MicroRNAs (miRNAs), small non-coding RNAs, regulate gene expression at the post-transcriptional level. Their aberrant expression occurs during the development of malignant diseases. Recently, miRNAs have been proposed as potential prognostic and predictive biomarkers for early diagnosis. To date, chemiluminescence (CL) and magnetic beads (MBs) have contributed the development of many useful nucleic acid detection methods. Herein, we take advantage of duplex-specific nuclease (DSN) to develop a novel miRNA detection method with the MB-CL platform. This strategy relies on: (1) target miRNA is firstly captured by biotinylated oligonucleotides immobilized on MB surface; (2) alkaline phosphatase (AP) is linked to the MB complex by biotin/streptavidin interaction; (3) CL labels (namely AP tags) are released when DNA/miRNA duplexes are cleaved off MBs by DSN; (4) the target miRNA is recycled, thus forming an isothermal signal amplification cycle which one target miRNA cleaves of thousands of CL labels during hybridization/DSN incubation; (5) the released labels are detected after mixing with adamantyl 1,2-dioxetanes (AMPPD). After optimization, the approach can detect as low as 2.5 fM miR-221 from serum samples without any reverse transcription and polymerase chain reaction (PCR) amplification. Consequently, the developed method demonstrates potential for early diagnosis based on miRNAs as biomarkers.

Direct sensing of microRNAs based on magnetic bead-chemiluminescence platform and duplex-specific nuclease signal amplification

251st National Meeting (2016)

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