Time-resolved Fluorescence Measurements Research Articles

Excited State Interaction of Ruthenium (II) Imidazole Phenanthroline Complex [Ru(bpy)2 ipH]2+ with 1,4-Benzoquinone: Simple Electron Transfer or Proton-Coupled Electron Transfer?

The unidirectional proton coupled electron transfer (PCET) from the excited state of Ru(II) imidazole phenanthroline complex [Ru(bpy)2 ipH]2+ to 1,4-benzoquinone, was studied by steady-state (SS) and time-resolved (TR) fluorescence and transient absorption (TA) measurements. The pKa (9.7) and pKa * (8.6) values of the complex suggest that it behaves as a photoacid on excitation. The difference in the quenching rates obtained from SS and TR fluorescence studies indicate participation of both dynamic quenching and static quenching involving the hydrogen bonded ipH ligand of [Ru(bpy)2 ipH]2+ with the 1,4-benzoquinone quencher, formed in the ground state. Within the hydrogen bonded complex, the ruthenium centre acts as the electron donor, while the ipH ligand acts as the proton donor to the hydrogen bonded 1,4-benzoquinone that acts simultaneously both as the electron and proton acceptor. It is proposed that the static quenching in the hydrogen bonded [Ru(bpy)2 ipH]2+ -1,4-benzoquinone pairs occurs involving the PCET mechanism, while the dynamic quenching occurs through the simple ET mechanism, on diffusional encounter of the isolated 1,4-benzoquinone with the excited [Ru(bpy)2 ipH]2+ complex. The occurrence of broad TA bands around 420-430 nm suggests formation of both 1,4-benzoquinone radical anion as well as the 1,4-benzosemiquinone radical by the interaction of excited [Ru(bpy)2 ipH]2+ with 1,4-benzoquinone, thus supporting the ET process in the studied system.

Plasmonic Enhancement of Two-Photon-Excited Luminescence of Single Quantum Dots by Individual Gold Nanorods.

Plasmonic enhancement of two-photon-excited fluorescence is not only of fundamental interest but also appealing for many bioimaging and photonic applications. The high peak intensity required for two-photon excitation may cause shape changes in plasmonic nanostructures, as well as transient plasmon broadening. Yet, in this work, we report on strong enhancement of the two-photon-excited photoluminescence of single colloidal quantum dots close to isolated chemically synthesized gold nanorods. Upon resonant excitation of the localized surface plasmon resonance, a gold nanorod can enhance the photoluminescence of a single quantum dot more than 10 000-fold. This strong enhancement arises from the combined effect of local field amplification and the competition between radiative and nonradiative decay rate enhancements, as is confirmed by time-resolved fluorescence measurements and numerical simulations.

Tetronic Star Block Copolymer Micelles: Photophysical Characterization of Microenvironments and Applicability for Tuning Electron Transfer Reactions.

Detailed photophysical investigations have been carried out using a probe dye, coumarin-153 (C153), to understand the microenvironments of micelles formed by the newly introduced Tetronic star block copolymers, T1304 and T1307, having the same poly(propylene oxide) (PPO) block size but different poly(ethylene oxide) (PEO) block sizes. Ground state absorption, steady-state fluorescence, and time-resolved fluorescence measurements have been used to estimate the micropolarity, microviscosity, and solvation dynamics within the two micelles. To the best of our knowledge, this is the first report on these important physicochemical parameters for this new class of the star block copolymer micelles. Our results indicate that T1307 micelle offers a relatively more polar and less viscous microenvironment in the corona region, compared to T1304. The effect of the two micellar systems has subsequently been investigated on the bimolecular photoinduced electron transfer (ET) reactions between coumarin dyes (electron acceptors) and aromatic amines (electron donors). On correlating the energetics and kinetics of the ET reactions, clear Marcus inversion (MI) behavior is observed in both of the micellar media. Interestingly, the ET rates for all of the donor-acceptor pairs are much higher in T1307 than in T1304, and the onset of MI also appears at a relatively higher exergenocity (-Δ G0) in the former micelle (∼0.45 eV for T1307) than the latter (∼0.37 eV for T1304). The effect of added NaCl salt studied selectively in T1307 micelle shows that the ET rate decreases significantly along with a shift in the onset of MI toward lower exergenocity region, so that in the presence of 2 M NaCl the system becomes quite comparable to T1304. On the basis of the observed results, it is realized that the micropolarity and hence the dynamics of the ET process can be tuned very effectively either by changing the constitution of the star block copolymer or by using a suitable additive as a modifier of the micellar microenvironment.

