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.     

A novel coumarin‐based fluorescent probe for selective detection of cysteine over homocysteine

A coumarin‐based fluorescent probe containing both acrylate moiety and an allyl substituent was developed for distinguishing cysteine from homocysteine. The maximum absorption wavelength of the probe showed a bathochromic shift upon addition of cysteine and the colour changed from colourless to yellow. The maximum fluorescence emission at 462 nm of the probe was dramatically enhanced upon addition of cysteine in aqueous solution. In HEPES buffer (pH = 7.4, EtOH/H₂O = 9:1, v/v), both of the fluorescence emission bands of the probe at ca. 390 and 462 nm were intensely enhanced upon the addition of cysteine, while other amino acids, namely, homocysteine, asparaginic acid, methionine, glycine, phenylalaninase, valine, tryptophan, serine, glutamic acid and glutathione, did not cause a remarkable change. The probe can be used for selectively colorimetric and ratiometric fluorescent detection of cysteine over homocysteine and other common amino acids in both organic media and aqueous solution. The concentration of cysteine can be estimated by titration of the probe in aqueous solution, and the detection limit of the probe towards cysteine was 2 μmol/L.     

A fluorescence resonance energy transfer probe for sensing pH in aqueous solution

A fluorescence resonance energy transfer (FRET) sensor, which has a FRET donor and an acceptor attached to each chain end of pH-sensitive polysulfonamide, is synthesized and its pH sensitivity is examined in terms of the FRET efficiency. This polymeric sensor exhibits an instantaneous conformation change from coil to globule at a specific pH, which results in the drastic on-and-off FRET efficiency. To detect a specific pH region, sulfadimethoxine and sulfamethizole are selected among various sulfonamides since their pK_a values are in the physiological pH. For tuning the emission color arising from FRET, 7-hydroxy-4-bromomethyl coumarin and coumarin 343 are used as a FRET donor and an acceptor, respectively, for a blue-to-green FRET sensor, and fluorescence amine isomer I and rhodamine B are used for a green-to-red FRET sensor. Each sensor shows a distinct color change from the emission wavelength of FRET donor to the emission wavelength of FRET acceptor, which well explains their feasibility as a useful optical sensor.     

A Coumarin‐Labeled Vinyl Sulfone as Tripeptidomimetic Activity‐Based Probe for Cysteine Cathepsins

A coumarin‐tetrahydroquinoline hydride 8 was synthesized as a chemical tool for fluorescent labeling. The rigidified tricyclic coumarin structure was chosen for its suitable fluorescence properties. The connection of 8 with a vinyl sulfone building block was accomplished by convergent synthesis thereby leading to the coumarin‐based, tripeptidomimetic activity‐based probe 10 , containing a Gly‐Phe‐Gly motif. Probe 10 was evaluated as inactivator of the therapeutically relevant human cysteine cathepsins S, L, K, and B: it showed particularly strong inactivation of cathepsin S. The detection of recombinant and native cathepsin S was demonstrated by applying 10 to in‐gel fluorescence imaging.     

A fluorescent probe for the dual detection of mercury ions and thiols based on a simple coumarin derivative

A fluorescent probe based on a simple coumarin derivative could recognise mercury ions (Hg(ii )) and thiols selectively in aqueous solution. The addition of Hg(ii ) induced a blue shift of the fluorescence emission band of the probe while the fluorescence was almost quenched by the addition of p‐toluenethiol or cysteine (Cys). The detection limit of the probe towards Hg(ii ) and Cys was 8 µmol/L. The probe could be used for the detection of Hg(ii ) and thiols by the naked eye under ultraviolet light irradiation.     

Thermochromic microgels and core‐shell microgels based on fluorescence resonance energy transfer

Microgels exhibiting thermochromic behavior based on fluorescence resonance energy transfer (FRET) are prepared. The FRET microgels consist of poly(N‐isopropylacrylamide) (PNiPAm) networks with fixed fluorescein and rhodamine moieties and exhibits volume phase transition (VPT) at 34–35°C. A critical decrease in their sizes during the VPT enhances the efficiency of FRET between fluorescein as a donor and rhodamine as an acceptor. Therefore, emission from fluorescein (523 nm) and that of rhodamine (579 nm) is dominant at temperatures below and above the VPT temperature, respectively, when fluorescein is excited. We also prepare thermochromic core‐shell FRET microgel exhibiting two‐step color change. The microgels consist of a PNiPAm core and a poly(N‐isopropylacrylamide‐co‐N,N‐diethylacrylamide) shell and exhibit dual temperate‐responsiveness at 19 and 33°C. The fluorescence spectrum of the microgels also changes in two steps at these temperatures. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Detecting Cysteine in Bioimaging with a Near-Infrared Probe Based on a Novel Fluorescence Quenching Mechanism

