Reaction-based near-infrared fluorescent probes for monitoring NADH-associated redox remodeling in ferroptosis.

Nicotinamide adenine dinucleotide (NADH) plays a central role in cellular redox balance and metabolic regulation, yet its dynamic behavior during ferroptosis remains insufficiently understood. Herein, we report reaction-based fluorescent probes, QX-I and QX-II, for imaging NADH-associated redox changes. Two probes were constructed by conjugating a responsive group (methyl-quinolinium salt) with an electron-withdrawing Xanthene moiety. The conjugated Xanthene structure not only shifts the emission wavelength of the probes into the near-infrared region, but also endows probe QX-I (modified with a single N-ethyl group) with advantages such as high fluorescence enhancement, rapid response, high sensitivity, and good selectivity. This probe enables visualization of endogenous NADH fluctuations in HepG2 and 4 T1 cells under metabolic regulation. Notably, QX-I was further employed to investigate NADH-related dynamic changes during ferroptosis. Cell and in vivo experiments demonstrated a decoupling relationship between lipid peroxidation and NADH-related redox states during ferroptosis inhibition. Overall, this study provides an effective tool for monitoring NADH-related redox remodeling and offers new insights into ferroptosis-associated redox metabolism.