Time-resolved Fluorescence Measurements Research Articles

Dual lanthanide-doped complexes: the development of a time-resolved ratiometric fluorescent probe for anthrax biomarker and a paper-based visual sensor

In this work, a novel time-resolved ratiometric fluorescent probe based on dual lanthanide (Tb: terbium, and Eu: europium)-doped complexes (Tb/DPA@SiO2-Eu/GMP) has been designed for detecting anthrax biomarker (dipicolinic acid, DPA), a unique and major component of anthrax spores. In such complexes-based probe, Tb/DPA@SiO2 can serve as a stable reference signal with green fluorescence and Eu/GMP act as a sensitive response signal with red fluorescence for ratiometric fluorescent sensing DPA. Additionally, the probe exhibits long fluorescence lifetime, which can significantly reduce the autofluorescence interferences from biological samples by using time-resolved fluorescence measurement. More significantly, a paper-based visual sensor for DPA has been devised by using filter paper embedded with Tb/DPA@SiO2-Eu/GMP, and we have proved its utility for fluorescent detection of DPA, in which only a handheld UV lamp is used. In the presence of DPA, the paper-based visual sensor, illuminated by a handheld UV lamp, would result in an obvious fluorescence color change from green to red, which can be easily observed with naked eyes. The paper-based visual sensor is stable, portable, disposable, cost-effective and easy-to-use. The feasibility of using a smartphone with easy-to-access color-scanning APP as the detection platform for quantitative scanometric assays has been also demonstrated by coupled with our proposed paper-based visual sensor. This work unveils an effective method for accurate, sensitive and selective monitoring anthrax biomarker with backgroud-free and self-calibrating properties.

Fluorescence quenching of α-tocopherol by graphene dispersed in aqueous surfactant solutions

The fluorescence of α-tocopherol in different solutions of zwitterionic (lauryl sulfobetaine, LSB), non-ionic (polyoxyethylene-23-lauryl ether, Brij L23) and cationic (hexadecyltrimethylammonium bromide, CTAB) surfactants, as well as in graphene (G) dispersions in these surfactants at different weight ratios has been comparatively investigated. A quenching phenomenon of the vitamin fluorescence in the micellar dispersions of G has been found, the effect being more pronounced for dispersions in Brij L23. This surfactant causes the highest degree of G exfoliation and is the most effective in stabilizing G dispersions. The fluorescence intensity ratio, calculated in the absence and in the presence of G, increases linearly with G concentration up to 30mgL−1 for dispersions in 5mM Brij L23 and in 5mM CTAB with and without NaCl. A more prominent fluorescence quenching effect is found at low surfactant concentrations, ascribed to a stronger interaction between α-tocopherol and G when the nanomaterial is less covered by the surfactant. Time-resolved fluorescence measurements indicate a static quenching mechanism between α-tocopherol and G. The fluorescence intensity and α-tocopherol concentration in 5mM Brij L23 aqueous solutions and in G (2.0wt%) dispersions in this surfactant follow a linear relationship, as well as the differences of the intensities measured in each case.

Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure.

Fluorescence spectroscopy, relying on intrinsic protein fluorophores, is one of the most widely used methods for studying protein folding, protein-ligand interactions, and protein dynamics. Tryptophan is usually the fluorophore of choice, given its sensitivity to its environment and having the highest quantum yield of the natural amino acids; however, changes in tryptophan fluorescence can be difficult to interpret in terms of specific structural changes. The introduction of quenchers of tryptophan fluorescence can provide information about specific structures, particularly if quenching is short-range; however, the most commonly employed quencher is histidine, and it is effective only when the imidazole side chain is protonated, thus limiting the pH range over which this approach can be employed. In addition, histidine is not always a conservative substitution and is likely to be destabilizing if inserted into the hydrophobic core of proteins. Here we illustrate the use of a Trp-selenomethionine (MSe) pair as a specific probe of protein structure. MSe requires a close approach to Trp to quench its fluorescence, and this effect can be exploited to design specific probes of α-helix and β-sheet formation. The approach is illustrated using equilibrium and time-resolved fluorescence measurements of designed peptides and globular proteins. MSe is easily incorporated into proteins and provides a conservative replacement for hydrophobic side chains, and MSe quenching of Trp fluorescence is pH-independent. The oxidized form of MSe, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.

A Spectroscopic and Molecular Simulation Approach toward the Binding Affinity between Lysozyme and Phenazinium Dyes: An Effect on Protein Conformation.

