A Redox-Sensitive Green Fluorescent Protein (roGFP) Sensing Strategy for Dynamic Analysis of Metal Oxide Nanoparticle-Induced Oxidative Stress.

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.