Composite Fluorescence Encoding Technology Based on Tetrahedral DNA Framework for Logical Distinction and Multiplexed Recognition

Multiplexed bioanalysis and deciphering their regulatory relationships have great significance for the diagnosis and treatment of diseases. In this study, a composite fluorescence encoding technology (CFET) combining spectrum and intensity was developed with tetrahedral DNA frameworks (TDFs) as carriers and four Alexa Fluor dyes (AF₄₀₅, AF₄₈₈, AF₅₄₆, and AF₆₄₇) as tags. Sixty-nine CFET codes could be established by altering the types and numbers of AF dyes at the vertexes of TDF. Distinct fluorescence spectra were measured by spectrophotometer, and unique fluorescence colors were observed after the incubation of CFET code samples with MCF-7 cells for 4 h. The CFET 0110 probe was constructed based on CFET 0110 to simultaneously detect miRNA-21 and miRNA-31 in vitro. The excellent specificity and sensitivity performance (LOD = 1 nM) was verified in the multiplexed detection. The multiplexed detection ability of CFET probes was successfully demonstrated through easy visual recognition of five miRNAs. By constructing logic gates based on different expression levels of miRNA-21 and miRNA-31, three breast cell lines (MCF-10A, MCF-7, and MDA-MB-231) could be precisely distinguished by their unique fluorescence imaging output. The identities of five miRNAs at the single-cell level were successfully decoded through the distinct merged fluorescence colors of the CFET probes. Overall, the CFET provides a novel avenue for multiplexed labeling in vitro and in live cells, demonstrating enormous potential for cell differentiation and biomolecular multiplex recognition.