A comparative study of binding interaction between Safranin O (SO) and Neutral Red (NR) with lysozyme (Lyz) has been reported using several spectroscopic methods along with computational approaches. Steady-state fluorescence measurements revealed static quenching as the major quenching mechanism in Lyz-SO and Lyz-NR interaction, which is further supported by time-resolved fluorescence and UV-vis measurements. Additionally, binding and thermodynamic parameters of these interactions are calculated from temperature dependent fluorescence data. Moreover, conformational changes of protein upon binding with SO and NR are provided by synchronous and circular dichroism (CD) measurements. Molecular docking study provided the exact binding location of SO and NR in lysozyme. Along with this study, molecular dynamics simulation is carried out to measure the stability of Lyz, Lyz-SO, and Lyz-NR complex. The present study revealed the strong binding affinity of dyes with lysozyme, and this study would be helpful toward medical and environmental science.

Oxadiazole-carbazole polymer (POC)-Ir(ppy)3 tunable emitting composites

POC polymer is an oxadiazole-carbazole copolymer we have previously synthetized and established as light emitting material in Organic Light Emitting Devices (OLEDs), although POC quantum yield emission efficiency and color purity still need to be enhanced. On the other hand, tris[2-phenylpyridinato-C2,N]iridium(III) (Ir(ppy)3) complexes, namely Ir(ppy)3 are among the brightest luminophores employed in green light emitting devices. Our aim, in this work, is to take advantage of Ir(ppy)3 bright emission by combining the Ir complex with blue emitting POC to obtain tunable light emitting composites over a wide range of the visible spectrum.Here we have investigated the optical proprieties POC based nanocomposites with different concentrations of Ir(ppy)3, ranging from 1 to 10 wt%. Both spectral and time resolved fluorescence measurements show an efficient energy transfer from the polymer to the dopants, resulting in white-emitting composites. The most intense and stable emission has been found when POC was doped with about 5 wt% concentration of Ir(ppy)3.

Ultrafast Electronic Relaxation through a Conical Intersection: Nonadiabatic Dynamics Disentangled through an Oscillator Strength-Based Diabatization Framework.

We employ surface hopping trajectories to model the short-time dynamics of gas-phase and partially solvated 4-(N,N-dimethylamino)benzonitrile (DMABN), a dual fluorescent molecule that is known to undergo a nonadiabatic transition through a conical intersection. To compare theory vs time-resolved fluorescence measurements, we calculate the mixed quantum-classical density matrix and the ensemble averaged transition dipole moment. We introduce a diabatization scheme based on the oscillator strength to convert the TDDFT adiabatic states into diabatic states of La and Lb character. Somewhat surprisingly, we find that the rate of relaxation reported by emission to the ground state is almost 50% slower than the adiabatic population relaxation. Although our calculated adiabatic rates are largely consistent with previous theoretical calculations and no obvious effects of decoherence are seen, the diabatization procedure introduced here enables an explicit picture of dynamics in the branching plane, raising tantalizing questions about geometric phase effects in systems with dozens of atoms.

Slow and Fast Fluorescence Quenching of LHCII in Chlamydomonas Reinhardtii Cells

In this work we have studied the process of non-photochemical quenching (NPQ) in vivo in the model green alga C. reinhardtii. NPQ is the process that protects algae and plants from high light damage by dissipating large part of the absorbed energy as heat and which is activated by the low lumenal pH. Despite a large research effort the NPQ mechanism remains debated. The main obstacle to understand NPQ resides in the gap between in vitro and in vivo studies. On the one hand, the complexity of the thylakoid membrane makes it very difficult to obtain molecular information from in vivo experiments. On the other hand, a good in vitro system for the study of the quenching is not available. This has generated to a series of contrasting models that cannot be validated. We have developed a “minimal NPQ cell” which allows us to study the effect of the individual NPQ players in the membrane of living cells. We show that LHCII, the main antenna complexes of algae and plants, exists in different quenching states in the membrane depending on the growth conditions. This difference is suggested to be due to a difference in crowding. However, we also show that LHCII is not able to switch to the quenched conformation in response to pH. Instead, the presence of a very small amount of the protein LHCSR1 is sufficient to induce a large quenching (50%) in a membrane that contains only LHCII when the pH drops to 5.5. The quenching is very fast and completely reversible. Time-resolved fluorescence measurements at room and low temperature show that the quenching occurs in 360 ps and is not induced by additional clustering of LHCII in the membrane. Based on these results we propose a new model that links NPQ to long term acclimation responses.

Self-Assembled Gold Nanoparticle-Fluorescent Protein Conjugates as Platforms for Sensing Thiolate Compounds via Modulation of Energy Transfer Quenching.

