Nanosecond Time-resolved Fluorescence Research Articles

Ultrafast proton coupled electron transfer between tryptophan and tyrosine in peptides Trp-Pron-Tyr

A series of model peptides (Trp-Pron-Tyr, WPnY, n=O, 1, 2, 3, 5), which contain tryptophan (Trp, W), tyrosine (Tyr, Y), and proline (Pro, P), have been studied under three typical pH conditions (3, 7, and 10) by steady-state absorption and fluorescence spectroscopy, nanosecond time-resolved fluorescence and femtosecond time-resolved transient absorption spectroscopy. When the peptide’s chain length is increased, Trp fluorescence quenching is expected to be gradually weakened. However, Trp fluorescence in WPY is strongly quenched and reveals even stronger quenching with increasing pH values, whose hypochromicity is clearly different from other model peptides. Transient absorption spectra also demonstrate that the excited state decay of WPY is much faster than that of other model peptides, especially at pH = 10. It is attributed to the efficient proton coupled electron transfer (PCET) between Trp and Tyr. Moreover, due to the very short distance between Trp and Tyr in WPY, this PCET process could be achieved by “direct transfer”, contrasted with the slow and long-range PCET process in other model peptides. Our results of the dipeptides WY and WP further suggest that Trp may also have more complex interactions with the peptide backbone or proline in those peptides. This work provides an experimental evidence for the electron transfer mechanism in WY dyads, which can help ones to understand how to reduce Trp radicals in proteins.

Interaction of Cucurbit[7]uril With Protease Substrates: Application to Nanosecond Time-Resolved Fluorescence Assays.

We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a “dark quencher.” The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.

Retention of CdS/ZnS Quantum Dots (QDs) on the Root Epidermis of Woody Plant and Its Implications by Benzo[a]pyrene: Evidence from the in Situ Synchronous Nanosecond Time-Resolved Fluorescence Spectra Method.

The retention of CdS/ZnS QDs on the epidermis has been confirmed to be one of the core procedures during the root uptake process. However, the retention mechanisms of QDs on the epidermis of woody plant were poorly understood for lacking of an appropriate QD quantitative method. In this study, a novel method for in situ determination of CdS/ZnS QDs retained on the root epidermis was established using synchronous nanosecond time-resolved fluorescence spectroscopy. No correlations between Kf values of oleylamine-CdS/ZnS QDs retained on the epidermal tissues and the surface/bulk composition of mangrove root were observed (p > 0.05) due to the existence of endocytosis mechanisms during the QD uptake processes. Moreover, the difference of the CdS/ZnS QDs in water and further translocated to xylem/phloem of root rather than the combination with cell wall/membranes was the predominant reason that caused the Kf values to follow the sequence of PEG-COOH-CdS/ZnS QDs < PEG-NH2-CdS/ZnS QDs ≪ oleylamine-CdS/ZnS QDs.

Sensitive detection of intracellular environment of normal and cancer cells by autofluorescence lifetime imaging

Intracellular fluorescence lifetime images of the endogenous fluorophores of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are well known as autofluorescence chromophores, were obtained from rat normal fibroblast cells (WFB) and H-ras oncogene-transfected cancer cells among WFB (W31). The average lifetime of the NADH and FAD autofluorescence was shorter in cancer cells than in normal cells, indicating that the difference in metabolism between healthy and cancer cells alters the conditions for coenzymes such as NADH and FAD and that the autofluorescence lifetime measurement of NADH and FAD is applicable for the noninvasive diagnosis of cancer cells. The pico- and nano-second time-resolved fluorescence spectra of NADH obtained with different time windows were similar in normal and cancer cells, indicating that every fluorescence decay component gives the same spectrum in both cell types. These results as well as the fluorescence lifetime images of exogenous fluorophores stained with sodium pheophorbide a in normal and cancer cells suggest that the difference in the fluorescence lifetime between normal and cancer cells cannot be attributed to a difference in the intracellular pH or refractive index but to the difference in the bound condition between proteins and NADH or FAD under the different intracellular environments of normal and cancer cells.

