Steady-state Fluorescence Spectra Research Articles

Addressing the interaction of stem bromelain with different anionic surfactants, below, at and above the critical micelle concentration (cmc) in phosphate buffer at pH 7: Physicochemical, spectroscopic, & molecular docking study

The structural stability and therapeutic activity of Stem Bromelain (BM) have been explored by unravelling the interaction of stem BM in presence of two different types of anionic surfactants namely, bile salts, NaC and NaDC and the conventional anionic surfactants, SDDS and SDBS, below, at and above the critical micelle concentration (cmc) in aqueous phosphate buffer of pH 7. Different physicochemical parameters like, surface excess (Γcmc), minimum area of surfactants at air water interface (Amin) etc. are calculated from tensiometry both in absence and presence of BM. Several inflection points (C1, C2 and C3) have been found in tensiometry profile of surfactants in presence of BM due to the conformational change of BM assisted by surfactants. Similar observation also found in isothermal titration calorimetry (ITC) profiles where the enthalpy of micellization (ΔH0obs) of surfactants in absence and presence of BM have calculated. Further, steady state absorption and fluorescence spectra monitoring the tryptophan (Trp) emission of free BM and in presence of all the surfactants at three different temperatures (288.15 K, 298.15 K, and 308.15 K) reveal the nature of fluorescence quenching of BM in presence of bile salts/surfactants. Time resolved fluorescence studies at room temperature also support to determine the several quenching parameters. The binding constant (Kb) of BM with all the surfactants and free energy of binding (∆G0 of bile salts/surfactants with BM at different temperatures have been calculated exploiting steady state fluorescence technique. It is observed that, the binding of NaC with BM is greater as compared to other surfactants while Stern-Volmer quenching constant (KSV) is found greater in presence of SDBS as compared with others which supports the surface tension and ITC data with the fact that surface activity of surfactant(s) is decreasing with the binding of the surfactants at the core or binding pocket of BM. Circular Dichroism (CD) study shows the stability of secondary structure of BM in presence of NaC and NaDC below C3, while BM lost its structural stability even at very low surfactant concentration of SDDS and SDBS which also supports the more involvement of bile salts in binding rather than surfactants. The molecular docking studies have also been substantiated for better understanding the several experimental investigations interaction of BM with the bile salts/surfactants.

Near-IR-Absorbing Bis-Donor Functionalized Aza-BODIPY Derivatives: Synthesis and Photophysical Study by Using Transient Optical Spectroscopy.

A series of near-IR absorbing 2,6-diarylated BF2-chelated aza-boron-dipyrromethenes (aza-BDPs) derivatives bearing different electron donors (benzene, naphthalene, phenanthrene, phenothiazine and carbazole) were designed and synthesized. The effect of different electron donor substitutions on the photophysical properties was studied by steady-state UV-vis absorption and fluorescence spectra, electrochemical, time-resolved nanosecond transient absorption (ns-TA) spectroscopy and theoretical computations. The UV-vis absorption spectra of AzaBDP-PTZ and AzaBDP-CAR (λabs=710 nm in toluene) showed a bathochromic absorption profile compared with the reference AzaBDP-Ph (λabs=685 nm in toluene), indicating the non-negligible electronic interaction at the ground state between donor and acceptor moieties. Moreover, the fluorescence is almost completely quenched for AzaBDP-PTZ/AzaBDP-CAR (fluorescence quantum yield, ΦF=0.2-0.7 % in toluene) as compared with the AzaBDP-Ph (ΦF=27 % in toluene). However, the apparent intersystem crossing ability of these compounds is poor, based on the singlet oxygen quantum yield (ΦΔ=0.3-1.5 %). The ns-TA spectral study showed typical Bodipy localized triplet state transient features, short-lived excited triplet state for AzaBDP-Ph (τT=53.2 μs) versus significantly long-lived triplet state for AzaBDP-CAR (τT=114 μs) was observed under deaerated experimental conditions. These triplet state lifetimes are much longer than that obtained with diiodoAzaBDP (intramolecular heavy atom effect, τT=1.5~7.2 μs). These information are useful for molecular structure design of triplet photosensitizers, for which longer triplet state lifetimes are usually desired. Theoretical computations displayed that the triplet state is mainly localized on the AzaBDP core, moreover, it was found that the HOMO/LUMO energy gap decreased after introducing donor moieties to the skeleton as compared with the reference.

