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.

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

A series of near-infrared fluorescent probes, labeled A to E, were developed by combining electron-rich thiophene and 3,4-ethylenedioxythiophene bridges with 3-quinolinium and various electron deficient groups, enabling the sensing of NAD(P)H. Probes A and B exhibit absorptions and emissions in the near-infrared range, offering advantages such as minimal interference from autofluorescence, negligible photo impairment in cells and tissues, and exceptional tissue penetration. These probes show negligible fluorescence when NADH is not present, and their absorption maxima are at 438 nm and 470 nm, respectively. In contrast, probes C-E feature absorption maxima at 450, 334 and 581 nm, respectively. Added NADH triggers the transformation of the electron-deficient 3-quinolinium units into electron-rich 1,4-dihydroquinoline units resulting in fluorescence responses which were established at 748, 730, 575, 625 and 661 for probes AH-EH, respectively, at detection limits of 0.15 µM and 0.07 µM for probes A and B, respectively. Optimized geometries based on theoretical calculations reveal non-planar geometries for probes A-E due to twisting of the 3-quinolinium and benzothiazolium units bonded to the central thiophene group, which all attain planarity upon addition of hydride resulting in absorption and fluorescence in the near-IR region for probes AH and BH in contrast to probes CH-EH which depict fluorescence in the visible range. Probe A has been successfully employed to monitor NAD(P)H levels in glycolysis and specific mitochondrial targeting. Furthermore, it has been used to assess the influence of lactate and pyruvate on the levels of NAD(P)H, to explore how hypoxia in cancer cells can elevate levels of NAD(P)H, and to visualize changes in levels of NAD(P)H under hypoxic conditions with CoCl2 treatment. Additionally, probe A has facilitated the examination of the potential impact of chemotherapy drugs, namely gemcitabine, camptothecin, and cisplatin, on metabolic processes and energy generation within cancer cells by affecting NAD(P)H levels. Treatment of A549 cancer cells with these drugs has been shown to increase NAD(P)H levels, which may contribute to their anticancer effects ultimately leading to programmed cell death or apoptosis. Moreover, probe A has been successfully employed in monitoring NAD(P)H level changes in D. melanogaster larvae treated with cisplatin.

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.

An effective fluorescent probe for detection of phosgene based on naphthalimide dyes in liquid and gaseous phases.

A fluorescent probe was developed for the detection of phosgene based on 1,8-naphthalimide, of which o-diaminobenzene was used as the recognition moiety. The probe does not fluoresce due to nonradiative decay. The probe reacts rapidly with phosgene via an intramolecular cyclization reaction, which induces large fluorescence due to increased rigidity in the resulting molecule and a low detection limit (0.23 nM). This probe has excellent selectivity for phosgene against competing interference analytes and, in the form of probe-loaded test paper, is an extremely sensitive method for phosgene sensing in the gas phase below 1 ppm concentrations.

Near-Infrared Fluorescent Probes with Amine-Incorporated Xanthene Platforms for the Detection of Hypoxia.

Three fluorescent probes A, B, and C that function in the near-infrared wavelengths and utilize pseudo xanthene platforms with an oxygen atom at the 10-position replaced by a [Me-N]2- group have been created to identify hypoxia via nitroreductase determinations at the 0.04, 0.10, and 0.19 ng/mL levels. Theoretical calculations suggest that the probes are not planar due to steric interactions. Absorptions of photons result in the transition of electron density from the indoline moieties to delocalized orbitals on the anthranilic section, ending up on the nitro groups of the electron-poor (i.e., nonreduced) probes (i.e., A, B, and C), whereas those for the more electron-rich (i.e., reduced) probes consisted of movement from the indoline groups to the right side of the anthranilic sections, resulting in a shift in absorption.

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.

A two-photon fluorogenic probe based on a coumarin schiff base for formaldehyde detection in living cells.

A novel two-photon fluorogenic probe has been developed to detect formaldehyde with fast response, low cytotoxicity, and excellent selectivity. This probe exhibits a strong turn-on fluorescence response to formaldehyde under excitation at 370 nm and has been successfully applied to detect formaldehyde in living cells. Theoretical calculations at the B3LYP/6-31 + G(d,p), APFD/6-311++G, and APFD/6-311 + G(2d,p) levels of theory for the absorption and emission wavelengths of the probes were in agreement with those obtained experimentally.

A ratiometric near-infrared fluorescent probe based on a novel reactive cyanine platform for mitochondrial pH detection.

