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

Abstract

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 2,3dimethylnaphtho[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 CoCl 2 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.

Sensitive monitoring of NAD(P)H levels within cancer cells using mitochondria-targeted near-infrared cyanine dyes with optimized electron-withdrawing acceptors

“…In contrast, cyanine dyes lacking this thiophene bridge connection demonstrate much quicker response times, typically within 6 minutes. 35 Probe C initially exhibited an absorption peak at 441 nm in the absence of NADH, with no detectable fluorescence under these conditions (Fig. S16, ESI†).…”

“…Suitable models and conditions for the theoretical calculations were derived and conducted as reported previously. 35 Calculated absorptions are listed in Table 1 with representations of the molecules in Fig. 4 and Fig.…”

Syntheses and Applications of Coumarin-Derived Fluorescent Probes for Real-Time Monitoring of NAD(P)H Dynamics in Living Cells across Diverse Chemical Environments

“…9−24 As a result, fluorescence imaging with fluorescent probes is a powerful tool for studying the molecular mechanisms underlying various physiological and pathological processes, as well as for developing new therapeutic strategies. [9][10][11][12][13][14][15][16][17][18][19][20][21]23,24 Developing fluorescent probes with longer emission wavelengths is critical for reducing the interference from the intrinsic fluorescence properties of NAD(P)H, minimizing signal attenuation, enhancing imaging resolution through deeper tissue penetration of light, and improving the accuracy and sensitivity of fluorescence imaging in live cells. [9][10][11][12][13][14][15][16][17][18][19][20][21]23,24 Our probe formulations are based upon coumarins which are a large family of 2H-chromen-2-one-containing compounds that are widely used as fluorescent fluorophores due to their unique optical and chemical properties, including broad excitation and emission wavelengths, high quantum yield, chemical stability, low toxicity, and ease of modification.…”

“…[9][10][11][12][13][14][15][16][17][18][19][20][21]23,24 Developing fluorescent probes with longer emission wavelengths is critical for reducing the interference from the intrinsic fluorescence properties of NAD(P)H, minimizing signal attenuation, enhancing imaging resolution through deeper tissue penetration of light, and improving the accuracy and sensitivity of fluorescence imaging in live cells. [9][10][11][12][13][14][15][16][17][18][19][20][21]23,24 Our probe formulations are based upon coumarins which are a large family of 2H-chromen-2-one-containing compounds that are widely used as fluorescent fluorophores due to their unique optical and chemical properties, including broad excitation and emission wavelengths, high quantum yield, chemical stability, low toxicity, and ease of modification. 25−29 As a result, coumarins are utilized as a desirable and ideal fluorophore platform for a wide range of applications, such as biological imaging, sensing, and diagnostics for cations, 30 anions, 27,29,31 pH, 32−34 NADH, 17 H 2 S, 27,29,31 cysteine, homocysteine, and glutathione (GSH), 27,29,…”

“…Developing sensitive and specific methods to detect and quantify NAD(P)H in live cells has broad implications for improving our understanding of the molecular mechanisms that underlie various physiological and pathological processes. − These methods can also aid in early disease diagnosis and prognosis, and the development of targeted therapeutic strategies. − Ultimately, advances in the detection and quantification of NAD(P)H can lead to improved patient outcomes and a better understanding of the molecular mechanisms underlying various diseases. − Fluorescence imaging with fluorescent probes is a valuable technique for assessing NAD(P)H in live cells. − This technique has several advantages over other methods, including high sensitivity, specificity, and noninvasiveness. It also allows for live-cell imaging, enabling the monitoring of changes in NAD(P)H in real-time. − Furthermore, fluorescence imaging with fluorescent probes enables multiplexing, which means that multiple targets can be detected simultaneously. − This technique also allows for high throughput screening, which can identify compounds that affect NAD(P)H metabolism. − As a result, fluorescence imaging with fluorescent probes is a powerful tool for studying the molecular mechanisms underlying various physiological and pathological processes, as well as for developing new therapeutic strategies.…”