An ultrasensitive one-pot Cas13a-based microfluidic assay for rapid multiplexed detection of microRNAs.

Aberrant microRNA expression is associated with tumor progression in various organs. Detecting microRNAs as clinical cancer biomarkers can facilitate early cancer diagnosis and monitoring. However, the rapid and accurate quantification of microRNAs from biological samples remains a significant challenge. Here we developed a one-pot isothermal assay utilizing a molecular circuit with CRISPR/Cas13a (CRISPR-circuit) to rapidly convert, amplify and report different microRNAs within 15 min at the attomolar (aM) level. Then the full process was performed on an active centrifugal microfluidic chip and its corresponding portable equipment for parallel detection of multiple microRNAs, including miR-21, miR-141, miR-196a, and miR-1246. We also demonstrated its application for identifying cell lines and clinical samples of cancer patients with varying microRNA levels, which showed a strong correlation with the RT-qPCR. The assay can be easily adapted for the detection of any microRNA by simply modifying the converter primer, thereby holding significant potential for accurate disease detection and clinical diagnosis.