Abstract
Accurate quantifications of cellular miRNAs are important not only for accelerating them becoming reliable diagnostics biomarkers but also for deeply understanding their influence on central signaling pathways. Although single-molecule miRNA imaging permits quantifying biomolecules at the single-molecule level, it is limited by the sensitivity and specificity of hybridization-based probes. We report a miRNA single-molecule imaging method by using conjugated polymer nanoparticle (CPN) labeled short DNA probe termed as a nanoflare. The transient hybridization of the nanoflares and target miRNAs yields a featured single-molecule kinetics signal rendering high single-molecule sensitivity and specificity. miRNA can be detected with a remarkable detection limit of 1 fM without using any amplification steps. The discrimination capability of homologous miRNAs was also demonstrated. Taking advantage of the featured single-molecule signal of the nanoflare, we can directly count single miR-21 molecules in single cells by using highly inclined and laminated optical sheet (HILO) microscopy. The statistics of the counting reveals miR-21's cell-to-cell fluctuation and differential expression of tumor cells and normal cells.
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