INTERACTION OF COPPER OXIDE NANOPARTICLES WITH BOVINE SERUM ALBUMIN BY SPECTROSCOPIC STUDIES

Objective: Since structural changes of adsorbed protein are necessary for cellular uptake of nanoparticles (NPs) it is of prime importance to know about structural changes of bovine serum albumin (BSA) when it interacts with CuO NPs–a potential new antitumor drug.Methods: CuO NPs prepared by sol-gel technique were characterized by x-ray diffraction (XRD) and tunneling electron microscope (TEM) techniques. The conformational changes induced by CuO NPs on BSA were studied by various spectroscopic techniques such as steady state and time-resolved fluorescence measurements. The changes in fluorescence emission parameters such as fluorescence intensity, fluorescence emission maximum and lifetimes of fluorescent residues in BSA were studied.Results: XRD analysis showed the average particle size as 32 nm. The TEM micrograph showed particles of different size varying from 10 to 45 nm. Fluorescence quenching was confirmed due to a decrease in fluorescence intensity of CuO NPs–BSA complex. The analysis of lifetime measurements indicated BSA contained two tryptophan (trp) residues that fluoresced in different environments. Static quenching mechanism was confirmed by time-resolved measurements when BSA interacted with CuO NPs.Conclusion: Minor structural changes of BSA protein were observed during the interaction studies.

Femtosecond Time-Resolved Raman Spectroscopy Reveals Structural Evidence for meta Effect in Stilbenols.

The meta effect in substituted aromatics plays a crucial role in their excited-state photophysical properties. Meta-substituted hydroxyarenes such as naphthols, stilbenols, and chromophoric constituents of green fluorescent proteins show unusual photoacidity and enhanced fluorescence lifetime and quantum yield when compared to their para-derivatives. Variation in the excited state features of the meta-derivatives when compared to the para-derivatives in stilbenols has been attributed to the enhanced torsional barrier for interconversion between the planar and the twisted perpendicular forms. Herein, we employed femtosecond time-resolved Raman spectroscopy to provide the direct structural evidence for the enhanced torsional barrier in meta-stilbenol. The Raman band profiles of the olefinic C═C stretch related to the torsional motion are found to decay with time constants of ∼750 and ∼13 ps in meta-stilbenol and para-stilbenol respectively, unraveling the structural evidence for the observed enhanced photoacidity originating from enhanced rates of excited-state proton transfer. Further, time-resolved fluorescence measurements are performed to elucidate the relaxation pathways of the excited states of the stilbenols.

Crystallization and Aggregation-Induced Emission in a Series of Pyrrolidinylvinylquinoxaline Derivatives

Aggregation-induced emission (AIE) has been meanwhile observed for many dye classes and particularly for fluorophores containing propeller-like groups. Herein, we report on the AIE characteristics of a series of four hydrophobic pyrrolidinylvinylquinoxaline (PVQ) derivatives with phenyl, pyrrolyl, indolyl, and methoxythienyl substituents used to systematically vary the torsion angle between this substituent at the quinoxaline C2 position and the planar PVQ moiety. These molecules, which are accessible via four- or five-component one-pot syntheses, were spectroscopically studied in organic solvents and solvent–water mixtures, as dye aggregates, solids, and entrapped in polystyrene particles (PSP). Steady-state and time-resolved fluorescence measurements revealed a strong fluorescence enhancement for all dyes in ethanol–water mixtures of high water content, accompanying the formation of dye aggregates with sizes of a few hundred nm, overcoming polarity and H-bonding-induced fluorescence quenching of the cha...