Host-guest interaction of cytidine in β-cyclodextrin microcavity: Characterization and docking study

Cytidine plays key roles in phosphoinositide signaling and the synthesis of lipids. Thus, in this research paper, we describe a study on the inclusion complex formation between cytidine and beta-cyclodextrin (β-CD). The formation of 1:1 a stoichiometry ratio of host-guest inclusion complex of Cytidine (CY) with a nano hydrophobic cavity of Beta-cyclodextrin (β-CD) in the solution phase is studied by a UV–visible spectrophotometer, fluorescence spectroscopy and time-resolved fluorescence. The binding constant and the stoichiometric ratio of inclusion complex have been calculated by using Benesi-Hildebrand plot. And by using the binding constant values, the thermodynamic parameter, (ΔG) is also calculated, which represents the inclusion process is spontaneous at 303K. A nanosecond time-resolved fluorescence study established that CY exhibits in bi-exponential decay in an aqueous medium and tri-exponential decay in the β-CD medium indicating the formation of a 1:1 inclusion complex. The host–guest solid inclusion complex of Cytidine (CY) with beta-cyclodextrin (β-CD) is prepared by physical mixture, kneading method and co-precipitation method and characterized by using Fourier transform infrared radiation, Thermo gravimetric analyses, differential scanning calorimetry and X-ray diffraction. A part of the CY molecule is entrapped in the β-CD microcavity confirmed by the spectral shifts. The most possible structure of the inclusion complex of CY and β-CD is confirmed by a molecular docking test.

In situ investigation into surfactant effects on the clearance of polycyclic aromatic hydrocarbons adsorbed onto soybean leaf surfaces

The partitioning of polycyclic aromatic hydrocarbons (PAHs) in agricultural crop leaves, contributes to the exposure of organisms to these chemicals through the dietary pathway. To precisely predict the fate of PAHs and crop safety, the clearance of three-ringed phenanthrene (Phe) and four-ringed pyrene (Pyr) adsorbed individually onto living soybean leaf surfaces, as well as the effects of two surfactants, namely, an ionic surfactant (sodium dodecylbenzene sulfonate, SDBS) and a non-ionic surfactant (polyoxyethyleneglycol dodecyl ether, Brij35), were investigated in situ using the laser-induced nanosecond time-resolved fluorescence (LITRF) method. The effects varied significantly with surfactant types primarily in terms of the elimination rates and the final residues of PAH chemicals. With increasing SDBS and Brij35 concentrations, volatilization rate constants (kC) of both Phe and Pyr initially decreased at fast rates and then at more moderate rates later on, resulting from the plasticizing effect of surfactants adsorbed on leaf surfaces. In addition, the photolysis rate constants (kP) decreased with the presence of SDBS but increased with the presence of Brij35. Overall, the total clearance rates of PAHs (kT) adsorbed onto living soybean leaf surfaces were inhibited by the presence of SDBS but promoted by the presence of Brij35. These observations show that surfactants may significantly alter the clearance of PAHs in agricultural systems, and the potential impact of surfactants on crop safety is closely related to surfactant types in natural environments.

Fluorescence of A100 MOF and Adsorption of Water, Indole, and Naphthalene on A100 by the Spectroscopic, Kinetic, and DFT Studies

Metal–organic frameworks (MOFs) are promising materials for adsorption and separations. It is important to understand the details of chemical bonding between the adsorbate and structural units in the MOFs. In A100 MOF, the near-UV–visible fluorescence is found to be the intralinker fluorescence. Naphthalene and indole form the stoichiometric “host-guest” π–π adsorption complexes with A100 that contain one adsorbate molecule per two BDC linkers, and adsorption of indole causes a strong quenching of the intralinker fluorescence. The excitation wavelength dependent steady-state fluorescence spectra, the nanosecond time-resolved fluorescence spectra, and DFT calculations indicate the strong π–π interactions between adsorbed indole and naphthalene and aromatic ring of the BDC linker, as well as hydrogen bonding between adsorbed indole and COO group of the linker. Activated A100 adsorbs up to four water molecules per BDC linker. Kinetic study of adsorption of naphthalene and indole from n-alkane on hydrated A10...

