An integrated target recognition and polymerase primer probe for microRNA detection.

Extremely sensitive and visual measurements of microRNA (miRNA) in situ for early detection and monitoring of diseases remains a major challenge. To address this issue, this work reports a rapid, highly sensitive and selective microRNA (miRNA) biosensing strategy based on isothermal circular strand-displacement polymerization (ICSDP), and miRNA imaging was performed inside cells. In this work, a double hairpin DNA probe (HP1/HP2 complex) embedded with a sensing region and polymerase primer region was designed. Briefly, after the specific binding of target miRNA with the HP1/HP2 probe, HP1/HP2 itself can function as a primer to initiate the ICSDP with the help of Klenow Fragment (KF), yielding target miRNA for new rounds of ICSDP. In this process, one target can produce multiple signal outputs (1: n), achieving low abundance of miRNA detection. Under optimized conditions, the proposed strategy showed high sensitivity with a detection limit of 5 pM within 15 min and can also easily distinguish the control miRNA from the target miRNA. This method can be further applied to image the intracellular miRNA of interest in situ inside the cancer cells.