Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.

The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.