Efficient and tunable white light emitting from Sb3+, Tb3+, and Sm3+ Co-doped Cs2NaInCl6 double perovskite via multiple energy transfer processes

Inorganic lead-free double perovskites have the advantages of low toxicity, broadband emission, and good stability, which make them promising luminescent materials for lighting applications. However, due to the limited regulation of their self-trapped exciton emission, it is still greatly challenging to achieve white light emitting from a single double perovskite host. Herein, efficient and tunable white light is realized in Cs2NaInCl6: Sb3+, Tb3+, Sm3+ double perovskite by controlling the ratios of the doped three ions with blue, green, and red emissions, respectively. The steady-state and transient fluorescence spectra of singly- and doubly-doped double perovskites reveal the existence of multiple energy transfer channels in the triply-doped phosphors, including from Sb3+ to Tb3+, Sb3+ to Sm3+, and Tb3+ to Sm3+. Benefiting from these channels, the color coordinates of the triply-doped phosphors can cross the whole white light area of the CIE chromaticity diagram by adjusting the ratios of the three dopants, and the maximum internal quantum yield of the white light phosphors is 66.61%. The white emission phosphors show the characteristic of being independent of excitation wavelength within 310–360 nm. Furthermore, the emission intensity at 430 K of the white light phosphor Cs2NaInCl6: 0.01Sb3+, 0.65Tb3+, 0.20Sm3+ remains 50% of that at room temperature. A WLED device fabricated with the phosphor and a 365 nm LED chip exhibits a high color rendering index of 90.9, correlated color temperature of 5469 K, and CIE coordinates of (0.333 and 0.328). The results indicate that the as-prepared double perovskite materials are promising candidates in the solid-state lighting field.

Spectral characteristics of the flavones and anthocyanins present in passionflower (Passiflora incarnata).

Rich in antioxidants with a variety of flavones and anthocyanins, passionflower/fruit has been extensively used in food, beverage, medicinal, and natural dyes industries. The individual components present in passionflower are identified by extracting them in methanol, partitioning them between ethyl acetate and aqueous layers, and recording their ESI mass spectrometric data. The steady-state absorption and fluorescence spectra of the extract in methanol and dimethyl sulfoxide are recorded and the lifetime of the fluorescing species is reported. The pH dependence of the absorption spectrum confirms the presence of the anthocyanins.

Truxene-to-Fluorenone Energy Transfer in a Robust Mesoporous Zn-MOF.

A new metal-organic framework (MOF; [Zn4O(hett)4/3(fluo)1/2(bdc)1/2]n; TFT-MOF) constructed on chromophoric ligands 5,5',10,10',15,15'-hexaethyltruxene-2,7,12-triacetate (hett), 9-fluorenone-2,7-dicarboxylate (fluo), terephthalate (bdc), and the Zn4O node has been prepared and identified by powder X-ray diffraction. This luminescent MOF exhibits large mesoporous pores of 2.7 nm based on computer modeling using density functional theory (DFT) calculations. The steady-state and time-resolved fluorescence spectra and photophysical parameters of TFT-MOF have been investigated and compared with those of the free ligands and their basic chromophores. All in all, TFT-MOF exhibits particularly efficient singlet-singlet energy-transfer processes described as 1(hett)* → (fluo) and 1(bdc)* → (fluo), leading to fluorescence arising for the fluo lumophore operating only through Förster resonance energy transfer (FRET) with an efficiency of transfer of up to >95%. This experimental conclusion was corroborated by DFT and time-dependent DFT (TDDFT). For the 1(hett)* → (fluo) process, the approximated overall rate constant of energy transfer was evaluated to be at most 2.04 × 1010 s-1 (using a Stern-Volmer approach of solution data and the relationship between distance and concentration). This process was analyzed using the Förster theory, where two intrapore energy transfer paths of center-to-center distances of 13 and 25 Å have been identified. TFT-MOF photosensitizes the formation of singlet oxygen (1O2 (1Σg)) as detected by its phosphorescence signal at 1275 nm.

Decay-Associated Fluorescence for Boron Determination in Uranium-Based Nuclear Fuels.

Herein is reported a novel as well as simple, sensitive, and cost-effective method for determination of boron by time-resolved fluorescence spectrometry in uranium-based nuclear fuels. Boron is complexed with fluorescent ligand chromotropic acid, and the complex formed is a measure of boron. Since the steady-state fluorescence spectra of excess ligand and complex are overlapping, the developed method emphasizes the power of time resolution. The signatory fluorescence decay times of ligand and complex are employed to derive their decay-associated spectra (DAS) and, thereby, spectroscopically eliminate the high background of ligand fluorescence. The calibration plot has a wide linear dynamic range of 5-100 ppb with r2 better than 0.998. Precision is better than 5% at the 10 ppb level and 4% at the 50 ppb level (n = 9). The detection limit is 1.5 ppb, and recovery of spiked boron (25 ppb) from the uranium samples was better than 94%. The developed method was validated by analyzing U3O8-based ILCE Standards and applied to enriched uranium fuel samples. The main advantage of the developed method is a reduction in sample size requirement due to better sensitivity and selectivity. This in turn reduces the load of uranium recovery from analytical waste, especially in the case of enriched uranium samples. Additionally, it eliminates the need of organic solvents/medium.

