Multi-Dimensionally Extended Functionalization Innovates to an Entropy-Driven Detection of Multi-miRNAs for One-Step Cancer Screening and Diagnosis in Living Cells.

Compared with tedious multi-step detections, multi-functional nanoprobes are effective for one-step screening and diagnosis of cancers by multi-detection of microRNAs (miRNAs). However, limited probe density, spatial mutual interference, and low target-triggered hybridization efficiency of nanoprobes will hinder intracellular applications. Here, for obtaining high loading density but low spatial mutual interference between functional biomolecules on nanoprobes, an extended biofunctionalization in three dimensions (the two-dimensional surface and a special "height" direction) is designed. Therefore, a multi-functional probe is constructed for one-step detection of multi-miRNAs for cancer screening and diagnosis. With linker-bridged multiple single-stranded DNAs swung out rigidly, multi-dimensionally extended upconversion nanorods (ME-UCNRs) covered by chitosan are constructed to load and deliver multiple biomolecules into living cells. Escaping from endolysosomes, ME-UCNRs maintain good biological activities of functionalized DNAs for effective detection of multi-miRNAs in living cells. Thereby, with multiple targets of miRNAs, toehold-mediated entropy-driven strand displacements are employed to give respectively changed fluorescent signals via fluorescence resonance energy transfer. Thus, a universal cancer biomarker of miR-21 and two specific liver-cancer biomarkers (miR-199a and miR-224) are efficiently detected through a one-step detection. By discriminating cancer cells from normal ones and determining liver-cancer cells simultaneously, this work innovates an efficient and definite one-step strategy for fast screening and early cancer diagnosis.