Enzyme incorporated microfluidic device for in-situ glucose detection in water-in-air microdroplets

Droplet generating microfluidic systems can provide miniaturized bioanalytical tools by using the homogenous and high-throughput droplets as nanoreactors. In this study, we demonstrated a sensitive and in-situ glucose monitoring system using water-in-air droplets in an enzyme incorporated microfluidic device. A thin film structure of a glucose oxidase (GOx) enzyme immobilized hydrogel was constructed in the middle of the microfluidic channel, and nanoliter scaled water-in-air droplets which contain a glucose sample, horseradish peroxidase (HRP), and an Amplex Red substrate were generated by flow focusing of water phase with air. Once the droplets passed through the enzyme trapped hydrogel, the droplets temporarily halted and a GOx mediated catalytic reaction with glucose proceeded, resulting in producing fluorescent resorufin products in the droplets. With optimized conditions such as the thickness of a hydrogel film and the size and flowing rate of droplets, fluorescence intensities of the released droplets linearly increased in proportional to the glucose concentration up to 3mM, and the limit of detection was calculated as 6.64µM. A spiked glucose in a real urine sample was also successfully analyzed, and the functionality of the proposed enzyme immobilized microfluidic chip was maintained for at least two weeks without loss of enzymatic activity and detection sensitivity. Thus, our methodology suggests a novel droplet based glucose sensing chip which can monitor glucose in a real-time and high-throughput manner.