Uphill energy transfer in photosystem I from Chlamydomonas reinhardtii. Time-resolved fluorescence measurements at 77\xa0K

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~ 12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~ 675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.

Supramolecular Control over the Interparticle Distance in Gold Nanoparticle Arrays by Cyclodextrin Polyrotaxanes.

Amphiphilic nonionic ligands, synthesized with a fixed hydrophobic moiety formed by a thiolated alkyl chain and an aromatic ring, and with a hydrophilic tail composed of a variable number of oxyethylene units, were used to functionalize spherical gold nanoparticles (AuNPs) in water. Steady-state and time-resolved fluorescence measurements of the AuNPs in the presence of α-cyclodextrin (α-CD) revealed the formation of supramolecular complexes between the ligand and macrocycle at the surface of the nanocrystals. The addition of α-CD induced the formation of inclusion complexes with a high apparent binding constant that decreased with the increasing oxyethylene chain length. The formation of polyrotaxanes at the surface of AuNPs, in which many α-CDs are trapped as hosts on the long and linear ligands, was demonstrated by the formation of large and homogeneous arrays of self-assembled AuNPs with hexagonal close packing, where the interparticle distance increased with the length of the oxyethylene chain. The estimated number of α-CDs per polyrotaxane suggests a high rigidization of the ligand upon complexation, allowing for nearly perfect control of the interparticle distance in the arrays. This degree of supramolecular control was extended to arrays formed by AuNPs stabilized with polyethylene glycol and even to binary arrays. Electromagnetic simulations showed that the enhancement and distribution of the electric field can be finely controlled in these plasmonic arrays.

Plasma-assisted CO2 conversion: optimizing performance via microwave power modulation

Significant improvement in the energy efficiency of plasma-assisted CO2 conversion is achieved with applied power modulation in a surfaguide microwave discharge. The obtained values of CO2 conversion and energy efficiency are, respectively, 0.23 and 0.33 for a 0.95 CO2 + 0.05 N2 gas mixture. Analysis of the energy relaxation mechanisms shows that power modulation can potentially affect the vibrational–translational energy exchange in plasma. In our case, however, this mechanism does not play a major role, likely due to the low degree of plasma non-equilibrium in the considered pressure range. Instead, the gas residence time in the discharge active zone together with plasma pulse duration are found to be the main factors affecting the CO2 conversion efficiency at low plasma pulse repetition rates. This effect is confirmed experimentally by the in situ time-resolved two-photon absorption laser-induced fluorescence measurements of CO molecular density produced in the discharge as a result of CO2 decomposition.

Aberration in the structural paradigm of lens protein α crystallin by UV-C irradiation

The conformation of lens protein α crystallin was investigated using different spectroscopic techniques under normal and UV-C-irradiated condition. The structural elucidation of commercially available lens protein α crystallin under the effects of UV-C irradiation has never been reported earlier. To study the effects of irradiation on the lens protein, we used UV–visible spectroscopy, CD spectroscopy, and steady-state and time-resolved fluorescence measurements along with FTIR study, under increasing doses of UV-C irradiation. Using the secondary and tertiary structural changes as parameters for detecting conformational perturbation, we investigated the structural paradigm shift in the lens protein α crystallin. Increasing doses of UV-C radiation resulted in decreasing β sheet content of α crystallin from 30 to 10%. The fluorescence profile confirmed the formation of ROS species in the protein upon extensive exposure to UV-C irradiation. These results inferred UV-C irradiation may induce alteration of secondary structure of the lens protein leading to impaired biological functioning.

