Microchip electrophoretic sensing of multiplex microRNAs based on dual nucleic acid amplification

MicroRNAs (miRNAs) are potential biomarkers for the early diagnosis of lung cancer. Evidence suggests that a group of miRNAs, called an "miRNA fingerprint", is more specific and informative than a single one. However, the simultaneous and accurate detection of multiple miRNAs in biological samples is challenging because of their high similarity and trace amounts. In this study, dual nucleic acid amplification driven by a duplex-specific nuclease (DSN)-mediated cycle and bipedal DNA walker integrated with microchip electrophoresis (MCE) was developed to simultaneously detect lung cancer-associated miRNAs (miR-23 and miR-21). The capture probe identified miRNAs, underwent DSN-mediated target recycling, and released the bipedal walking legs, which subsequently initiated the assembly of the bipedal DNA walker. Thus, two stages of signal amplification were achieved. The unique design of the walker improved its walking efficiency to generate a detectable and distinguishable signal for the quantification of miR-23 and miR-21 by MCE. This method demonstrates excellent accuracy and specificity for single base mismatches, enabling the sensitive detection of miRNAs with a low limit of detection (LOD) of 0.2 fM (S/N = 3) and a wide linear range of 1 fM to 10 pM. This method has been successfully applied to human lung tumor cells, exhibiting notable advantages such as high sensitivity, high speed and good specificity, thereby holding great promise for application in tumor diagnosis.