Multiplexed miRNA imaging using spherical nucleic acids and catalytic hairpin assembly for screening the differential immunotoxicity of deoxynivalenol and its acetylated derivatives in RAW264.7 cells.

Deoxynivalenol (DON) and its acetylated derivatives are type-B trichothecene mycotoxins produced by Fusarium species. Even low-level exposure can trigger immune and inflammatory responses, posing a substantial risk to human and animal health. Therefore, establishing toxicity screening techniques for low-dose exposure is essential for early food-safety risks. miR-210 and miR-155 are representative indicators of cellular stress in macrophages. Elucidating their dynamic profiles is critical for understanding inflammation triggered by low-dose mycotoxin exposure. Capitalizing on this, this research developed a spherical nucleic acids (SNAs) probe-driven catalytic hairpin assembly (CHA) to quantify mycotoxin-triggered immunotoxicity in RAW264.7 cells. On the one hand, SNAs can serve as ideal carriers for functional nucleic acids; on the other hand, the enzyme-free CHA amplification acts as an in-built signal amplifier. Under optimized conditions, DON and its acetylated derivatives induce a time-dependent increase in intracellular miR-155 and miR-210 levels, which in turn activate the CHA reaction, resulting in a progressive increase in cellular fluorescence intensity. As the concentration of DON, 3-Ac-DON, and 15-Ac-DON increased (0.05-2 μmol/L) and exposure time increased (0-12 h), the fluorescence signal within the cells gradually increased. Intracellular fluorescence intensity exhibited a positive, dose-dependent correlation with toxin concentration, and can serve as a reliable indicator of the cytotoxic effects induced by varying toxin doses in RAW264.7 cells. The miRNA imaging method established in this study reveals the dynamic changes of miRNAs during low-dose toxin exposure, thereby offering a theoretical foundation and technical support for the early screening of the immunotoxicity of DON and its derivatives.