Label-free microRNA detection using a locked-to-unlocked transforming system assembled by microfluidics

MicroRNA (miRNA) is a potential biomarker for the early screening and diagnosis of cancers and is widely present in human blood, urine and saliva. Here, we report a microfluidics-assembled tool for miRNA detection based on the regulation of DNA locked and unlocked states and explore its application in complex samples. Microfluidic techniques are used to continuously assemble the locked-to-unlocked transforming system using a rapid one-step method. It only takes 2 min to produce enough locked-to-unlocked systems for a miRNA detection experiment. DNA molecules with a recognition sequence and a G-rich reporter sequence (G4m) are locked by attaching both ends to the surface of magnetic beads (MBs) in microchannels. The presence of the target miRNA can initiate the specific cleavage of one end of G4m by duplex-specific nuclease, resulting in the transition of G4m from a locked state to an unlocked state. This transition enables G4m to freely fold into a G-quadruplex, which can participate in the catalysis of ABTS oxidation and result in a turquoise color. During the whole process, the target miRNA remains intact and continuously initiate specific cleavage, facilitating signal amplification. Magnetic separation steps are employed to assist in miRNA enrichment and interference reduction. As a proof of concept, we quantified miRNA-21 using the locked-to-unlocked system. The assay allows specific detection of miRNA-21 in the range of 3.2-570 pM with a detection limit of 2.01 pM (S/N = 3). Furthermore, the locked-to-unlocked system is used to analyze miRNA-spiked urine, saliva and serum samples and shows robust performance in different matrices.