A near-infrared reactive cyanine platform (probe A) was prepared by condensation of 9-chloro-1,2,3,4-tetrahydro-10-methyl-acridinium iodide with Fisher's aldehyde. A near-infrared fluorescent probe (probe B) was prepared by modifying a reactive chlorine atom of probe A with tert-butyl(2-aminoethyl)carbamate through a substitution reaction. The deprotection of the Boc group of probe B was achieved under an acidic condition, affording an amine-functionalized cyanine dye (probe C). A near-infrared ratiometric fluorescent probe (probe D) for mitochondrial pH detection was synthesized by conjugating a FRET coumarin donor to a FRET cyanine acceptor (probe C) through an amide bond connection. Probe A shows low fluorescence of 2% due to an electron-withdrawing chlorine atom, while probes B-D display high fluorescence quantum yields of 60%, 32%, and 35% in aqueous solutions containing 10% ethanol, respectively. Probes B-D show strong fluorescence with push-pull molecular structures in neutral and basic pH conditions. However, protonation of the probe's second amine at the 9-position under acidic condition disrupts the push-pull feature of the probes, resulting in fluorescence quenching of the new cyanine fluorophores. The probes can selectively stain mitochondria, while probe D was employed to detect pH changes in HeLa cells and Drosophila melanogaster first-instar larvae.

Ratiometric Detection of Glutathione Based on Disulfide Linkage Rupture between a FRET Coumarin Donor and a Rhodamine Acceptor.

Abnormal levels of glutathione, a cellular antioxidant, can lead to a variety of diseases. We have constructed a near-infrared ratiometric fluorescent probe to detect glutathione concentrations in biological samples. The probe consists of a coumarin donor, which is connected through a disulfide-tethered linker to a rhodamine acceptor. Under the excitation of the coumarin donor at 405 nm, the probe shows weak visible fluorescence of the coumarin donor at 470 nm and strong near-infrared fluorescence of the rhodamine acceptor at 652 nm due to efficient Forster resonance energy transfer (FRET) from the donor to the acceptor. Glutathione breaks the disulfide bond through reduction, which results in a dramatic increase in coumarin fluorescence and a corresponding decrease in rhodamine fluorescence. The probe possesses excellent cell permeability, biocompatibility, and good ratiometric fluorescence responses to glutathione and cysteine with a self-calibration capability. The probe was utilized to ratiometrically visualize glutathione concentration alterations in HeLa cells and Drosophila melanogaster larvae.

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria.

Ratiometric near-infrared fluorescent probes (AH+ and BH+) have been prepared for pH determination in mitochondria by attaching dithioacetal and formal residues onto a hemicyanine dye. The reactive formyl group on probe BH+ allows for retention inside mitochondria as it can react with a protein primary amine residue to form an imine under slightly basic pH 8.0. Probes AH+ and BH+ display ratiometric fluorescent responses to pH changes through the protonation and deprotonaton of a hydroxy group in hemicyanine dyes with experimentally determined pKa values of 6.85 and 6.49, respectively. Calculated pKa values from a variety of theoretical methods indicated that the SMDBONDI method of accounting for solvent and van der Waals radii plus including a water molecule located near the site of protonation produced the closest overall agreement with the experimental values at 7.33 and 6.14 for AH+ and BH+ respectively.

A near-infrared fluorescent probe based on a hemicyanine dye with an oxazolidine switch for mitochondrial pH detection.

A near-infrared fluorescent probe (AH+) has been prepared by incorporating an oxazolidine switch into a near-infrared hemicyanine dye. The probe shows fast and sensitive responses to pH from an oxazolidine switch to the hemicyanine dye upon pH decreases from 10.0 to 5.0. The probe shows good photostability, low cytotoxicity, and reversible fluorescence responses to pH changes with a pKa value of 7.6. It has been successfully used to determine pH changes in mitochondria.

Cell Membrane-Specific Fluorescent Probe Featuring Dual and Aggregation-Induced Emissions.

A cell membrane-specific fluorescent probe was prepared by conjugating a coumarin dye with a tetraphenylethene (TPE) derivative through an α,ß-unsaturated ketone connection. The probe has two absorptions: one from the TPE moiety at 300 nm and a second one due to the coumarin moiety at 458.5 nm. The probe fluoresces at 470 nm in tetrahydrofuran (THF) solution. The probe exhibits a useful aggregation-induced emission (AIE) property. A gradual increase in the water content of a THF solution causes a significant decrease and 12 nm red shift in the fluorescence peak at 470 nm, giving rise to a new strong fluorescence peak at 591 nm at a 95% water content. The probe is hydrophobic with an AIE property and binds to cell membranes, resulting in 591 nm fluorescence upon implantation into cells. The probe possesses a long retention time despite the lack of a long, cell membrane-anchored hydrophobic alkyl chain, which is typical for traditional membrane-specific probes. Our probe also displays low cytotoxicity and excellent photostability.

Ratiometric fluorescent probes based on through-bond energy transfer of cyanine donors to near-infrared hemicyanine acceptors for mitochondrial pH detection and monitoring of mitophagy.

