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, A, and 3,4-ethylenedioxythiophene, B, bridges are detailed. We synthesized probes C and D, replacing the thiophene connection in probe A with phenyl and 2,1,3-benzothiadiazole units, respectively. Probe E was prepared by substituting probe A'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 A and B, respectively, and generate absorbance signals at 690.6 nm and 685.9 nm. In contrast, probes C and D displayed pronounced interference from NADH fluorescence at 450 nm, whereas probe E exhibited minimal fluorescence alterations in response to NAD(P)H. Pre-treatment of A549 cells with glucose in the presence of probe A led to a significant increase in fluorescence intensity. Additionally, subjecting probe A 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 A in sensing changes in NAD(P)H levels within a whole organism.

Highly Sensitive Cyanine Dyes for Rapid Sensing of NAD(P)H in Mitochondria and First-Instar Larvae of Drosophila melanogaster.

We have developed two highly sensitive cyanine dyes, which we refer to as probes A and B. These dyes are capable of quick and sensitive sensing of NAD(P)H. The dyes were fabricated by connecting benzothiazolium and 2,3-dimethylnaphtho[1,2-d]thiazol-3-ium units to 3-quinolinium through a vinyl bond. In the absence of NAD(P)H, both probes have low fluorescence and absorption peaks at 370 and 400 nm, correspondingly. This is because of their two electron-withdrawing acceptor systems with high charge densities. However, when NAD(P)H reduces the probes' electron-withdrawing 3-quinolinium units to electron-donating 1,4-dihydroquinoline units, the probes absorb at 533 and 535 nm and fluoresce at 572 and 586 nm for A and B correspondingly. This creates well-defined donor-π-acceptor cyanine dyes. We successfully used probe A to monitor NAD(P)H levels in live cells during glycolysis, under hypoxic conditions induced by CoCl2 treatment and after treatment with cancer drugs, including cisplatin, camptothecin, and gemcitabine. Probe A was also employed to visualize NAD(P)H in Drosophila melanogaster first-instar larvae. We observed an increase in NAD(P)H levels in A549 cancer cells both under hypoxic conditions and after treatment with cancer drugs, including cisplatin, camptothecin, and gemcitabine.

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

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.

Near-infrared rhodol dyes bearing salicylaldehyde moieties for ratiometric pH sensing in live cells during mitophagy and under hypoxia conditions.

We describe a simple but efficient approach to make fluorescent probes A and B based on rhodol dyes incorporated with salicyaldehyde moiety for monitoring pH changes in mitochondria under oxidative stresses and hypoxia conditions, and for tracking mitophagy processes. Probes A and B possess pKa values (pKa ≈ 6.41 and 6.83 respectively) near physiological pH and exhibit decent mitochondria-targeted capabilities, low cytotoxicity, and useful ratiometric and reversible pH responses, which make the probes appropriate for monitoring pH fluctuations of mitochondria in living cells with built-in calibration feature for quantitative analysis. The probes have been effectively useful for the ratiometric determination of pH variations of mitochondria under the stimuli of carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), hydrogen peroxide (H2O2), and N-acetyl cysteine (NAC), and during mitophagy triggered by cell nutrient deprivation, and under hypoxia conditions with cobalt chloride (CoCl2) treatment in living cells. In addition, probe A was efficient in visualizing pH changes in the larvae of fruit flies.

Near-infrared fluorescent probe based on rhodamine derivative for detection of NADH in live cells.

A near-infrared fluorescent probe was prepared for selective detection of reduced nicotinamide adenine dinucleotide (NADH) in live cells. The probe turns off the fluorescence with a closed spironolactone switch. However, reduction of the probe by NADH turns on fluorescence at 740 nm. Theoretical calculations suggest a more planar arrangement between the rhodamine and quinoline moieties with increased π-delocalization resulting from reduction.