Self-priming Dual-hairpin probe-mediated Primer Exchange Reaction for Label-free Fluorescent detection of MicroRNA in Alzheimer’s Disease

Abstract

The accurate detection of microRNA (miRNA) biomarkers associated with Alzheimer’s disease (AD) is critical for early diagnosis and therapeutic monitoring. Herein, we report a label-free fluorescent bio-sensing platform that integrates a self-priming dual‑hairpin probe with a primer exchange reaction (PER) for sensitive miRNA analysis in AD. Target binding triggers a conformational rearrangement of the probe, activating a self-priming hairpin that initiates enzyme‑driven strand displacement amplification (SDA). Through cyclical extension and cleavage, the SDA process displaces a second hairpin structure, denoted as the PER template probe. Release of this probe enables primer binding and initiates PER, yielding large amounts of G‑rich single‑stranded DNA products. These sequences fold into G‑quadruplex structures that selectively bind thioflavin T, leading to a strong turn‑on fluorescence response. The assay achieves a broad linear detection range from 0.5 fM to 100 pM, with an ultralow detection limit of 87 aM. Furthermore, this strategy offers a cost‑effective, label‑free fluorescence method for SDA‑based detection and exhibits promising applicability in complex biological matrices.