A highly sensitive MiRNA detection sensor powered by CRISPR/Cas13a and entropy-driven amplification

Abstract

MicroRNAs (miRNAs) are critical regulators of numerous physiological and pathological processes, influencing gene expression and playing essential roles in cellular development, differentiation, and disease progression. Their sensitive and specific detection is vital for advancing biomedical research and clinical diagnostics, particularly for early disease detection and biomarker discovery. However, traditional miRNA detection methods often face significant challenges, such as limited sensitivity, insufficient specificity, and the inability to detect low-abundance miRNAs in complex biological samples. To overcome these limitations, we present a novel miRNA detection electrochemiluminescence (ECL) platform that integrates entropy-driven amplification with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated RNA cleavage. The entropy-driven amplification strategy exploits the thermodynamic advantages of nucleic acid hybridization, driving spontaneous molecular reorganization to amplify detection signals and achieve ultralow detection limits. CRISPR/Cas13a, an RNA-targeting nuclease, provides exceptional sequence specificity by recognizing and binding to target miRNA sequences, activating a collateral cleavage mechanism. This activity cleaves hairpin (HP) structure, generating a signal that further triggers EDA over DNA tetrahedron (DT) to induce a vigorous ECL response. Based on this strategy, we achieve rapid and precise quantification of miR-17 at femtomolar levels. Experimental results demonstrate high sensitivity, specificity, and the ability to analyze complex biological samples in the laboratory. This innovative approach holds great promise for advancing molecular diagnostics and personalized medicine.