Wang Yizhu, Ju Rui, Fu Yan, Zhang Fengqi, Yin Ziyue, Lv Mengyuan, Zhu Yanbo, Song Zhiqiang, Li Xiaonan, Wang Na
China Medical University-The Queen's University of Belfast Joint College (CQC), China Medical University, No.77 Puhe Road, Shenyang, Liaoning Province 110122, P.R. China.
Research Institute of Biomedical and Advanced Materials, College of Life and Health, Dalian University, No.10 Xuefu Street, Dalian, Liaoning Province 116622, P. R. China.
ACS Omega. 2025 Apr 9;10(15):15587-15597. doi: 10.1021/acsomega.5c00774. eCollection 2025 Apr 22.
The induction of oxygen radicals and oxidative stress are major pathways through which nanomaterials cause adverse health effects. Dynamic monitoring of redox processes in living cells exposed to nanomaterials is currently limited due to the inadequacy of conventional methods. Herein, we construct a Grx1-roGFP2 (glutaredoxin 1 fused with redox-sensitive Green Fluorescent Protein 2) protein sensor expressed in Madin-Darby Canine Kidney (MDCK) cells that allows dynamic analysis of metal oxide (MOx) nanoparticle-induced oxidative stress. We selected eight representative MOx as test objects, ranking their toxicity potentials according to the overlap degree of their band gap energies with cellular redox potentials and their ability to release metal ions to catalyze the generation of oxygen radicals. The sensor demonstrates high sensitivity in detecting MOx-induced intracellular redox fluctuations, operating within a 6-200 mg/mL range and a 30-min response time, while maintaining sustained sensitivity over 24 h. The sensor utilizes an oxidation/reduction ratio curve to precisely characterize the unique pattern of oxidative stress induced by each MOx, encompassing the stress's intensity (curve slope), amplitude (curve plateau), features (curve shape), and accumulation of oxygen radicals (curve area integral). These results highlight that the developed Grx1-roGFP2 sensor holds more advantages over traditional probes, showing extensive application prospects in higher standards of nanotoxicological evaluation.
氧自由基的诱导和氧化应激是纳米材料产生不良健康影响的主要途径。由于传统方法的不足，目前对暴露于纳米材料的活细胞中氧化还原过程的动态监测受到限制。在此，我们构建了一种在犬肾上皮细胞（MDCK）中表达的Grx1-roGFP2（与氧化还原敏感的绿色荧光蛋白2融合的谷氧还蛋白1）蛋白传感器，用于动态分析金属氧化物（MOx）纳米颗粒诱导的氧化应激。我们选择了八种代表性的MOx作为测试对象，根据它们的带隙能量与细胞氧化还原电位的重叠程度以及释放金属离子以催化氧自由基生成的能力对它们的毒性潜力进行排名。该传感器在检测MOx诱导的细胞内氧化还原波动方面表现出高灵敏度，在6-200 mg/mL范围内和30分钟响应时间内运行，同时在24小时内保持持续灵敏度。该传感器利用氧化/还原比曲线精确表征每种MOx诱导的氧化应激的独特模式，包括应激的强度（曲线斜率）、幅度（曲线平台）、特征（曲线形状）和氧自由基的积累（曲线面积积分）。这些结果表明，所开发的Grx1-roGFP2传感器比传统探针具有更多优势，在更高标准的纳米毒理学评估中显示出广泛的应用前景。
