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.     

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 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.     

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     

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking,Ratiometric pH Sensing,and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pK_a, off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.     

A naphthalimide-rhodamine ratiometric fluorescent probe for Hg based on fluorescence resonance energy transfer

On the basis of fluorescent resonance energy transfer from 1,8-naphthalimide to rhodamine B, a new fluorophore dyads (4) containing rhodamine B and a naphthalimide moiety was synthesized as a ratiometric fluorescent probe for detecting Hg²⁺ with a broad pH range 5.7-11.0. The selective fluorescence response of 4 to Hg²⁺ is due to the Hg²⁺-promoted desulfurization of the thiocarbonyl moiety, leading to the ring-opening of rhodamine B moiety of 4. When 4 was employed at 0.1 μM with the slit size being 20 nm/20 nm, a low level of Hg²⁺ (up to 3 × 10⁻⁸ M) can be detected using the system.     

FRET-mediated pH-responsive dual fluorescent nanoparticles prepared via click chemistry

Herein, we report an easy preparation of azide-coated polystyrene-based nanoparticles (15 nm in diameter) and their surface functionalization via CuAAC with fluorophores in water. Resultant dual fluorescent nanoparticles coated with dansyl and pH-sensitive fluorescein moieties as the donor/acceptor FRET pair show a ratiometric response to pH upon excitation at a single wavelength.     

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.     

Broadband luminescence and emission enhancement

Three novel quinoline– and fluorene–coumarin hybrids were prepared and characterised by Fourier Transform–infrared spectroscopy, proton and carbon nuclear magnetic resonance spectroscopy, mass spectrometry, elemental analysis and ultraviolet–visible spectrophotometry. Their photoluminescence properties were investigated. All of these hybrid molecules exhibited a broadband emission from ca. 450 to 800 nm when excited by a 325 nm helium–cadmium laser at room temperature. Importantly, the fluorene–coumarin hybrid shows a very bright red emission at ca. 650 nm and displays up to a 10‐fold increase in fluorescence emission intensity, as compared with the other samples. The experimental results confirm that remarkable enhancements in the fluorescence emission intensity can be obtained by introducing a fluorene group into the azo‐bridged coumarin‐based π‐conjugated framework. The geometry structures and frontier orbitals are calculated by the ab initio Hartree–Fock method.     

甘氨酸-氟硼荧近红外荧光分子探针的合成及性能测试

将一种具有近红外荧光性能的氟硼荧染料（BDDIPY）作为显影基团,制成新型的检测肿瘤细胞的近红外荧光分子探针。首先以氟硼二吡咯烷（BOD）为起始原料,通过缩合反应,将具有强供电子作用的N-苯基亚氨基二苄引入到氟硼荧中,得到了荧光光谱处于近红外区的氟硼荧化合物,接着用N-羟基琥珀酰亚胺为活化剂,将具有肿瘤亲和力的甘氨酸引入到荧光基团-氟硼荧（BDDIPY）,最终得到甘氨酸-氟硼荧近红外荧光探针,并对其结构进行了核磁和质谱表征,并进行了光学和生物学性能测试。实验结果显示：甘氨酸-氟硼荧近红外荧光分子探针的荧光发射波长大于700 nm,与肺癌细胞（GLC-82）具有较好的亲和力,达到23.65,是一个具有良好应用前景的检测肿瘤细胞的近红外荧光分子探针。     

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%).     

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.     

The synthesis of a novel 1,8-naphthalimide based PAMAM-type dendron and its potential for light-harvesting

The photophysical characteristics of a PAMAM-type dendron that utilizes a 4-ethoxy-1,8-naphthalimide donor and 4-piperidine-1,8-naphthalimide acceptor in nine organic solvents of different polarities were investigated using absorption and fluorescent spectroscopy. The 1,8-naphthalimide acceptor can be indirectly excited by the donor 1,8-naphthalimide at 372 nm whereby the only emission observed is that due to the acceptor at 517 nm; no acceptor emission at 435 nm was observed. The efficiency of energy transfer was ～98%.     