Near-infrared (NIR) fluorescent probes are very significant for detecting cysteine in biological systems. Herein, we report a highly selective and sensitive NIR turn-on fluorescent probe (BDP-NIR) based on BODIPY with large Stokes shift (105 nm) for detecting Cys. We clarified the sensing mechanism based on the different thiol-induced S_NAr substitution/rearrangement reaction of the probe with cysteine and homocysteine/glutathione, which leads to the corresponding amino- and thiol-BODIPY dyes with distinct photophysical properties. Moreover, a novel mechanism of fluorescence quenching was demonstrated by density functional theory calculation. The reason for the fluorescence quenching of the probe might be intersystem crossing (from singlet to triplet excited state). Moreover, BDP-NIR had a high linear dynamic range of 0–500 μM, which was promising for detecting cysteine quantificationally. Significantly, BDP-NIR was capable of sensing endogenous cysteine in living cells and in vivo.     

Fluorescence properties of fluorescein and rhodamine supported on alumina nanowire films

In this paper, alumina nanopore array films and nanowire films were prepared by anodic oxidation method, and fluorescein and rhodamine molecules were loaded onto their surfaces to fabricate composite fluorescent films. The effects of morphologies and annealing temperatures of alumina films on the fluorescence properties of the composite films were investigated. When loaded on alumina nanowire films, the properties of rhodamine B (RB), fluorescein (FL), rhodamine 110 (R110), rhodamine 6G (R6G) and the FRET (fluorescence resonance energy transfer) systems they constituted were studied in detail. Results show that the fluorescence emission of RB is stronger on alumina nanowire films than on alumina nanopore array films. The molecular structure of donor and acceptor can affect the energy transfer efficiency of FRET system (E), the fluorescence emission enhancement multiple of acceptor (I_A(DA)/I_A) and the transferred energy utilization factor (η). Compared to the systems of FL-R110, FL-R6G, R110-R6G, R110-RB and R6G-RB, FL-RB has the best comprehensive performance with the largest I_A(DA)/I_A (4.90) and η (6.42), despite its medium E (76.3%).     

Fluorescence enhancement of coumarin–quinoline by transition metal ions: Detection of paramagnetic Ni2+ and Co2+

A readily available coumarin–quinoline was employed as a novel fluorescent probe for paramagnetic Co²⁺ and Ni²⁺ ions, which are well-known fluorescence quenchers. NMR and IR indicated that the enhanced fluorescence response was attributable to coordination of the paramagnetic Ni²⁺ or Co²⁺ ions with the coumarin–quinoline compound. Given the difficulty of designing enhanced fluorescent probes for paramagnetic Co²⁺ and Ni²⁺ ions, the coumarin–quinoline compound may inspire the further development of more sophisticated sensing constructs for the fluorescence detection of these transition metal ions.     

Emission under hypoxia: one-electron reduction and fluorescence characteristics of an indolequinone-coumarin conjugate

A characteristic feature of the reactivity of indolequinone derivatives, substituents of which can be removed by one-electron reduction under hypoxic conditions, was applied to the development of a new class of fluorescent probes for disease-relevant hypoxia. A reducing indolequinone parent molecule conjugated with fluorescent coumarin chromophores could suppress efficiently the fluorescence emission of the coumarin moieties by an intramolecular electron-transfer quenching mechanism and a conventional internal-filter effect. Under hypoxic conditions, however, the conjugate, denoted IQ-Cou, underwent a one-electron reduction triggered by X irradiation or the action of a reduction enzyme to release a fluorescent coumarin chromophore, whereupon an intense fluorescence emission with a maximum intensity at 420 nm was observed. The one-electron reduction of IQ-Cou was suppressed by molecular oxygen under aerobic conditions. IQ-Cou also showed intense fluorescence in a hypoxia-selective manner upon incubation with a cell lysate of the human fibrosarcoma cell line HT-1080. The IQ-Cou conjugate has several unique properties that are favorable for a fluorescent probe of hypoxia-specific imaging.     

