Programmable strand displacement-based magnetic separation for simultaneous amplified detection of multiplex microRNAs by chemiluminescence imaging array

High throughput analysis of miRNAs is of great significance to clinical and biomedical applications. In this work, we have developed a highly sensitive and selective chemiluminescence imaging array (CLIA) for simultaneous detection of three miRNAs with high throughput, easy operation and low cost. In this assay, three kinds of hairpins that can specially recognize respective miRNAs (miR-155, miR-let-7a and miR-141) are parallel modified on the magnetic beads (MBs). The employment of MBs enables quick sorting of multiplex targets in high yield and purity with high throughput. Upon introduction of the target miRNAs, they hybridize to the corresponding hairpins and initiate the DNA machines with the assistance of Kienow fragment exo- DNA polymerase and Nb.BbvCI NEase, achieving exponential amplification of targets. After that, the toehold mediated strand displacement (TMSD) reactions are performed through sequential addition of displacement probes under magnetic separation, resulting in the release of HRP-tagged DNA hybrids in solution for CL imaging and recovery of MBs for repetitive use. This CLIA method demonstrates ultrahigh sensitivity with detection limits down to fM level, wide linear range over 5 orders of magnitude, excellent selectivity to distinguish one-base mismatched target miRNA, and good performance in real sample analysis. Together with the advantages of high throughput, easy operation, acceptable accuracy, and good recyclability and reproducibility, the proposed multiplex CLIA method holds great potential in practical applications, such as profiling the pattern of miRNA expression, disease screening, biomedical research, and so on.