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MicroRNAs (miRNAs) regulate the expression of mRNAs in cells. The determination of subcellular miRNAs in single living cells is essential for understanding the subcellular localizations and functions of miRNAs as well as the microheterogeneity of cells. However, current approaches fail either to keep the cells alive or maintain their original cellular composition or are unable to provide subcellular resolution. Herein, we presented a new approach, called plasmonic affinity sandwich assay (PASA), for rapid and ultrasensitive probing of trace cytoplasmic and nuclear miRNAs in single living cells. It combined efficient in vivo subcellular extraction with ultrasensitive plasmon-enhanced Raman scattering (PERS) detection. By virtue of a micromanipulator, target miRNAs in the cytoplasm and nucleus were first specifically extracted from single living cells by gold thinlayer-coated glass microprobes modified with a half complementary sequence to the target miRNA and then, they were labelled with silver nanotags modified with a Raman reporter and the other half complement. Sandwich-like complexes of extraction sequence-target miRNA-labelling sequence were formed on the extraction microprobe, which were subjected to PERS detection. The subcellular resolution of this approach was confirmed with miR-29b (predominantly localized in the nuclei) and miR-29a (mainly located in the cytoplasm), whereas the quantitative capability was verified with three cytoplasmic miRNAs including miR-21, miR-155 and miR-203. This approach obviated tedious steps such as subcellular fractionation and enzymatic amplification, and it required only 10 min. It could be a promising tool to provide insights into subcellular localizations, functions and microheterogeneity of miRNAs.

Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay