrogel microstructures, and a set of microchannels) that were hierarchically integrated into a microfluidic device. The quantum dot (QD)–enzyme conjugates were entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning process. Glucose oxidase (GOX) and alcohol oxidase (AOX) were chosen as the model oxidase enzymes, conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the hydrogel microstructures, which resulted in a fluorescent hydrogel microarray that was responsive to glucose or alcohol. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H2O2, which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50μM of glucose and 70μM of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using our novel microfluidic device composed of multiple microchannels.