Bead-enriched catalyzed hairpin assembly for the flow cytometric detection of microRNA via FRET signal readout

The accurate quantification of microRNAs (miRNAs) is of great significance for various biomedical applications. Herein, a facile bead-enriched catalyzed hairpin assembly (CHA) assay for flow cytometric miRNA analysis via fluorescence resonance energy transfer (FRET) signal readout is successfully developed. Different from the traditional homogeneous CHA strategies, the CHA reaction is confined on magnetic beads (MBs), so that even low levels of fluorescent dye can be enriched on the minuscule space around the MBs and then sensitively investigated by flow cytometry (FCM), resulting in the improvement of sensitivity. What's more, with the help of a robust FCM-based FRET signal output, the potential detection inaccuracy caused by the fluctuation of experimental conditions and instrument settings can be greatly decreased by recording the fluorescence intensity ratio of FAM and Cy3 instead of a single fluorescent dye. In this way, precise miRNA sensing with a detection limit of 0.7 pM is achieved, and the amount of miRNA target in complex total RNA is successfully analyzed. This work achieves FCM-assisted ratiometric fluorescence analysis of miRNAs, providing a new route for the DNA machine-based biosensing system.