新型香豆素类Hg^2+荧光探针的合成及性能研究

以4-二乙胺基水杨醛、三氟乙酰乙酸乙酯以及2-噻吩甲酰肼为原料合成了新型含氟香豆素类硫代酰腙型荧光探针化合物,经1HNMR、13CNMR、19FNMR、HR-MS对化合物结构进行了确认。通过荧光光谱法研究了化合物在乙腈溶液中对金属离子的识别性能。在pH 4.5~9.5的范围内,Hg^2+的加入使得化合物在464 nm处的荧光发射峰发生猝灭,其他金属离子的存在未对该现象造成明显干扰。Hg^2+浓度越高,猝灭效果越明显。因此,化合物对Hg^2+具有良好的荧光选择性和抗干扰性。Job′s曲线表明,化合物与Hg^2+的配合比为1∶1。     

A novel rhodamine-based fluorescent and colorimetric “off-on” chemosensor and investigation of the recognizing behavior towards Fe

A novel rhodamine based probe has been synthesized as an “off-on” chemosensor for Fe³⁺. Upon coordination with Fe³⁺, the probe displayed good brightness and fluorescent enhancement. A linear relationship was observed to exist between the relative fluorescence intensity of this probe and the concentration of Fe³⁺ in the range of 5 μM-20 μM with a detection limit of 5 μM. It offered highly sensitive toward Fe³⁺ over other ions. The recognizing behaviors toward Fe³⁺ have been investigated both experimentally and computationally. It can be expected that Fe³⁺ coordinated with the N atom of thiazole moiety in the probe accompanied by the transferring of electrons of the phenylthiazole resulted in the opening of the spiro-ring.     

Molecular diffusion in ternary poly(vinyl alcohol) solutions

The diffusion kinetics of a molecular probe—rhodamine B—in ternary aqueous solutions containing poly(vinyl alcohol), glycerol, and surfactants was investigated using fluorescence correlation spectroscopy and dynamic light scattering. We show that the diffusion characteristics of rhodamine B in such complex systems is determined by a synergistic effect of molecular crowding and intermolecular interactions between chemical species. The presence of glycerol has no noticeable impact on rhodamine B diffusion at low concentration, but significantly slows down the diffusion of rhodamine B above 3.9% (w/v) due to a dominating steric inhibition effect. Furthermore, introducing surfactants (cationic/nonionic/anionic) to the system results in a decreased diffusion coefficient of the molecular probe. In solutions containing nonionic surfactant, this can be explained by an increased crowding effect. For ternary poly(vinyl alcohol) solutions containing cationic or anionic surfactant, surfactant–polymer and surfactant–rhodamine B interactions alongside the crowding effect of the molecules slow down the overall diffusivity of rhodamine B. The results advance our insight of molecular migration in a broad range of industrial complex formulations that incorporate multiple compounds, and highlight the importance of selecting the appropriate additives and surfactants in formulated products.     

Synthesis and fluorescence properties of polystyrene via atom transfer radical polymerization using a coumarin derivate as initiator

Well-defined polystyrene was successfully synthesized via atom transfer radical polymerization, using a novel coumarin derivate, 2-bromo-2-methyl-propionic acid 4-methyl-2-oxo-2H-chromen-7-yl ester (BMP), synthesized in our lab as an initiator, and CuBr/N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) as the catalyst at 110 °C. The kinetics of the polymerization was studied in detail. The fluorescence properties of the resultant polystyrenes were studied in N,N-dimethylformamide (DMF). It is very interesting that the adding of Fe³⁺ has significant effect on the fluorescence intensities, which decreased with increasing the concentration of iron in solutions. The obtained polystyrenes can be used as fluorescent probe molecules for Fe³⁺.     

Upconversion nanoparticles for sensitive and in-depth detection of Cu(2+) ions

Detection of Cu(2+) ions and study of their subcellular distribution in physiological processes are of considerable significance because of their potential environmental and biological applications. Some fluorescence based sensors have been developed for selective detection of Cu(2+) ions, based on organic fluorescent probes that specifically bind to Cu(2+) ions. However, these sensors are not suitable for detection in biological samples due to the short penetration depth of UV/visible light used to excite the fluorescent probes. The use of near-infrared (NIR) light can afford penetration depths of an order of magnitude greater than that of visible light, however, a material that can convert NIR light to visible light is required. A facile method has been developed for in-depth detection of Cu(2+) ions based on fluorescence upconversion. A mesoporous silica shell is coated on upconversion nanoparticles (UCNPs) and a Cu(2+) ion sensitive fluorescent probe, rhodamine B hydrazide, is incorporated into the mesoporous silica. Upon excitation by a NIR light, the UCNPs emit visible light to excite the Cu(2+)-sensitive fluorescent probe. Because of the unique optical properties of UCNPs and their ability to convert NIR light to visible light, this is a feasible method for sensitive and in-depth detection of Cu(2+) ions in a complex biological or environmental sample due to the low autofluorescence and the high penetration depth of NIR light.     

