Ratiometric fluorescent biosensor for microRNAs imaging in living cells

Abstract Two-dimensional nanomaterials have been widely used as fluorescent quenching platforms and nanocarriers of fluorophore-labelled nucleic acid probes to design nanoprobes for fluorescence imaging of intracellular microRNAs (miRNAs). However, the detection result was simply dependent on single fluorescent signal of fluorophore-labelled nucleic acid probes. Herein, a ratiometric fluorescent biosensor was proposed by utilizing protonated phenyl-doped carbon nitride (PDCN) nanosheets not only as a fluorescent quenching platform and nanocarrier of ROX-ssDNA probes but also as an internal fluorescent reference. After specific recognition and hybridization of target miRNAs, the fluorescence of ROX-ssDNA probes was recovered while the fluorescence of protonated PDCN nanosheets remained unchanged. By using the fluorescence ratio of ROX-ssDNA probes and protonated PDCN nanosheets as the response signal, this biosensor was used to detect DNA analog of miRNA-224 over a linear range of 1– 50 nmol L−1 with a limit of detection 0.2 nmol L−1. Finally, this ratiometric fluorescent biosensor was applied for fluorescence imaging of miRNA-224 in HeLa cells and HepG2 cells, which indicated this biosensor possessed a great potential for quantitative detection of miRNAs by fluorescence imaging in living cells.