Activatable Naphthalimide-Based Fluorescent Probe for Hydrogen Peroxide Detection: Visualizing Microcystin-LR Induced Oxidative Stress in Zebrafish and Hepatocyte Injury Mechanism

Abstract

Microcystin-LR (MC-LR), a toxic cyanotoxin, accumulates in fish through uptake, posing severe threats to fisheries safety and human health. The interaction between MC-LR and aquatic fish induces oxidative stress, which triggers excessive production of reactive oxygen species (ROS), notably hydrogen peroxide (H2O2). Abnormal ROS fluctuations are closely linked to the onset of various diseases, making the development of in vivo H₂O₂ detection and regulation methods critically important. Herein, we designed and synthesized an activatable fluorescent probe using naphthalimide as the fluorophore. The probe’s performance was optimized by tuning the hydrophilicity of its 2-(2-aminoethoxy) ethanol substituent and introducing a phenylborate moiety for specific H2O2 recognition. This novel probe exhibited exceptional analytical performance for H₂O₂ determination, including high selectivity against interfering substances and remarkable sensitivity with a detection limit of 53.2 nmol/L. In biological applications, the probe successfully achieved intracellular H₂O₂ imaging in HeLa cells and real-time monitoring of endogenous H₂O₂ generation in zebrafish exposed to MC-LR. Molecular docking simulations revealed that MC-LR forms a stable binding complex with the OATP1d1 protein via hydrogen bonds and hydrophobic interactions, providing a molecular basis for its cellular uptake and accumulation. Furthermore, this study elucidated the mechanistic association between MC-LR induced hepatocyte injury and the subsequent oxidative stress storm. Notably, garlic extract was identified as an effective exogenous intervention to downregulate ROS levels in MC-LR exposed zebrafish, offering a potential strategy for mitigating cyanotoxin-induced oxidative damage. These results demonstrate that the developed fluorescent probe can visually reveal the link between MC-LR accumulation and its hepatotoxicity at the cellular and molecular levels. Thus, this probe serves as a powerful tool for environmental monitoring, food safety, and human health protection, holding broad application prospects in related fields.