Enzyme-Free and Product-Accelerated DNA Walker Cascade for Ultrasensitive Detection of Tic Disorder-Associated MicroRNA

Abstract

Tic disorders (TDs) are prevalent neuropsychiatric conditions in children, yet early diagnosis remains challenging due to limited availability and high sequence homology of associated microRNA biomarkers. Existing detection methods often suffer from insufficient sensitivity, background interference, and operational complexity. Here, we report an enzyme-free, product-accelerated DNA walker cascade for ultrasensitive detection of tic disorder-associated miRNA. The biosensor integrates three synergistic amplification modules: a conformation-switching DNAzyme probe for catalytic target recycling, a self-locking DNAzyme tracker for background suppression, and a product-accelerated DNA walker for enhanced signal generation. Upon target recognition, the DNAzyme probe cyclically converts miRNA into initiator strands, which unlock surface-immobilized trackers and activate DNAzyme cleavage of fluorescent substrates. Each cleavage event generates Cy5-labeled fragments that actively displace initiator strands, creating a positive feedback loop that enables a single initiator to trigger multiple walking cycles. This product-accelerated mechanism overcomes kinetic constraints of conventional DNA walkers, achieving substantially amplified signal output. The method operates without protein enzymes or costly reagents, achieving a detection limit of 2.04 fM with a linear range from 5 fM to 50 pM. It exhibits high specificity against mismatched sequences and excellent recovery rates (97.4%–104.2%) in serum samples, with strong agreement with qRT-PCR results. With its simplicity, cost efficiency, and reliable performance, this enzyme-free platform holds great promise for early diagnosis and point-of-care applications.