MiRNA analysis has played an important role in precise diagnosis, treatment and prognosis of cancer, especially the multiplexed miRNAs imaging. In this work, a novel fluorescence emission intensity (FEI) encoding strategy was developed based on tetrahedron DNA framework (TDF) carrier and the FRET effect between Cy3 and Cy5. Six FEI-encoded TDF (FEI-TDF) samples were constructed by tuning the labeling number of Cy3 and Cy5 at vertexes of TDF. For fluorescence characterization in vitro, distinct FEIs in the spectra and different colors under ultraviolet (UV) irradiation of FEI-TDF samples were observed. By dividing ranges of FEI of samples, the stability of FEIs was highly improved. Based on ranges of FEIs in each sample, five codes with good discrimination were finally developed. Before the application of intracellular imaging, the excellent biocompatibility of TDF carrier was proved by CCK-8 assay. The barcode probes based on samples 12, 21 and 11 were designed as example models to realize multiplexed imaging of miRNA-16, miRNA-21 and miRNA-10b in MCF-7 cells with obvious different fluorescence merge colors. FEI-TDFs provide a new research perspective for the development of fluorescence multiplexing strategies in the future.

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