The synthesis,photophysical properties and energy transfer of a coumarin-based bichromophoric compound

The energy transfer between two coumarin compounds having donor emission/acceptor absorption spectra overlap was studied firstly in solution by applying an excess of the acceptor. The observed transfer was then confirmed in the bichromophoric compound resulting from the covalent binding of the two fluorophores via a short alkyl chain.     

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway.     

Synthesis and characterization of novel hyperbranched fluorescent polymers that can be precisely used for metal ion detection and quantification

Four novel hyperbranched polymers with 4 or 5 ring-closed rhodamine units in each were achieved through RAFT polymerization followed by modification with rhodamine moieties. The solubility, thermostability, and photophysical properties of the polymers were studied. The polymers showed high selectivity and sensitivity to Fe³⁺ among various metal ions in CH₃CN/H₂O (75/25, v/v) and could signal Fe³⁺ through multichannels: emerging a new absorption around 558 nm, over 30 nm fluorescence redshift and significant fluorescence enhancement (including 33–37 folds in intensity and 8.3–12.8 folds in quantum yield), accompanied by visual and fluorescent color changes. The polymers could be applied in the analysis of Fe³⁺ in real water samples. © 2020 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48933.     

Cytosine detection by a fluorescein-labeled probe containing base-discriminating fluorescent nucleobase

We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base-discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine ((ND)A). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of (ND)A showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of (ND)A was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of (ND)A overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400-500 nm. Thus, we designed FRET-BDF probes containing (ND)A as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET-BDF probes was observed only when the complementary base of (ND)A is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the (ND)A fluorescence by a G/C base pair flanking (ND)A.     

Subpicosecond Spectroscopy of Charge Separation in Rhodobacter capsulatus Reaction Centers

The time-evolution of the near-infrared absorption changes accompanying the conversion of the excited primary electron donor, P*, to the radical pair state, P⁺BPh_L⁻, has been examined in detail in Rhodobacter capsulatus reaction centers. In a series of spectra spanning the time interval from about 600 fs to 15 ps, two isosbestic points occurring at 765 run and 798 nm are maintained throughout. The finding of an isosbestic point at 798 nm in this series of spectra, which encompass the time during which P* decays and BPh_L⁻ forms, places severe constraints on the possible reduction of the 800-nm-absorbing monomeric BChl_L in the initial stage of the charge-separation process. The near-infrared P* spectrum, which is revealed clearly only when P is excited directly in its long-wavelength absorption band, contains bleaching only of the 855-nm band and a broad featureless transient absorption below 810 nm. This spectrum shows that P does not contribute any significant oscillator strength to the 800-nm ground state absorption band.     

Embedding carbon dots in Eu3+-doped metal-organic framework for label-free ratiometric fluorescence detection of Fe3+ ions

Fluorescent detection is a new spectroscopic measurement for ions sensing due to the advantages of real-time determination with high selectivity, accuracy, and low cost. However, chemosensors based on fluorescent detection are usually determined by absolute intensity from a monochromatic emission signal, which is easy to be fluctuated by the external environment, especially for Fe³⁺ detection in complex fluids. Herein, we rationally design a dual-emission Eu³⁺: CDs@ZIF-8 to construct a ratiometric fluorescent sensor with self-calibrating ability for Fe³⁺ determination. High efficient carbon dots (CDs) are embedded in europium ions (Eu³⁺)-doped MOF by simple stirring preparation at room temperature. The label-free ratiometric fluorescent probe (I_CDs@ZIF-8/I_Eu) exhibits simultaneous blue and red emission under the same excitation at 365 nm. Remarkably, Eu³⁺: CDs@ZIF-8 displays the superiority of high selectivity to Fe³⁺, which shows ratiometric fluorescence characteristics (I₀/I) in a range of 0-6 μmol\L with a low limit of detection (LOD) of 0.897 μmol\L. Besides, the CDs-MOF nanocomposite holds good aqueous dispersibility and low cytotoxicity, which shows great potential applications in medical aid including biological detection and clinical diagnosis.