Exponential Amplification for Chemiluminescence Resonance Energy Transfer Detection of MicroRNA in Real Samples Based on a Cross-Catalyst Strand-Displacement Network

Abstract

An exponential amplification strategy for ultrasensitive detection of microRNA (miRNA) in biological extracts is developed based on a cross-catalyst strand-displacement reaction (CC-SDR). Functionally, the system consists of one upstream circuit and two downstream circuits, each of which comprises a three-stranded substrate complex and a single-stranded fuel. Importantly, the exponential amplification process does not require a polymerase or a nicking endonuclease. The whole network is activated by a miRNA trigger in the upstream circuit, which regenerates the miRNA trigger to catalyze another new upstream circuit and release two specified DNA outputs to further act as catalysts of the two downstream circuits, respectively. During each cross-catalyst network, two "mimic trigger" DNA are generated, which in turn catalyze the upstream system. Finally, the exponentially produced luminol-reduced AuNPs (lumAuNPs) and fluorescein-tagged signals are sensitively read out in the form of luminol-H(2)O(2)-horseradish peroxide (HRP)-fluorescein chemiluminescence resonance energy transfer (CRET) triplex probes by employing magnetic nanoparticles to reduce high background, achieving a detection limit of let-7a miRNA as low as 0.68 fM. Moreover, the proposed strategy exhibits an excellent specificity to discriminate one-base differences among the let-7 miRNA family and is successfully applied in real sample assay: let-7a miRNA in total RNA samples extracted from human lung tissue, and let-7b miRNA from human lung cancer cells and cervical adenocarcinoma cells, respectively. To the best of our knowledge, this is the first study to use the chemiluminescence technique for miRNA detection, which can be expected to provide a new and ultrasensitive platform for amplified detection and subsequent analysis of miRNA.