Two ratiometric near-infrared fluorescent probes have been developed to selectively detect mitochondrial pH changes based on highly efficient through-bond energy transfer (TBET) from cyanine donors to near-infrared hemicyanine acceptors. The probes consist of identical cyanine donors connected to different hemicyanine acceptors with a spirolactam ring structure linked via a biphenyl linkage. At neutral or basic pH, the probes display only fluorescence of the cyanine donors when they are excited at 520 nm. However, acidic pH conditions trigger spirolactam ring opening, leading to increased π-conjugation of the hemicyanine acceptors, resulting in new near-infrared fluorescence peaks at 740 nm and 780 nm for probes A and B, respectively. This results in ratiometric fluorescence responses of the probes to pH changes indicated by decreases of the donor fluorescence and increases of the acceptor fluorescence under donor excitation at 520 nm due to a highly efficient TBET from the donors to the acceptors. The probes only show cyanine donor fluorescence in alkaline-pH mitochondria. However, the probes show moderate fluorescence decreases of the cyanine donor and considerable fluorescence increases of hemicyanine acceptors during the mitophagy process induced by nutrient starvation or under drug treatment. The probes display rapid, selective, and sensitive responses to pH changes over metal ions, good membrane penetration, good photostability, large pseudo-Stokes shifts, low cytotoxicity, mitochondria-targeting, and mitophagy-tracking capabilities.

Near-infrared fluorescent probes with BODIPY donors and rhodamine and merocyanine acceptors for ratiometric determination of lysosomal pH variance.

Three fluorescent probes have been developed by conjugating three different BODIPY donors to rhodamine and merocyanine acceptors for ratiometric determination of lysosomal pH variations. Probe A consists of a 1,3,5,7-tetramethyl-BODIPY donor and a near-infrared rhodamine acceptor bearing a lysosome-targeting morpholine residue. Probe B is composed of a 3,5-dimethyl-BODIPY donor and a near-infrared rhodamine acceptor modified with an o-phenylenediamine residue. Probe C contains a 3-styrene-functionalized BODIPY donor with longer wavelength emission and a near-infrared merocyanine acceptor containing a morpholine residue. Under neutral or basic pH conditions, the probes only show fluorescence from the BODIPY donors under BODIPY excitation because the rhodamine and merocyanine acceptors maintain closed spirolactam configurations. However, excitation at BODIPY absorption wavelengths concomitant with gradual pH decrease results in fluorescence decreases with the BODIPY donors and fluorescence increases from the rhodamine and merocyanine acceptors due to through-bond energy transfer from the donors to the acceptors. This is because the spirolactam ring opens under more acidic conditions and fluorescence of the acceptors results from significantly improved π-conjugation. These experimental results are substantiated with theoretical calculations on models of the different probes. The probes have all been used to determine lysosome pH variations in HeLa cells. Probe B was further utilized to successfully detect pH fluctuations in HeLa cells under oxidative stress and with treatment of NH4Cl and chloroquine.

Near-infrared fluorescent probes based on TBET and FRET rhodamine acceptors with different pK a values for sensitive ratiometric visualization of pH changes in live cells.

Three near-infrared ratiometric fluorescent probes (A-C) based on TBET and FRET near-infrared rhodamine acceptors with different pK a values were designed and synthesized to achieve sensitive ratiometric visualization of pH variations in lysosomes in visible and near-infrared channels. Tetraphenylethene (TPE) was bonded to near-infrared rhodamine dyes through short electrical π -conjugation linkers to prevent an aggregation-caused quenching (ACQ) effect and allow highly efficient energy transfer of up to 98.9% from TPE donors to rhodamine acceptors. Probes A-C respond to pH variation from 7.4 to 3.0 in both buffer solutions and live cells with significant decreases of donor fluorescence and concomitant extraordinary increases of rhodamine acceptor fluorescence because of highly efficient energy transfer. In addition, probe C is capable of determining pH fluctuations in live cells treated with chloroquine. The probes show good photostability, excellent cell membrane permeability, high selectivity to pH, and two well-resolved emission peaks to ensure accurately comparative and quantitative analyses of intracellular pH changes.

Fluorescent probes with high pKa values based on traditional, near-infrared rhodamine, and hemicyanine fluorophores for sensitive detection of lysosomal pH variations.

Sterically hindered fluorescent probes (A-C) have been developed by introducing 2-aminophenylboronic acid pinacol ester to a traditional, A, a near-infrared rhodamine dye, B, and a near-infrared hemicyanine dye, C, forming closed spirolactam ring structures. Probe A was non-fluorescent under basic pH conditions whereas probes B and C were moderately fluorescent with fluorescence quantum yields of 9% and 5% in pH 7.4 PBS buffer containing 1% ethanol, respectively. With all probes increasing acidity leads to significant increases in fluorescence at 580 nm, 644 and 744 nm for probes A, B and C with fluorescence quantum yields of 26%, 21% and 10% in pH 4.5 PBS buffer containing 1% ethanol, respectively. Probes A, B and C were calculated to have pKa values of 5.81, 5.45 and 6.97. The difference in fluorescence under basic conditions is ascribed to easier opening of the closed spirolactam ring configurations due to significant steric hindrance between the 2-aminophenylboronic acid pinacol ester residue and an adjacent H atom in the xanthene derivative moiety in probe B or C. The probes show fast, reversible, selective and sensitive fluorescence responses to pH changes, and are capable of sensing lysosomal pH variations in living cells.

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria.

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.