Fӧrster resonance energy transfer between MoS2 quantum dots and polyaniline for turn-on bovine serum albumin sensing

Fӧrster resonance energy transfer (FRET) between MoS2 and polyaniline (PANI) is explored by steady state and time resolved fluorescence spectroscopy in the present study and FRET based turn-on sensing of bovine serum albumin (BSA) is also demonstrated. The intense fluorescence of prepared MoS2 QDs is quenched by addition of PANI. Stern-Volmer plot suggests that the quenching is dynamic one. The observed quenching is explained by FRET mechanism also. The efficiency of energy transfer and rate of energy transfer are found to be increasing with increasing concentration of PANI. Time resolved fluorescence measurements show the reduction of lifetime components of MoS2 (donor) upon increasing concentration of PANI (acceptor). The quenched fluorescence of MoS2 in the presence of PANI is gradually recovered by addition of BSA at nM level which demonstrates turn-on BSA sensing.

Ultrafast Exciton Delocalization, Localization, and Excimer Formation Dynamics in a Highly Defined Perylene Bisimide Quadruple π-Stack.

An adequately designed, bay-tethered perylene bisimide (PBI) dimer Bis-PBI was synthesized by Pd/Cu-catalyzed Glaser-type oxidative homocoupling of the respective PBI building block. This newly synthesized PBI dimer self-assembles exclusively and with high binding constants of up to 106 M-1 into a discrete π-stack of four chromophores. Steady-state absorption and emission spectra show the signatures of H-type excitonic coupling among the dye units. Broadband fluorescence upconversion spectroscopy (FLUPS) reveals an ultrafast dynamics in the optically excited state. An initially coherent Frenkel exciton state that is delocalized over the whole quadruple stack rapidly (τ = ∼200 fs) loses its coherence and relaxes into an excimer state. Comparison with Frenkel exciton dynamics in PBI dimeric and oligomeric H-aggregates demonstrates that in the quadruple stack coherent exciton propagation is absent due to its short length of aggregates, thereby it has only one relaxation pathway to the excimer state. Furthermore, the absence of pump-power dependence in transient absorption experiments suggests that multiexciton cannot be generated in the quadruple stack, which is in line with time-resolved fluorescence measurements.

Studies on the interaction between 7-(dimethyl amino)-4-(trifluoromethyl)-2H-1-benzopyran-2-one and cadmium sulfide quantum dots

Quantum dots (QDs) are well known for their optical properties which differ from those of bulk semiconductors. Herein, we have created an energy transfer platform that combines CdS QDs with a coumarin based dye C485 [7-(dimethyl amino)-4-(trifluoromethyl)-2H-1-benzopyran-2-one]. Spectroscopic studies of energy transfer between the dye donor and CdS QDs as acceptors reveal the occurrence of dynamic quenching. Analysis of the steady-state and time resolved fluorescence measurements of C485 in the presence of CdS QDs infers fluorescence resonance (Förster type) energy transfer (FRET) as responsible for the quenching phenomena. The energy transfer efficiency as well as energy transfer distance for the donor-acceptor pair is calculated using steady-state fluorescence method. Luminescence enhancement of CdS QDs play a critical role in device performance for solar applications and also in the field of biological applications.

Influence of Eugenol on the Organization and Dynamics of Lipid Membranes: A Phase-Dependent Study.

Eugenol is known for its antimicrobial effects against microorganisms responsible for infectious diseases in humans, food-borne pathogens, and oral pathogens. In spite of several reports on the antimicrobial function of eugenol by modulating the structural properties of cell membranes, there is limited information on the influence of eugenol in the lipid membrane. In this work, we explored the effect of eugenol on the organization and dynamics of large unilamellar vesicles (LUVs) of DMPC using the intrinsic fluorescence of eugenol and an extrinsic hydrophobic probe, DPH, in varying phases. The organization and dynamics of the bilayers of DMPC vesicles were monitored by utilizing varieties of steady-state and time-resolved fluorescence measurements. Our results show that eugenol stabilizes the gel phase and elevates the phase-transition temperature of DMPC in a concentration-dependent fashion. Fluorescence lifetime measurements demonstrate that higher eugenol-induced water penetration was observed in fluid-phase membranes. Time-resolved anisotropy measurements demonstrate that eugenol reduces the semiangle of DPH wobbling-in-a-cone in gel-phase membranes, whereas the semiangle remains unaffected in fluid-phase membrane. This implies that the eugenol further orders the gel-phase membrane, and this could be a plausible reason for the eugenol-dependent elevation of the phase-transition temperature of DMPC. We envisage that these results will contribute important information to understand the interaction of eugenol with biological membranes.