Fluorescence lifetime plate reader: resolution and precision meet high-throughput.

We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications.

Reversible photoswitching of triplet-triplet annihilation upconversion using dithienylethene photochromic switches.

Reversible photoswitched triplet-triplet annihilation upconversion (TTA UC) was demonstrated with dithienylethene (DTE) derivatives as the photochromic units, 2,6-diiodoBodipy as the triplet photosensitizer, and perylene as the triplet acceptor/emitter. The TTA UC is undisturbed by the open-form DTE but can be switched OFF upon photoirradiation of the mixture of the three components at 254 nm, i.e., by the closed-form DTE. Subsequent visible light irradiation restores the TTA UC. By studying the competitive triplet-state energy-transfer processes with nanosecond time-resolved transient difference absorption and fluorescence spectroscopy, we confirmed that the quenching of the perylene triplet excited state by closed-form DTE is dominant among the four possible quenching processes.

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10−5 s−1). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.

Investigation of inclusion complexes of sulfamerazine with α- and β-cyclodextrins: An experimental and theoretical study

Inclusion complexation behavior and binding ability of sulfamerazine (SMRZ) with α- and β-cyclodextrins (α-CD and β-CD) were investigated. The formation of inclusion complexes are studied by UV–visible, fluorescence, time-resolved fluorescence, 1H NMR, FT-IR, DSC, XRD, SEM, TEM and molecular modeling methods. Both experimental and PM3 results indicated that the SMRZ is partially encapsulated in the CD cavity. The different spectral shifts observed in both the CDs indicate that different types of inclusion complexes are formed. Nanosecond time-resolved fluorescence studies demonstrated that SMRZ exhibit biexponential decay in water and triexponential decay in CD solutions. The resonance of the aromatic protons of SMRZ showed remarkably upfield shift in the complexes suggested that the aniline ring deeply encapsulated in the CD cavity. The amino and amido stretching vibrations at 3483cm−1 and 3379cm−1 respectively are strongly affected in the inclusion complexes. DSC curves for the inclusion complexes exhibited a broad endothermic effect from 106.4°C, 123.8°C and 234.5 6°C for α-CD and 118.2°C and 231.4°C for β-CD. TEM images of both inclusion complexes are forms a nanochain like agglomerated structures with a width ranging from 40nm to 100nm. Thermodynamic parameters and binding affinity of the inclusion complex formation were determined and discussed.

Azo dye/cyclodextrin: New findings of identical nanorods through 2:2 inclusion complexes

Inclusion complexation behavior of 4-aminoazobenzene (AAB) and 4-amino-2,3′-dimethyl azobenzene (GBC, fast grant GBC) with α- and β-cyclodextrins (α-CD, β-CD) is analyzed by scanning electron microscope, transmission electron microscope, Fourier transform infrared spectroscopy, differential scanning calorimetry, powder X-ray diffraction, and proton nuclear magnetic resonance spectroscopy techniques. Transmission electron microscope analysis suggests that identical nanorods formed in AAB/CD inclusion complexes while different dimension nanostructures were observed in GBC/CD inclusion complexes. The nanostructures confirmed that the ratio of 2:2 (guest:host) inclusion complex has been developed to a miniature nanorod. Nanosecond time-resolved fluorescence studies indicated that AAB/GBC have fast life time in water, whereas slow life time in CDs corresponds to a higher-order structure of 2:2 complexes. Thermodynamic parameters and binding affinity of the inclusion complex formation were determined and discussed. van der Waals interactions are mostly responsible for enthalpy-driven complex formation of AAB and GBC with cyclodextrins.

