Split-crRNA Transcription Based Dual-Recognition Cascade Amplification Assay for Sensitive and Accurate Postoperative Delirium Related MicroRNA Analysis

Postoperative delirium (POD) represents a serious neuropsychiatric complication, underscoring the urgent need for sensitive and specific detection of circulating microRNAs (miRNAs) as potential biomarkers. Conventional isothermal amplification methods, however, often face a trade-off between sensitivity and specificity. Herein, we report a cascade amplification strategy that integrates catalytic hairpin assembly (CHA), split DNAzyme cleavage, T7 RNA polymerase-mediated transcription, and CRISPR-Cas12a trans-cleavage to achieve ultrasensitive and accurate miRNA detection. The design is centered on a dual-recognition mechanism: the same target miRNA molecule first initiates the CHA cascade to reassemble an active split DNAzyme, and subsequently acts as the spacer RNA to reconstitute a functional Cas12a/crRNA complex. This two-step, sequence-specific verification substantially enhances detection fidelity by minimizing off-target binding. Through multistage amplification combining CHA, DNAzyme catalysis, transcriptional amplification, and Cas12a-mediated trans-cleavage, the assay achieves exceptional sensitivity, with a detection limit as low as 0.52 fM for miRNA-193 and a wide linear range from 1 fM to 100 pM. It demonstrates high specificity by effectively discriminating single-base mismatches, and performs robustly in spiked human serum, yielding recovery rates of 97.3–107.2%. Moreover, results correlate closely with RT-qPCR, confirming reliability in complex matrices. This work presents a versatile and robust biosensing platform that not only offers a promising tool for POD-related miRNA analysis, but also establishes a modular framework adaptable for detecting diverse nucleic acid targets in point-of-care diagnostics.