An ultrasensitive microchip electrophoresis assay based on separation-assisted double cycling signal amplification strategy for microRNA detection in cell lysate.

Microchip electrophoresis (MCE) assay is an analysis technique with low consumption and high automation. It is a useful tool in biomedical research and clinical diagnosis. However, the low detection sensitivity limits its application in trace biomarker analysis because of its extremely small sample size. To address the need for high sensitivity in MCE, we have developed an ultrasensitive MCE method based on a separation-assisted double cycling signal amplification strategy for the detection of microRNA (miRNA) in cell lysate. In this method, two short single-stranded DNAs P1 and P2 complement each other to form a duplex DNA probe (P1/P2). In the presence of target miRNA, P2 in the P1/P2 probe can be displaced to form double-stranded miRNA/P1. Then, the degradation of P1 in miRNA/P1 by T7 Exo releases the miRNA, and the released miRNA participates in a displacement reaction with another P1/P2 probe to complete the first cycle. The displaced free P2 hybridizes with the hairpin fluorescence probe (MB) to form the P2/MB duplex, which can also be degraded by T7 Exo to release P2. The released P2 can bind with another MB probe to complete the second cycle. By using MCE-laser-induced fluorescence (LIF) as separation and detection platform and miRNA-141 as model analyte, the proposed MCE assay can detect miRNA-141 at concentrations as low as 8.0 fM, which is the highest sensitivity achieved to date for an MCE assay. This method for detecting trace miRNA holds great potential in biomedical research and clinical diagnosis.