Here, we report the one-step in situ detection of targeted miRNAs expression in single living cancer cells via MoS nanosheet-based fluorescence on/off probes. The strategy is based on the folic acid (FA)-poly(ethylene glycol)-functionalized MoS nanosheets with adsorbed dye-labeled single-stranded DNA (ssDNA). Once the nanoprobes are internalized into cancer cells, the hybridization between the probes and target miRNA results in the detachment of dye-labeled ssDNA from MoS nanosheets surface, leading to the green fluorescence recovery. In this nanoprobe, MoS nanosheets offer advantages of high fluorescence quenching efficiency and extremely low toxicity. The FA conjugation could protect the probes and improve cancer cell transfection efficiency. The ability of this nanoprobe for endogenous miRNA detection in single living cancer cells is demonstrated for two types of cancer cells with different miRNA-21 expressions (MCF-7 and Hela cells). This functionalized MoS nanosheet-based nanoprobes could provide a sensitive and real-time detection of intracellular miRNA detection platform.

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Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor‐associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in‐situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal‐organic framework nanosensor (Nano‐DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF‐based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual‐color fluorescence dye‐labeled DNA probes on MOF nanosheet surface. In the Nano‐DMFC, 2D MOF‐based nanoprobes are precisely microinjected into each single‐cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano‐DMFC platform successfully detects dual miRNAs at single‐cell resolution in 10 mixed positive MCF‐7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano‐DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC‐based cancer early diagnosis and prognosis.