Photoactivatable NPOM-caged split-activator CRISPR/Cas12a biosensor for amplification-free imaging of deoxynivalenol-induced miR-155 dynamics and lateral-flow sensing.

MicroRNA-155 (miR-155) is an immune-associated small RNA biomarker involved in macrophage responses to environmental stressors such as deoxynivalenol (DON). However, achieving amplification-free miRNA sensing with low background and precise temporal regulation remains challenging. Here, we develop a photoactivatable split-activator CRISPR/Cas12a biosensor for miR-155 detection without target nucleic acid amplification and for dynamic analysis of toxin-induced cellular responses. The sensing system integrates an NPOM-caged crRNA with a split-complementary activator, enabling optical control of Cas12a activation. Upon brief 365 nm irradiation, miR-155 and ssDNA jointly reconstitute an activation-competent Cas12a complex, enabling target-dependent reporter cleavage after optical triggering. After systematic optimization, the fluorescence biosensor achieved quantitative miR-155 detection over a range of 1 pM-5 nM with an LOD of 0.69 pM. In mouse serum, the LOD was 1.27 pM, with relative matrix responses of 83.4%-92.3% and recoveries of 103.5%-106.2%. For DON-stimulated ANA-1 macrophages, the biosensor successfully resolved dose- and time-dependent miR-155 dynamics through live-cell fluorescence imaging and cell-lysate analysis. The time-course CRISPR signals showed excellent agreement with RT-qPCR measurements (Pearson r = 0.996, R2 = 0.992). Orthogonal validation using miR-155 inhibition, NPOM/UV controls, component-deletion experiments, and flow cytometry further supported intracellular target responsiveness and sensing reliability. Furthermore, the same architecture was adapted to a lateral-flow assay with an LOD of 11.99 pM and applied as a portable visual/semi-quantitative readout for cell-derived and mouse serum samples. Therefore, this work establishes a programmable light-gated Cas12a biosensing platform for amplification-free miRNA quantification, live-cell molecular imaging, and portable analysis of toxin-induced immune responses.