Experimental and theoretical investigation of fluorescence solvatochromism of dialkoxyphenyl-pyrene molecules.

We investigated the fluorescence properties of dialkoxyphenyl-pyrene molecules experimentally as well as theoretically. Our experiments confirmed fluorescence solvatochromism in 2,5-dimethoxyphenyl-pyrene and, in contrast there was no significant solvent-effect on the emission properties of the isomers, 3,5- and 2,6-dimethoxyphenyl-pyrene. This clear difference in the solvent-dependence would reflect the difference in character of the excited-state between the isomers, which differ only in the substitution positions of the two methoxy groups. The positional effects of the di-substituted molecules are successfully explained theoretically by the topologies of the highest occupied molecular orbital of the phenyl group that are governed by the relative positions of the two substituents, though it is somewhat contradictory to the meta-effect for the mono-substituted molecules. Theoretical calculations were also used to analyze the character of the excited states; 2,5-dimethoxyphenyl-pyrene alone exhibited an intramolecular charge transfer character for the excited state, which was responsible for the solvatochromism effect. The dynamics of the excited states were analyzed using time-resolved fluorescence measurements, in which a characteristic increase of the fluorescence intensity was observed for 2,5-dialkoxyphenyl-pyrene; this observation was supported by the theoretical calculations as well.

Anomalous fluorescence enhancement and fluorescence quenching of graphene quantum dots by single walled carbon nanotubes.

We explore the mechanism of the fluorescence enhancement and fluorescence quenching effect of single walled carbon nanotubes (SWCNTs) on highly fluorescent graphene quantum dots (GQDs) over a wide range of concentrations of SWCNTs. At very low concentrations of SWCNTs, the fluorescence intensity of the GQDs is enhanced, while at higher concentrations, systematic quenching of fluorescence is observed. The nature of the Stern-Volmer plot for the latter case was found to be non-linear indicating a combined effect of dynamic and static quenching. The contribution of the dynamic quenching component was assessed through the fluorescence lifetime measurements. The contribution of static quenching is confirmed from the red shift of the fluorescence spectra of the GQDs after addition of SWCNTs. The fluorescence intensity is first enhanced at very low concentration due to improved dispersion and higher absorption by GQDs, while at higher concentration, the fluorescence of GQDs is quenched due to the complex formation and associated reduction of the radiative sites of the GQDs, which is confirmed from time-resolved fluorescence measurements. Laser confocal microscopy imaging provides direct evidence of the enhancement and quenching of fluorescence at low and high concentrations of SWCNTs, respectively. This study provides an important insight into tuning the fluorescence of GQDs and understanding the interaction between GQDs and different CNTs, which is important for bio-imaging and drug delivery applications.

Highly sensitive sensing of glutathione based on Förster resonance energy transfer between MoS2 donors and Rhodamine 6G acceptors and its insight

We report excitation dependent tunable emission property of MoS2 quantum dots (QDs) which can be applicable for enhanced energy transfer applications. Spectral overlap of fluorescence spectra of the prepared MoS2 QDs excited at different wavelengths with the absorption spectrum of Rhodamine 6G (R6G) shows the possibility of Förster resonance energy transfer (FRET) phenomena between MoS2 QDs and R6G. The calculated overlap integrals are found to be increased when the excitation wavelength is varied from 330 to 430 nm. Steady state and time resolved fluorescence measurements are carried out to study the FRET process between MoS2 QDs and R6G experimentally. The results show that the energy is transferred from MoS2 QDs (serve as a donor) to R6G (function as an acceptor). Enhancement in energy transfer efficiency and resonant energy transfer rate are found as a result of exciting at different wavelengths, which is attributed to the increased overlap integral as shown by the spectral overlap. The FRET between prepared MoS2 QDs and R6G at 330 and 430 nm excitation is used for glutathione sensing application.