Serum-Based Detection of Liver Pathology Using a Fluorogenic Alkaline Phosphatase Probe

Development of “ultrahigh contrast” fluorogenic probes for trapping alkaline phosphatase (ALP) activities in human serum is highly desirable for clinical auxiliary diagnosis for hepatobiliary diseases. However, the intrinsic dilemma of incomplete ionization of intramolecular charge transfer (ICT)-based ALP fluorophores and autofluorescence interference of serum result in low sensitivity and accuracy. Given that unique halogen effects could lead to a drastic decrease in the pKa value and a significant enhancement in the fluorescence quantum yield, herein we report an enzyme-activatable near-infrared probe based on a difluoro-substituted dicyanomethylene-4H-chromenep for achieving fluorescent quantification of human serum ALP. Rational design strategy is demonstrated by altering the substituted halogen groups to well regulate the pKa for meeting the physiological precondition. Owing to the complete ionization at pH 7.4 with tremendous fluorescence enhancement, the difluoro-substituted DCM-2F-HP manifests a linear relationship between the emission intensity and ALP concentration in both solution and serum samples. Along with measuring 77 human serum samples, the DCM-2F-HP based fluorescence method not only exhibits significant correlations with clinical colorimetry, but also distinguishes ALP patients from healthy volunteers, as well as assessing the progress of liver disease, thus providing a potential toolbox for quantitatively detecting ALP and warning the stage of hepatopathy.     

Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin 3

Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live-cell fluorogenic labeling of the photoactive yellow protein (PYP)-tag. On the basis of the photochemical mechanisms of coumarin and the probe–tag interactions, we introduced a hydroxy group into an environment-sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF–ON ratio than any previously developed PYP-tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high-contrast imaging enabled by our probe.     

Hydrogen-Bond Dynamics and Solvation of Electronically Excited States as Determined by Femtosecond Vibrational Spectroscopy

The ultrafast dynamics of site-specific hydrogen bonds between an organic chromophore serving as hydrogen acceptor and a hydrogen-donating species in solution is studied by femtosecond vibrational spectroscopy. This new method gives specific insight into microscopic structural changes of hydrogen bonds initiated by electronic excitation of the chromophore. We study H-bonded complexes of coumarin 102 with the solvent CHCl₃, and with phenol. Upon electronic excitation, the intermolecular hydrogen bond between proton donor and acceptor is cleaved within 200 fs, followed by a slower reorientation dynamics of the donor molecules extending into the picosecond regime. For CHCl₃, this slower rearrangement is interpreted in terms of polar solvation. In complexes consisting of a coumarin molecule and two phenol moieties, the slower dynamics is related to geometry changes of the phenol–phenol hydrogen bond. The consequences of those results for a microscopic picture of polar solvation are discussed.     

A novel near-infrared fluorescence sensor for detection of thrombin activation in blood

Thrombosis is an important pathophysiologic component of many cardiovascular diseases. Thrombin, a serine protease, plays a central role in thrombosis formation. Detection and imaging of thrombin activity may thus be of considerable biomedical interest. The goal of this study was to design, synthesize, and characterize a novel thrombin-activated near-infrared fluorescence (NIRF) probe. The probe consisted of a thrombin-cleavable peptide spacer (...D-Phe-Pip-Arg...; Pip=pipecolic acid) and contained a terminal fluorescence reporter which was quenched when conjugated to a biocompatible delivery vehicle. A control peptide spacer was synthesized that differed by one amino acid. Following thrombin addition, the probe was activated within minutes. The NIRF signal increased by a factor of 27-fold within 20 minutes, and was inhibited by hirudin, a specific thrombin inhibitor. NIRF optical imaging experiments confirmed rapid activation of the probe in both buffer and human blood. The control probe showed minimal activation in all experiments. In addition to potentially furthering our understanding of thrombin regulation in vivo, the thrombin-activated near-infrared probe may have broad clinical application to the diagnosis of arterial and venous thrombosis.     

Polymer concentration detection method based on fluorescent polymer to evaluate its retention and percolation

A new fluorescent polymer was prepared by copolymerization of acrylamide and a modified rhodamine B (RhB). Fourier transformation infrared spectrometer and nuclear magnetic resonance confirmed the successful modification of RhB and the synthesis of fluorescent polymer. The rheological and fluorescence properties were studied by fluorospectrophotometer and rotational rheometer. The adsorption retention and percolation properties were evaluated by sand pack displacement experiments. The results indicated that fluorescence intensity revealed a distinct linear relationship with polymer concentration within a certain concentration range. High temperature and strong acidic conditions could diminish fluorescence intensity whereas formation water ions exerted inconsiderable influence on it. Rheological results manifested that fluorescent polymer can be used as polymer flooding agent. The sand pack displacement results proved the feasibility of using fluorescent polymer to detect polymer concentration. By this means, polymer breakthrough time and concentration variations could be confirmed. Based on the curves of injection pressure and polymer concentration, the percolation state and retention of polymer were able to be analyzed. Significantly, this work can provide support and guidance for the practical applications of fluorescent polymer. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47468.