Thiophene-based organic dye with large Stokes shift and deep red emission for live cell NAD(P)H detection under varying chemical stimuli

Abstract

We report a novel method for synthesizing red and deep red cyanine dyes with large Stokes shifts, probes A and B, for live cell NAD(P)H detection. The probes were prepared using thiophene-based organic dyes featuring a π-conjugated bridge of thiophene and 3,4-ethylenedioxythiophene units linking the 1-methylquinolinium acceptor and formyl acceptor, respectively. These probes display weak absorption peaks at 315 nm (A) and 334 nm (B) and negligible fluorescence in the absence of NADH. However, upon the presence of NADH, new absorption and fluorescence peaks appear at 477 nm and 619 nm for probe A and at 486 nm and 576 nm for probe B, respectively. This is due to the NADH-facilitated reduction of the 1-methylquinolinium unit into 1-methyl-1,4-dihydroquinoline, which then acts as the electron donor for the probes, leading to the formation of well-defined electron donor–acceptor dye systems. Probe A has a large Stokes shift of 144 nm, which allows for better separation between the excitation and emission spectra, reducing spectral overlap and improving the accuracy of fluorescence measurements. The probes are highly selective for NAD(P)H, water-soluble, biocompatible, and easily permeable to cells. They are also photostable and were successfully used to monitor changes in NADH concentration in live cells during glycolysis in the presence of glucose, lactate, and pyruvate, treatment of FCCP and cancer drug cisplatin, and under hypoxia triggered by CoCl₂. Furthermore, the probes were able to image NAD(P)H in Drosophila melanogaster larvae. Notably, cisplatin treatment increased the NAD(P)H concentration in A459 cells over time. Overall, this work presents a significant advancement in the field of live cell imaging by providing a simple and cost-effective method for detecting changes in NAD(P)H concentration under varying chemical stimuli.