The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-histidine coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ∼95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction, and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (Kd-1) for these compounds. These findings combined indicate that the AuNP-fluorescent protein conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures.

Insights into the Photophysics and Supramolecular Organization of Congo Red in Solution and the Solid State.

Steady-state and time-resolved absorption and fluorescence measurements are reported for Congo Red (CR) in aqueous and dimethylsulfoxide (DMSO) solutions. The very low fluorescence quantum yield (≈10-4 ) for CR in dilute solutions together with the absence of a triplet state indicates that internal conversion is the dominant deactivation route with more than 99.99 % of the quanta loss (attributed to the energy gap law for radiationless transitions). Although no direct evidence for trans-cis photoisomerization was obtained from absorption or fluorescence data, the global analysis of fs-transient absorption data indicates the presence of a photoproduct with a lifetime of ≈170 ps that is suggested to be associated with such a process. Spectral data for more concentrated CR solutions indicate the presence of oblique or twisted J-type aggregates. These results are compared with spectra for CR in the solid state (sodium salt) and intercalated in a layered double hydroxide via a one-step co-precipitation route. Powder XRD and electronic spectral data for the nanohybrid indicate that the CR guest molecules are intercalated as a monolayer consisting of slipped cofacial J-type aggregates.

Plasmonic effects of gold colloids on the fluorescence behavior of dye-doped SiO2 nanoparticles

The interactions of dye molecules with gold nanoparticles are of great interest owing to the potential applications in the areas of bioimaging, sensing and photodynamic therapy applications. In many cases the distances between fluorophores and the metal particles can change during the experiment and the spectral features of the units are not taken into account. In this work, the fluorescence behaviour of two dyes with different spectral properties (Rhodamine B and 9-aminoacridine) are investigated in the presence of gold nanoparticles having diameters of 2 or 26nm and hence different plasmonic properties. In order to fix the distance between the dye and the gold nanoparticles, the dyes are entrapped in 20nm silica nanoparticles, and the metal colloids are adsorbed on the silica surface. The distance between the fluorescent units and the metal particles is tuned by growing additional silica layers on the pristine nanoparticles. Steady-state and time-resolved fluorescence measurements show that in the presence of gold nanoparticles, having 2nm diameter, a drastic quenching of the dye emission is observed, for all the prepared samples, despite the average dye-metal distances. When gold nanoparticles with 26nm diameters are used, their interactions with the dyes are strongly dependent on the averaged distances between the metal colloids and the dyes and on the overlap of their spectral properties. Indeed, an enhanced emission is observed for 9-aminoacridine while the fluorescence of longer wavelength emitting Rhodamine B is quenched. The steady state and time-resolved data are analysed to evaluate the plasmonic impact of the radiative and non-radiative rate constants of the dyes.

Investigation of Solvent Effects on Photophysical Properties of New Aminophthalimide Derivatives-Based on Methanesulfonate.

Novel aminophthalimide derivatives were synthesized starting from (3aR,7aS)-2-(2-hydroxypropyl)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione (9) , and solvent effects on the photo-physical properties of these newly synthesized aminophthalimide derivatives (compounds 14 and 15) were investigated using UV-Vis absorption spectroscopy, steady-state and time-resolved fluorescence measurements. Both absorption and fluorescence spectra exhibited bathochromic shift with the increased polarity of the solvents for both molecules. Solute-solvent interactions were analyzed using the Lippert-Mataga and Bakhshiev polarity functions, and Kamlet-Taft and Catalan multiple linear regression approaches. The results revealed that these two molecules experienced specific interactions. Furthermore, photo-physical parameters were calculated for both molecules in all of the solvents, such as the fluorescence quantum yield, fluorescence lifetime, radiative (kr) and non-radiative (knr) rate constant values. It was observed that the fluorescence quantum yield values decreased linearly with increasing solvent polarity. This study proved the new dyes including isopropyl methanesulfonate group displayed different behavior from previous studies of aminophthalimide derivatives in water. It was recommended that these new dyes having interesting properties by changing solvent can be used various applications such as environmentally sensitive fluorescent probes, labels in biology, laser industry.

Optically Detected Degradation of NaYF4:Yb,Tm-Based Upconversion Nanoparticles in Phosphate Buffered Saline Solution.

