Stem-loop-terminated dendrimer DNA enables the ratiometric fluorescent miRNA analysis by fluorescence resonance energy transfer via the proximity/local concentration effect.

MicroRNA-21 (miR-21), a short non-coding RNA, functions as a diagnostic early biomarker for cancers, cardiovascular diseases, and inflammatory disorders. Thus, the development of a rapid, convenient, and highly sensitive method for miR-21 detection is essential for accurate disease diagnosis through point-of-care testing. Herein, we designed the stem-loop-terminated fluorescent DNA dendrimer, comprising Y-DNAa and Y-DNAb, to create a proximity/local concentration effect for detecting miR-21 through fluorescence resonance energy transfer (FRET). Both Y-DNAa and Y-DNAb consists of a dendritic core and three hairpin sticky ends, which were differentially labeled, with Y-DNAa functionalized with Cy3 and Y-DNAb with Cy5. In detection system, the confined microenvironment enabled miR-21 to act as a reusable catalyst, efficiently and repeatedly triggering the catalytic hairpin assembly (CHA) of Y-DNAa and Y-DNAb. Simultaneously, the fluorescence resonance energy transfer (FRET) signal was generated between the Cy3 of Y-DNAa and the Cy5 of Y-DNAb due to the sticky-end hybridization of stem-loop. The detection system exhibited a linear response over the range of 0.5 to 40 nM, with two linear ranges of 0-3 nM and 3-40 nM, and a detection limit of 0.09 nM. Meanwhile, we also used this sensing platform for miR-21 detection in the serum samples. Spike-recovery experiments yielded recoveries ranging from 98.9% to 103.1% with relative standard deviations below 5.66%, demonstrating significant potential for nucleic acid-based clinical diagnostics.