Photodegradation of 4,4-Bis(4-hydroxyphenyl)valeric acid and its inclusion complex with β-cyclodextrin in aqueous solution

Abstract Nanosecond laser flash photolysis and time-resolved fluorescence were used to study photochemistry of 4,4-Bis(4-hydroxyphenyl)valeric acid (diphenolic acid, DPA) and complex of DPA with β-cyclodextrin (DPA-βCD) in aqueous solutions. For both systems the primary photochemical process was found to be photoionization with the formation of a hydrated electron–phenoxyl radical pair. Inclusion of DPA in cyclodextrin cavity leads to the great increase of photoionization and fluorescence quantum yield (from 0.01 to 0.14) as well as fluorescence lifetime (from 0.17 to 2.9 ns) due to decreasing of the quenching rate of the singlet excited state of complexed DPA by solvent molecules.

In situ Determination of Phenanthrene Adsorbed onto Surface of Mangrove Leaves Using a Laser-Induced Nanosecond Time-Resolved Fluorescence System

National Natural Science Foundation of China [21075102, 21177102]; Fundamental Research Funds for the Central Universities, China [CXB2011036]; NFFTBS, China [J1310024]

In situ determination mechanisms for the depuration of polycyclic aromatic hydrocarbons adsorbed onto the leaf surfaces of living mangrove seedlings

To further increase understanding of the mechanisms responsible for air-surface exchange processes, the depuration of adsorbed individual fluorene (Flu), anthracene (Ant), phenanthrene (Phe), fluoranthene (Fla), and pyrene (Pyr) from the leaf surfaces of living Aegiceras corniculatum (Ac) and Kandelia obovata (Ko) seedlings were in situ investigated in real time using laser-induced nanosecond time-resolved fluorescence (LITRF) system. Depuration of the PAHs from the leaf surfaces of the two mangrove seedlings included a rapid and a slow phase, and both of them followed first-order kinetics. Furthermore, significant inter-species and inter-chemical variability existed in terms of the elimination rates and the remaining PAHs residues during the two phases. The rapid phase mainly represented a fast volatilization, of which the volatilization rates moderately correlate with PAH molecular weight, while combined effect of volatilization and photolysis was the dominant mechanism for the slow phase. The retainment of PAHs on the leaf surfaces was associated with the plant species and physicochemical properties of PAHs, especially logKOA.

In situ investigation of the depuration of fluoranthene adsorbed on the leaf surfaces of living mangrove seedlings

A novel approach for the in situ determination of fluoranthene (Fla) that was adsorbed onto the leaf surfaces of Avicennia marina (Am), Kandelia obovata (Ko), Bruguiera gymnorrhiza (Bg), and Aegiceras corniculatum (Ac) was established using laser-induced nanosecond time-resolved fluorescence (LITRF). The detection limits for the in situ determination of the Fla adsorbed onto the Am, Ko, Bg and Ac leaf surfaces were 0.03, 0.14, 0.16 and 0.31ngspot−1, respectively. Using the LITRF method, the depuration of the adsorbed Fla from the leaf surfaces of the four selected mangrove species was investigated in situ. The results showed that the method was very simple and rapid to perform and it was applicable for a real-time in situ survey of the depuration processes of PAHs that were adsorbed onto the mangrove leaves. The depuration of Fla that was adsorbed onto the Am, Ko, Bg and Ac leaf surfaces followed fast and slow phases, both of which varied significantly between the mangrove species in terms of the elimination rate, the remaining Fla residues, and the effect of temperature on the remaining Fla residues on the leaf surfaces. Variations in leaf wax content and the leaf surface roughness among the four mangrove species were responsible for the differences in the rapid phase, while photolysis and penetration into the inner cuticle were dominant mechanisms for the slow depuration.

