In recent years, 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. Detection of miRNA is anticipated with the MB-CL platform. But the CL intensity is limited by the inner filter-like effect arising from dark MBs. To this end, we developed a highly sensitive miRNA detection approach based on MBs and enzyme label-released CL assay. In this design, miRNA is captured to MB surface and linked with CL labels by a sandwich strategy. CL/enzyme labels are released from MBs before CL detection in order to eliminate the negative effect. CL detection was performed using the system of alkaline phosphatase (AP) and adamantyl 1,2-dioxetanes (AMPPD). CL labels (namely AP tags) were conjugated to target molecule by biotin/streptavidin interaction, and then released from MBs after nucleic acid enzymolysis. RNase H and DNase I was respectively investigated for the release of enzyme labels, and the result proved that RNase H was superior to DNase I. After optimized, the developed technique demonstrated a low detection limit of 5 fM for miR-21. This detection could be achieved by directly sensing target miRNA in serum samples without reverse transcription and polymerase chain reaction (PCR) amplification. Consequently, the developed approach shows great potential for early cancer diagnosis based on miRNAs as biomarkers.

Direct detection of microRNAs based on magnetic beads and enzyme label-released chemiluminescence assay

251st National Meeting (2016)

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