In a proof-of-concept study, we assessed different analytical and spectroscopic parameters for stability screening of differently sized β-NaYF4:20 mol % Yb3+, 2 mol % Tm3+ upconversion nanoparticles (UCNPs) exemplarily in the bioanalytically relevant buffer phosphate buffered saline (PBS; pH 7.4) at 37 and 50 °C. This included the potentiometric determination of the amount of released fluoride ions, surface analysis with X-ray photoelectron spectroscopy (XPS), and steady-state and time-resolved fluorescence measurements. Based on these results, the luminescence lifetime of the 800 nm upconversion emission was identified as an optimum parameter for stability screening of UCNPs and changes in particle surface chemistry.

Probing the binding of Azilsartan to DNA by molecular docking, steady-state/time-resolved fluorescence, viscosity, infrared, and circular dichroism spectra

ABSTRACTAzilsartan, a new antihypertensive drug, has effects on the sympathetic nervous system and expression of genes. The interaction of Azilsartan with DNA was investigated using molecular docking and multi-spectral techniques. Molecular docking revealed that Azilsartan could interact with DNA via groove binding from a theoretical perspective. Time-resolved fluorescence measurements indicated that the quenching mechanism was static, and further analysis of quenching data demonstrated that the binding was spontaneous and mainly driven by hydrophobic forces. The results of interaction with denatured DNA, viscosity, infrared spectroscopy, and circular dichroism showed that Azilsartan could bind to DNA through groove binding, which was consistent with docking analyses.

Fluorescence property of photosystem II protein complexes bound to a gold nanoparticle.

Development of an efficient photo-anode system for water oxidation is key to the success of artificial photosynthesis. We previously assembled photosystem II (PSII) proteins, which are an efficient natural photocatalyst for water oxidation, on a gold nanoparticle (GNP) to prepare a PSII-GNP conjugate as an anode system in a light-driven water-splitting nano-device (Noji et al., J. Phys. Chem. Lett., 2011, 2, 2448-2452). In the current study, we characterized the fluorescence property of the PSII-GNP conjugate by static and time-resolved fluorescence measurements, and compared with that of free PSII proteins. It was shown that in a static fluorescence spectrum measured at 77 K, the amplitude of a major peak at 683 nm was significantly reduced and a red shoulder at 693 nm disappeared in PSII-GNP. Time-resolved fluorescence measurements showed that picosecond components at 683 nm decayed faster by factors of 1.4-2.1 in PSII-GNP than in free PSII, explaining the observed quenching of the major fluorescence peak. In addition, a nanosecond-decay component arising from a 'red chlorophyll' at 693 nm was lost in time-resolved fluorescence of PSII-GNP, probably due to a structural perturbation of this chlorophyll by interaction with GNP. Consistently with these fluorescence properties, degradation of PSII during strong-light illumination was two times slower in PSII-GNP than in free PSII. The enhanced durability of PSII is an advantageous property of the PSII-GNP conjugate in the development of an artificial photosynthesis device.

Cyclodextrin-assisted modulation of the photophysical properties and acidity constant of pyrene-armed calix[4]arene.

The supramolecular interaction of cyclodextrin homologues with a pyrene-armed calixarene has been investigated. p-tert-Butylcalix[4]arene-di-4-(pyren-1-yl)butanoic acid (PCX4) has been synthesized and characterized by 1H NMR, FT-IR and mass spectrometry. The modulation in the photophysical properties, especially the monomer and excimer emission behaviour of the pyrene group in PCX4 with β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD), has been explored in aqueous solutions by using optical absorption, steady-state and time-resolved fluorescence measurements. Steady-state fluorescence studies illustrate that upon gradual addition of γ-CD to the PCX4 solution, initially there is a decrease in the monomer emission (at 378, 398 and 420 nm) along with the appearance of weak excimer emission at 482 nm. Beyond a particular concentration of γ-CD (>1.2 mM), PCX4 shows strong excimer emission along with an increase in the monomer emission, indicating two types of interaction modes between PCX4 and γ-CD. Fluorescence anisotropy measurements confirm intramolecular excimer formation with a lower concentration of γ-CD, whereas it predominantly becomes an intermolecular excimer beyond 1.2 mM of γ-CD. On the other hand, the monomer emission of the pyrene group of PCX4 increases in the presence of β-cyclodextrin having lower cavity sizes due to the formation of a 1 : 1 complex. For the first time, the intrinsic fluorescence from the pyrene moiety of PCX4 is employed to determine the pKa value of PCX4 and it is ∼6.1. The cyclodextrin-encapsulated PCX4 brings a 1.2 unit downward pKa shift. Along with the pH-responsive interconversion of monomer to excimer emission of γ-CD complexed PCX4, this system can find applications in ratiometric sensing and optical supramolecular pH-sensors.