Exploring the effect of 5-Fluorouracil on conformation, stability and activity of lysozyme by combined approach of spectroscopic and theoretical studies

In this present work, a detailed investigation of the effect of an anticancer drug, 5-Fluorouracil (5-FU), on conformation, stability and activity of lysozyme (Lyz) was reported. The interaction between Lyz and 5-FU was reflected in terms of intrinsic fluorescence quenching and change in secondary structure of Lyz. The mode of quenching mechanism involved was evaluated by the steady-state and time-resolved fluorescence measurements. Synchronous and Circular Dichroism (CD) results revealed the conformational changes induced in Lyz upon complexation with 5-FU. Additionally, the effect of temperature and chemical denaturant on the stability of Lyz-5FU complex was carried out. As well as the activity of Lyz in the absence and presence of 5-FU were measured using Micrococcus luteus strain. To support our experimental findings, in vitro interaction between Lyz and 5-FU was done by theoretical studies. The current study will provide a better understanding on the nature of the interactions possible between proteins and drug molecules, which might create a bench mark in medical science in terms of the toxic effect or biological benefits of drug molecules on protein structure and conformation.

Resveratrol Interferes with an Early Step in the Fibrillization Pathway of Human Lysozyme and Modulates it towards Less-Toxic, Off-Pathway Aggregates.

The effect of resveratrol, a polyphenol in red wine, on the amyloid fibril formation of human lysozyme (HuL) was investigated, towards elucidating the mechanism of resveratrol action and probing its role as a possible modulator of lysozyme aggregation and toxicity. By using a number of biophysical tools, resveratrol was observed to alter the fibrillization kinetics of HuL and inhibit its fibrillization by binding with weak to moderate affinity to the conformations populated at the early stages of the pathway with concomitant stabilization of these initial conformations. The marginal decrease in the lifetime of HuL in the presence of resveratrol by time-resolved fluorescence measurements indicated the involvement of a static quenching mechanism in the interaction between HuL and resveratrol. Docking studies predicted the binding of resveratrol to aggregation-prone regions in HuL, and structure and activity analyses demonstrated the retention of much of the α-helical structure and activity of HuL in the presence of resveratrol. Resveratrol modulated the fibrillization pathway towards less-hydrophobic, less-toxic, off-pathway aggregates. These results demonstrate that binding of resveratrol to HuL could protect against the formation of pathogenic, cytotoxic aggregates formed in amyloidogenic disorders, such as systemic amyloidosis; thus suggesting its potential as a plausible therapeutic agent against lysozyme amyloidosis.

Ultrafast Dynamics of a Fluorescent Tetrazolium Compound in Solution.

Tetrazolium salts are exploited in various fields of research by virtue of their low reduction potentials. Increasingly, associated applications also attend to the photochemical and luminescence properties of these systems. Here, we investigate the photoinduced dynamics of phenyl-benzo[c]tetrazolo-cinnolinium chloride (PTC), one of the very few known fluorescent tetrazolium compounds, by using time-correlated single-photon counting, femtosecond fluorescence upconversion, and ultrafast transient absorption spectroscopy. PTC is generated photochemically by ultraviolet illumination of 2,3,5-triphenyl-tetrazolium chloride (TTC) in various alcohols. Time-resolved fluorescence measurements on PTC with different excitation wavelengths disclose biphasic solvation and vibrational relaxation dynamics. Depending on the solvent, the emission behavior of PTC is characterized by quantum yields on the order of several tens of percent and corresponding excited-state lifetimes of several hundreds of picoseconds. The radiative rate is basically constant for the studied alcohols, whereas the rate of the competing non-radiative process is sensitive to the solvent polarity. Hence, we discuss the possible involvement of intermediate radicals and further presumptive reaction pathways pursued after photoexcitation of PTC.