Development of a High-Fidelity EXPAR Platform and Its Application for Ultrasensitive Detection of Diabetic Foot Ulcer-Associated MicroRNAs

Diabetic foot ulcer (DFU), a severe complication of diabetes, necessitates sensitive detection of pathogenic microRNAs (miRNAs) as emerging diagnostic biomarkers. The exponential amplification reaction (EXPAR) offers high efficiency and rapid kinetics for miRNA detection, but its application is limited by persistent nonspecific amplification arising from spurious template-template interactions and interference from partially complementary sequences in complex samples. To address this, we present an integrated strategy combining a blocking probe prehybridized to the EXPAR template to sterically hinder nonspecific hybridization, magnetic bead-based isolation of target-bound probes to remove interferents, and a self-priming mechanism that regenerates the EXPAR primer only upon target recognition. This design ensures high specificity and minimizes primer-independent amplification. Coupling this optimized EXPAR platform with a G-quadruplex-based signal transduction system enables facile colorimetric readout for instrument-free visual detection. The method achieves a detection limit of 41.4 aM across a dynamic range of 100 aM to 50 pM for DFU-associated miRNAs, with excellent selectivity against nontarget miRNAs and consistent reproducibility in fibroblasts and serum. This work provides a generalizable solution to the specificity issue in EXPAR and delivers a reliable, sensitive platform for miRNA profiling in clinical and point-of-care settings.