Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging

Abstract

A single-molecule assay for multiple microRNA detection.

Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum

“…Single-molecule methods, such as fluorescence microscopy, can of course also be used for the detection of miRNAs. 21 , 22 However, the complexity dramatically increases as the number of biomarkers being detected is also increased. There will be a limit to how many fluorophores can be used, especially at the single-molecule level.…”

A controlled T7 transcription-driven symmetric amplification cascade machinery for single-molecule detection of multiple repair glycosylases

“…42 Recently, singlemolecule detection has attracted much attention due to its signicant advantages of high signal-to-noise ratio, high sensitivity, and low sample consumption. [43][44][45] In this research, we demonstrate the development of a controlled T7 transcription-driven symmetric amplication cascade machinery for single-molecule detection of multiple repair glycosylases by taking advantage of the intrinsic superiorities of T7-based transcription amplication and single-molecule detection. This method exhibits good specicity, high sensitivity, with a detection limit of 3.52 Â 10 À8 U mL À1 for hOGG1 and 3.55 Â 10 À7 U mL À1 for hAAG, and a large dynamic range with ve orders of magnitude.…”

Multicolor fluorescence encoding of different microRNAs in lung cancer tissues at the single-molecule level

“…This assay involves three consecutive amplification reactions which enables the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophores-encoded DNA products which can release large numbers of fluorescent molecules for the generation of amplified signals. Unlike the reported methods for miRNA assay with the requirement of the carefully designed probes (e.g., hairpin probes labelled with differentcolor quantum dots, 48 signal probes labelled with Cy3 / Cy5, 55 hairpin probes labelled with ferrocene / methylene blue, 49 and the magnetic DNA nanospheres-encoded probes labelled with ferrocene/methylene blue 56 ), this assay enables the simultaneous detection of miRNAs without the need of either miRNA purification or multiple fluorescent-labelled probes. Due to high efficiency of the cascade signal amplification and high signal-to-noise ratio of single-molecule detection, this assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay.…”