Near-infrared Absorption and Emission Probes with Optimal Connection Bridges for Live Monitoring of NAD(P)H Dynamics in Living Systems.

Two NAD(P)H-biosensing probes consisting of 1,3,3-trimethyl-3H-indolium and 3-quinolinium acceptors, linked by thiophene, , and 3,4-ethylenedioxythiophene, , bridges are detailed. We synthesized probes and , replacing the thiophene connection in probe with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe was prepared by substituting probe 's 3-quinolinium unit with a 1-methylquinoxalin-1-ium unit. Solutions are non-fluorescent but in the presence of NADH, exhibit near-infrared fluorescence at 742.1 nm and 727.2 nm for probes and , respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes and displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe led to a significant increase in fluorescence intensity. Additionally, subjecting probe to lactate and pyruvate molecules resulted in opposite changes in NAD(P)H levels, with lactate causing a substantial increase in fluorescence intensity, conversely, pyruvate resulted in a sharp decrease. Treatment of A549 cells with varying concentrations of the drugs cisplatin, gemcitabine, and camptothecin (5, 10, and 20 μM) led to a concentration-dependent increase in intracellular fluorescence intensity, signifying a rise in NAD(P)H levels. Finally, fruit fly larvae were treated with different concentrations of NADH and cisplatin illustrating applicability to live organisms. The results demonstrated a direct correlation between fluorescence intensity and the concentration of NADH and cisplatin, respectively, further confirming the efficacy of probe in sensing changes in NAD(P)H levels within a whole organism.