Dynamic DNA logic circuits regulating CRISPR-Cas12a trans-cleavage on MXene for high-throughput miRNAs detection

For the early diagnosis of malignancies, microRNAs (miRNAs) serve as crucial biomarkers. However, the direct application of the CRISPR-Cas system for multiplexed miRNA screening is hindered by the "one-crRNA-to-one-target" limitation. To address this challenge, we report a programmable paper-based microfluidic biosensor integrating computer-aided designed dynamic DNA circuits with CRISPR-Cas12a system on MXene. Within this sensing architecture, specific dual-miRNA targets independently initiate catalytic hairpin assembly cascades, and two DNA activators are generated to form a strict "AND" logic gate. Afterwards, the simultaneous presence of both activators triggers the robust trans-cleavage activity of Cas12a, cutting fluorophore-labeled single stranded DNA probes on MXene and yielding effective fluorescence recovery. Herein, MXene serves as both substrate and superior quencher, dramatically suppressing background noise to guarantee high signal-to-background ratio. Thus, the combination of catalytic hairpin assembly and Cas12a achieves exponential signal amplification. Furthermore, a paper-based microfluidic device is constructed for high-throughput miRNAs detection in human serum. This work provides a novel strategy for precise miRNA detection in early molecular cancer diagnostics and point-of-care testing.