DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA

作者：Rufei Cao, Shuji Wang, Qingxi Guo, Hongsheng Xie, Yongxiang Wang, Tao Li, Feifei Chang, Huahua Shi, Shijia Ding, Xun Min, Xiaolei Duan · 发表于：Analytica Chimica Acta · 年份：2025 · DOI：10.1016/j.aca.2025.344620 · 被引用次数：6 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、MicroRNA in disease regulation

Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference c...