Local environment inside a novel aromatic micelle investigated by steady-state and femtosecond fluorescence spectroscopy of an encapsulated solvatochromic probe.

The local environment within a recently developed anthracene-shelled micelle (ASM), which is a micelle-like nanocapsule composed of anthracene-embedded amphiphiles, was investigated by steady-state and time-resolved spectroscopy of an encapsulated solvatochromic fluorescent probe molecule, coumarin 153 (C153). The absorption maximum of encapsulated C153 (452 nm) is more red-shifted than that of free C153 in water, indicating a highly polar environment inside the micelle. Despite this, the fluorescence Stokes shift of encapsulated C153 (∼3700 cm-1) is substantially smaller than that of free C153 in water. Femtosecond time-resolved broadband fluorescence measurements further showed that the dynamic Stokes shift is completed within 1 ps, revealing that the reorganization of the micelle interior following photoexcitation of the C153 probe is characterized by a sub-picosecond, limited-amplitude response. The femtosecond fluorescence anisotropy data showed that the orientational diffusion of the host-guest complex is slower (860 ps) than that of the empty micelle (510 ps), suggesting that the micelle structure is flexible enough to expand when the guest molecule is accommodated and that the micelle rotates with the encapsulated guest molecule. This softness of the micelles further allows some of them to simultaneously encapsulate two C153 molecules, as evidenced by the appearance of blue-shifted, H-dimer-like absorption and fluorescence bands. Based on these steady-state and femtosecond time-resolved spectroscopic data, we discuss the electronic state of C153 and micelle structure as well as the host-guest interaction in this novel flexible synthetic nanocapsule.

Guest-Induced Modulation of the Energy Transfer Process in Porphyrin-Based Artificial Light Harvesting Dendrimers.

A series of dendritic multiporphyrin arrays (PZnTz-nPFB; n = 2, 4, 8) comprising a triazole-bearing focal zinc porphyrin (PZn) with a different number of freebase porphyrin (PFB) wings has been synthesized, and their photoinduced energy transfer process has been evaluated. UV/vis absorption, emission, and time-resolved fluorescence measurements indicated that efficient excitation energy transfer takes place from the focal PZn to PFB wings in PZnTz-nPFB's. The triazole-bearing PZn effectively formed host-guest complexes with anionic species by means of axial coordination with the aid of multiple C-H hydrogen bonds. By addition of various anionic guests to PZnTz and PZnTz-nPFB's, strong bathochromic shifts of PZn absorption were observed, indicating the HOMO-LUMO gap (ΔEHOMO-LUMO) of PZn decreased by anion binding. Time-resolved fluorescence measurements revealed that the fluorescence emission predominantly takes place from PZn in PZnTz-nPFB's after the addition of CN-. This change was reversible because a treatment with a silver strip to remove CN- fully recovered the original energy transfer process from the focal PZn to PFB wings.

Cubosomes for in vivo fluorescence lifetime imaging

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Controlled fluorescence in a beetle's photonic structure and its sensitivity to environmentally induced changes

The scales covering the elytra of the male Hoplia coerulea beetle contain fluorophores embedded within a porous photonic structure. The photonic structure controls both insect colour (reflected light) and fluorescence emission. Herein, the effects of water-induced changes on the fluorescence emission from the beetle were investigated. The fluorescence emission peak wavelength was observed to blue-shift on water immersion of the elytra whereas its reflectance peak wavelength was observed to red-shift. Time-resolved fluorescence measurements, together with optical simulations, confirmed that the radiative emission is controlled by a naturally engineered photonic bandgap while the elytra are in the dry state, whereas non-radiative relaxation pathways dominate the emission response of wet elytra.

Using ATTO Dyes To Probe the Photocatalytic Activity of Au–CdS Nanoparticles

Metal–semiconductor nanohybrids (or heterostructures), such as Au–CdS, have become an important class of materials because of their role in photochemical hydrogen production and in other catalytic reactions. Here we report the results of photophysical studies of the interactions of these particles with ATTO dyes (ATTO 590 and 655), which are used as fluorescent probes in a wide range of spectroscopic techniques, most notably super-resolution microscopies. The most important feature of the Au–CdS particles is that they provide the possibility of selective excitation at either their CdS or their Au domains, which absorb preferentially at wavelengths shorter or longer than 500 nm, respectively, thus making possible an excited-state charge transfer reaction from ATTO. Fluorescence quenching of ATTO is dominated by charge transfer to either the CdS domain (λex = 400 nm) or the Au domain (λex = 570 nm). This quenching is quantified by steady-state and time-resolved absorption and fluorescence measurements, and ...