A ratiometric and trans-cleavage-triggered feedback CRISPR/Cas12a cascade for amplification-free MicroRNA detection using a multifunctional switch

MicroRNAs (miRNAs), as crucial regulators of gene expression, play significant roles in cancer development. Among them, miRNA-21 has been recognized as a promising biomarker for the diagnosis and prognosis evaluation of multiple cancers. However, existing CRISPR/Cas12a-based miRNA detection methods generally require reverse transcription and pre-amplification steps, which complicate the operational workflow and are prone to introducing cross contamination. Furthermore, the dependence on single-fluorescence signal detection makes them vulnerable to environmental and instrumental fluctuations, which results in risk of false-positive outcomes. Herein, we developed a ratiometric and sensitive CRISPR/Cas12a system with trans-cleavage-triggered feedback signal amplification for amplification-free detection of miRNA-21. The strategy employs a novel multifunctional switch probe that integrates target recognition, signal transduction, and cascade amplification within a unified system. A trans-cleavage-triggered feedback signal amplification circuit was constructed to enhance detection sensitivity without the need for additional enzyme systems. Moreover, a ratiometric fluorescence signal was implemented by calculating the fluorescence intensity ratio between CY5 and Auramine O, which effectively minimizes interference from environmental and instrumental variations and substantially improves detection accuracy and reliability. Experimental results demonstrate that the method achieves a detection limit of 1.28 nM, along with excellent specificity, accuracy, and reproducibility. In real sample analysis, differential expression levels of miRNA-21 in lung adenocarcinoma cells (A549) and non-cancerous adult bronchial epithelial cells (BEAS-2B) was observed, which highlights its potential value for clinical diagnostics.