Ultrasensitive and specific imaging of circulating microRNA based on split probe, exponential amplification, and topological guanine nanowires

Herein a simple chemiluminescence imaging method was constructed for the detection of circulating microRNA (miRNA) using split probe, isothermal exponential amplification reaction (EXPAR) and topological assembly of G-quadruplex. The detection probe was tailored with a reorganization region at the 3′-end and an amplification region with two same repeated sequences and a nicking enzyme site at the 5′-end. The reorganization region was split by a short sequence in the middle and thus termed split probe. Then the specific reorganization and rapid response of the functional probe generated abundant G-quadruplex sequences, which further self-assembled to long and continuous guanine nanowires (G-wires) in high concentration of Mg2+ solution by taking advantage of their parallel structure. The exponential amplification of G-quadruplex through a feedback mechanism and the assembled G-wires resulted in a low detection limit down to 5:35 aM with six orders linearity magnitude. The split probe improved the detection specificity with a relatively larger energetic penalty method, and thus achieved an excellent discrimination capability for distinguishing mismatched targets, even with one base mutation. In addition to ultrasensitivity and high specificity, the imaging method is label-free and high-throughput, endowing it a simple and powerful platform for trace amount of miRNA detection in clinical diagnostics.