A label-free fluorescence assay for microRNAs based on linear enzymatic signal amplification

MicroRNAs (miRNAs) are regulators in various physiological and pathological processes with significant potential for disease diagnosis and therapeutic monitoring. However, their quantification remains a challenge due to their high degradability, short sequence length, and naturally low abundance in sample specimen. Herein, we report a fluorescence assay based on an innovative linear enzyme-assisted isothermal signal amplification strategy and label free fluorescence readout by means of an RNA-intercalating reagent. Poly(A) polymerase-catalyzed polyadenylation of target miRNA retained on the solid surface of magnetic beads resulted in a poly(A) tail of ∼150 bp added to the 3' end of miRNA. The elongation of the miRNA sequence enables robust fluorescence staining with SYBR Green II, thereby allowing highly sensitive fluorescence detection. The assay proposed allows quantification of target miRNAs with a detection limit of 66.4 fM and a linear calibration relating fluorescence intensity to miRNA concentration (R² = 0.998). Its applicability was demonstrated by quantifying target miRNAs in cellular samples. With its high sensitivity and accuracy, the assay successfully detected paclitaxel-induced downregulation of miRNA-146a-5p in MDA-MB-231 breast cancer cells. Collectively, these results highlight the potential of this assay for quantitative miRNA analysis in biomedical and clinical applications.