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Photoactivatable NPOM-caged split-activator CRISPR/Cas12a biosensor for amplification-free imaging of
Kuan Chang1, Jiadi Sun1, Tao Liu1
1School of Food Science and Technology, International Joint Laboratory on Food Safety, Synergetic Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; Key Laboratory of Screening, Prevention, and Control of Food Safety Risks, State Administration for Market Regulation, Wuxi, Jiangsu, 214122, PR China; Institute of Future Food Technology, JITRI, Yixing, Jiangsu, 214200, PR China.
Abstract:
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.
