Abstract
Ultrasensitive PCR-free microRNA (miR) analysis based on biosensors with enzyme-free nucleic acid amplification and reusable surface has great clinical significance in cancer prognosis. However, building such a biosensing strategy has long been challenging due to uncontrollable miR-triggered cascade amplifiers and insufficient sensing surface regeneration capability. To meet the challenge, for the first time, a general approach, named enzyme-free multitag-regulated cascade reaction (MCR), is developed to fabricate reliable trace miR biosensors. As a proof of concept, miR let-7a is detected on an evanescent wave fluorescent optical-fiber biosensing platform. The size and morphology of well-formed MCR assemblies (∼1 μm in length) are characterized by atomic force microscopy. This MCR method achieves a 30 000-fold improved sensitivity (detection limit 0.8 fM) compared to the MCR-free system and can detect abnormal urinary miR levels in lung cancer patients. Moreover, the biosensor is robust enough to be reused for over 100 cycles, which greatly reduces the cost of single detection. In sum, MCR is developed as a generalizable ultrasensitive miR biosensing approach for cancer prognosis, which opens a broad field for facile enzyme-free biosensing applications by nucleic acid assembling regulation.
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