Dual-Accelerated Signal Amplification in Biosensing via Spatial Confining Catalytic Hairpin Assembly-Activated Spherical CRISPR/Cas12a System for Trans-Cleavage of Hairpin DNA Reporters

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and are implicated in various diseases, including cancer. Due to their critical role in diagnostics, there is a growing need for sensitive, specific, and rapid detection methods for miRNAs. In this study, we present a dual-accelerated signal amplification platform for miRNA biosensing, which integrates spatial confining catalytic hairpin assembly (SC-CHA) with spherical CRISPR/Cas12a (S-CRISPR/Cas12a) system for (SC-CHA@S-CRISPR/Cas12a) trans-cleavage of hairpin DNA reporters. The method employs a biotinylated palindrome-rich assembly sequence (PAS) to form DNA nanoballs, which serve as a scaffold for the operation of SC-CHA upon miRNA binding. The SC-CHA products bind with crRNA and Cas 12a protein, activating S-CRISPR/Cas12a system to cleave the hairpin DNA reporter and generate a detectable fluorescence signal. The uniqueness of this system lies in the combined use of DNA nanoballs and hairpin DNA reporters, both of which significantly accelerate reaction kinetics, resulting in rapid signal generation. Additionally, the spherical DNA nanostructure, integrated with the S-CRISPR/Cas12a system, greatly enhances biostability and accelerating reaction kinetics. These features enable the platform to exhibit high sensitivity, with a limit of detection (LOD) as low as 13.75 fM, and excellent specificity, successfully distinguishing miRNA-21 from other miRNAs. The assay is also biostable, demonstrating reliable performance in complex biological samples such as human serum. This dual-acceleration approach offers a promising solution for sensitive, rapid, and specific miRNA biosensing, with potential applications in early cancer diagnosis and clinical monitoring.