Spatially Resolved, Error‐Robust Multiplexed MicroRNA Profiling in Single Living Cells

Simultaneous imaging of multiple microRNAs (miRNAs) in individual living cells is challenging due to the lack of spectrally distinct encoded fluorophores and non-cytotoxic methods. We describe a multiplexed error-robust combinatorial fluorescent label-encoding method, termed fluorophores encoded error-corrected labels (FluoELs), enabling multiplexed miRNA imaging in living cells with error-correcting capability. The FluoELs comprise proportional dual fluorophores for encoding and a constant quantitative single fluorophore for error-corrected quantification. Both are embedded in 260 nm core-shell silica nanoparticles modified with molecular beacon detection probes. The FluoELs are low cytotoxic and could accurately quantify and spatially resolve nine breast-cancer-related miRNAs and evaluate their coordination. The FluoELs enabled a single-cell analysis platform to evaluate miRNA expression profiles and the molecular mechanisms underlying miRNA-associated diseases.