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A versatile single-molecule counting-based platform by generation of fluorescent silver nanoclusters for sensitive detection of multiple nucleic acids.

Manshu Peng,Z. Fang,Na Na,Ouyang Jin

Published 2019 in Nanoscale

ABSTRACT

The good photostability and strong brightness of individual DNA-templated silver nanoclusters (DNA-AgNCs) have been confirmed by single-molecule imaging in this work and DNA-AgNCs as a new class of outstanding fluorophores are applied in the construction of single-molecule counting-based probes for the first time. Based on the fluorescent AgNC-generating molecular beacons (AgNC-MBs), we present a versatile method for simultaneous analysis of multiple nucleic acids. Distinct from previous designs in which a AgNC stabilizing sequence is incorporated into the stem of a hairpin DNA to form the AgNC-MB, we prepared a nicked MB in which the AgNC stabilizing sequence is hybridized with the longer stem of a single-stranded DNA (ssDNA) with a stem-loop structure. Our proposed AgNC-MB is activated by probe-target hybridization then releasing the AgNC stabilizing sequence via a toehold-mediated strand displacement reaction, the versatility of which has been greatly improved because bases in the target-binding region are not involved in the formation of DNA-AgNCs. As a proof of concept, the simultaneous detection of two breast cancer-related MicroRNAs (miR-21 and let-7a miRNA) has been achieved with total internal reflection fluorescence (TIRF)-based imaging and the detection sensitivity of our method has been demonstrated to be improved by at least two orders of magnitude compared with conventional AgNC-MBs. Furthermore, in the single-nucleotide mutation identification assay, the simultaneous detection strategy introduces a competitive reaction between the two probe-target hybridizations, resulting in the excellent discrimination ability of the AgNC-MB sensing platform and the mutant-type targets can be successfully detected at low abundance. The new AgNC-MB sensing platform demonstrated potential to make AgNCs an attractive alternative to conventional organic dyes for single-molecule studies.

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