Construction of a Dual-Response Fluorescent Probe for Potential Detection of Sulfites and NAD(P)H

Abstract Sulfites, which serve as significant multifunctional food additives, can act as potential pollutants or toxins in vivo when present in excess and can be reduced in association with NAD(P)H. While NAD(P)H is a central redox coenzyme that fuels mitochondrial ATP production, sustains cellular redox balance, and regulates diverse signaling pathways, it would be invaluable to monitor the rapid subcellular fluctuations of NAD(P)H and sulfites for a comprehensive understanding of metabolic syndromes, neurodegenerative disorders, and ischemia-related injuries by constructing a dual-response fluorescent probe. Herein, FL-D was designed and synthesized as an excitation-gated dual-response fluorescent probe that decouples the optical detection of sulfites from cellular reducing power within a single molecular scaffold. Built on a functionalized quinolinium platform, FL-D exhibited spectroscopically orthogonal response modes governed by the excitation wavelength. Under 378 nm excitation, FL-D functioned as a ratiometric sensor for bisulfite and sulfite (HSO3–/SO32–), utilizing intrinsic emission bands at 434 and 510 nm to display a pronounced ratiometric shift with high selectivity against competing biothiols. In contrast, under 500 nm excitation, FL-D entirely bypassed the sulfur-responsive spectral window, operating as a specific turn-on fluorescent reporter for NADH and generating a strong emission enhancement at 577 nm under physiological conditions (up to 100-fold variation), which could potentially monitor fluctuations in NADH and sulfites. Such profound excitation-gated orthogonality should enable independent yet simultaneous interrogation of sulfur metabolism and nicotinamide redox status without reliance on probe cocktails or complex spectral unmixing for further investigation.