Microfluidic circulating reactor system for sensitive and automated duplex-specific nuclease-mediated microRNA detection.

Duplex-specific nuclease signal amplification (DSNSA) is a promising microRNA (miRNA) quantification strategy. However, existing DSNSA based miRNA detection methods suffer from costly chemical consumptions and require laborious multi-step sample pretreatment that are prone to sample loss and contamination, including total RNA extraction and enrichment. To address these problems, herein we devised a pneumatically automated microfluidic reactor device that integrates both analyte extraction/enrichment and DSNSA-mediated miRNA detection in one streamlined analysis workflow. Two flow circulation strategies were investigated to determine the effects of flow conditions on the kinetics of on-chip DSNSA reaction in a bead-packed microreactor. With the optimized workflow, we demonstrated rapid, robust on-chip detection of miR-21 with a limit-of-detection of 35 amol, while greatly reducing the consumption of DSN enzyme to 0.1 U per assay. Therefore, this microfluidic system provides a useful tool for many applications, including clinical diagnosis.