Low-Temperature Spectroscopic Properties of the Peridinin–Chlorophyll a–Protein (PCP) Complex from the Coral Symbiotic Dinoflagellate Symbiodinium

The spectroscopic properties of the peridinin-chlorophyll a-protein (PCP) from the coral symbiotic dinoflagellate Symbiodinium have been characterized by application of various ultrafast optical spectroscopies including femto- and nanosecond time-resolved absorption and picosecond time-resolved fluorescence (TRF) at 77 K. Excited state properties of peridinin and Chl a and their intramolecular interaction characteristics have been obtained from global fitting analysis and directed kinetic modeling of the data sets and compared to their counterparts known for the PCP from Amphidinium carterae. The lifetimes of the excited state of peridinin show close agreement with those known for the counterpart PCP, demonstrating that molecular interactions have the same characteristics in both complexes. More variances have been recorded for the excited state properties of Chl a including elongation of both the intramolecular energy transfer time between Chl's within the pair in the protein monomer and the excited state lifetime of the long wavelength form of Chl a (terminal acceptor). Kinetic modeling of formation of the peridinin triplet state has shown that the PCP is protected from potential photodamage due to an extremely fast peridinin triplet state formation of kTT = (14.4 ± 2.3) × 10(9) s(-1) ((70 ± 12)(-1) (ps)(-1)) that guarantees instantaneous depletion of Chl a triplets and prevents formation of harmful singlet oxygen ((1)ΔgO2).

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.

In situ Determination of Phenanthrene Adsorbed onto Surface of Mangrove Leaves Using a Laser-Induced Nanosecond Time-Resolved Fluorescence System

Developing new methods for in situ determination of polycyclic aromatic hydrocarbons(PAHs) in plant leaves is of great significance to further achieve the field detection of PAHs in actual leaf samples.A set of laser-induced nanosecond time-resolved fluorescence(LITRF) system was employed to complete the in situ quantitative determination of the phenanthrene(Phe) that was adsorbed onto the leaf surfaces of three kinds of mangrove species,named Kandelia obovata(Ko),Bruguiera gymnorhiza(Bg) and Aegiceras corniculatum(Ac).Experimental results showed that the linear dynamic ranges for the in situ determination of the Phe that was adsorbed onto the leaf surfaces of Ko,Bg and Ac were 2-1400 ng/spot,1-1000 ng/spot and 4-2000 ng/spot,with the detection limits of 0.20,0.14 and 0.42 ng/spot,respectively.The relative standard deviations were less than 6.0%(n=9).The experimental recoveries for the Phe in Ko,Bg,and Ac were 89.6%-108.1%,78.2%-92.4% and 93.2%-112.9%,respectively.Satisfactory results were obtained for the in situ determination of the Phe that was adsorbed onto the surfaces of the lab-exposure mangrove leaf samples contaminated by the Phe.The sensitivity of the established method was much higher than that of the previously established fiber optical fluorimetry,and the linear dynamic range got improved.The LITRF method marked a significant step toward realizing the field detection of PAHs in vegetation.

Radiative exciton recombination dynamics in QD-tagged polystyrene microspheres

Fluorescent polystyrene microspheres are prepared by the incorporation of fluorescent CdSe/CdS core/shell semiconductor nanocrystals (quantum dots, QDs) using the emulsification/solvent evaporation method. The radiative exciton recombination dynamics is investigated by nanosecond time-resolved fluorescence spectroscopy at ambient conditions. The time constants of fast and slow fluorescence decay in QDs, dispersed in toluene, were 3.5 and 17.8 ns, respectively. For the QD-tagged microspheres, the time constants of fast and slow processes were ~2–3 and ~11–12 ns, respectively, and did not depend significantly on the QD-content of the microspheres. The fast decay component could be attributed to the recombination of delocalized exciton in the internal core states, and the slow component was attributed to the localized exciton in the surface states. It was found that the ratio of amplitudes of the fast and slow processes also changed after incorporation of QDs in microspheres. The observed differences in fluorescence decay between non-entrapped QDs and QD-tagged microspheres were probably due to energy transfer between the nanocrystals, which were in close proximity inside the microspheres. The obtained fluorescent QD-tagged microspheres are characterized by the other methods as well, which makes them of value for various applications as optical materials.