Synergistic effect of dual-site occupation and activators confinement realizing an efficient ultra-broadband NIR emitter

Broadband near-infrared (NIR) phosphor-conversion light-emitting diode (pc-LED) devices have an extensive application prospect in night vision monitoring, nondestructive testing, and biomedical imaging. In this work, the ultra-broadband NIR emission produced by the chromium-activated NaZn(PO3)3 phosphor with two-site occupation peaks at around 900 nm with a full width at half-maximum (FWHM) of 218 nm. Time-resolved spectroscopic techniques and steady-state fluorescence spectra coupled with crystal structure confirm the multiple occupations of the chromium ion. Notably, the NaZn(PO3)3: Cr3+ phosphors exhibit an internal quantum efficiency (IQY) of 80.6 % and an external quantum efficiency (EQY) of 35.4 %, which may originate from the activators confinement, direct band-gap property, and high absorbance. To better comprehend the connection between structure and luminescence capabilities, the band structure, crystal field strength parameters, fluorescence lifetime, and thermal stability were all thoroughly investigated. Finally, the optimal sample has demonstrated its application in the quantitative analysis of ethanol/water mixtures. The results provide a novel perspective for the rational design of desired emitters.

How good can a simple artificial neural network predict the medium reorganization energy and the free energy gap from a steady-state fluorescence spectrum?

Medium reorganization energy, Erm, and free energy gap, ΔG, are key characteristics in determining the photoinduced charge transfer rate. They are strongly related to the width and position of the steady-state fluorescence and absorption spectra of organic dyes in solvents. Steady-state spectroscopy is one of the most standard techniques for studying organic dyes. Here, we present a simple machine learning model for cost-effective prediction the medium reorganization energy and the free energy gap from the steady-state fluorescence spectra. Fluorescence spectra with corresponding reference values of physical quantities are calculated using an explicit mathematical expression that addresses electronic-vibrational transitions, intramolecular and solvent reorganizations. An approach to the separate formation of data sets for training and validation is proposed. Gaussian noise is used in training to improve predictions. This study shows that Erm and ΔG can be predicted with the mean absolute error ∼0.024 eV and ∼0.009 eV, respectively. Robustness testing is performed on the experimental spectra of coumarin-153, coumarin-307, and coumarin-522B in a series of solvents.

Exploring the binding mechanism and adverse toxic effects of degradation metabolites of pyrethroid insecticides to human serum albumin: Multi-spectroscopy, calorimetric and molecular docking approaches.

Pyrethroid insecticides (PIs), a class of structurally similar non-persistent organic pollutants, can be degraded and metabolized to more toxic, and longer half-life products. In this study, the binding interaction mechanisms between human serum albumin (HSA) and the main degradation metabolites of PIs, 3-phenoxybenzoic acid (3-PBA) and 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA), were studied by theoretical simulation and experimental verification. Steady state fluorescence spectra showed that the fluorescence quenching mechanism was static. According to the binding constant, 4-F-3-PBA (1.53×105Lmol-1) was bound more strongly to HSA than 3-PBA (1.42×105Lmol-1) in subdomain ⅡA (site I). It was found by isothermal titration calorimetry that the metabolites and HSA spontaneously combined mainly through hydrogen bond and van der Waals interaction. Ultraviolet absorption spectra and circular dichroism spectra showed that the metabolites caused slight changes in the microenvironment and conformation of HSA. The above results were proved by molecular docking. The toxicity properties of the metabolites were further analyzed by software, and 4-F-3-PBA was found to be more toxic than 3-PBA. Considering the high exposure level of these metabolites in food, the environment and human body, it is necessary to further explore the toxicity of PIs metabolites.

Photophysical properties of 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride and its Zn(II) complex

The spectral and photophysical properties of two porphyrins, 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (1) and its Zn(II) complex 2 were investigated in different solvents by absorption, steady-state and time-resolved fluorescence spectroscopy, and laser flash photolysis. The findings were corroborated by computations at the TD-B3LYP/6-31++G(d,p) level of theory. The absorption spectra of 1 and 2 are the typical for porphyrins and their Zn complexes with the Soret bands at ≈ 420 nm and 430 nm, respectively, and the Q-bands at 515–650 nm, and 560–605 nm. Smaller differences in the absorption spectra compared to the symmetric porphyrins were assigned to more pronounced HOMO and HOMO-1 energy levels splitting in the asymmetric molecules. The temperature dependence of the absorption spectra with hypsochromic shifts suggests the formation of H-aggregates. The emission spectra are situated at 600–700 nm, and the vibronic resolution of the spectra for 1 depends on the solvent and the excitation wavelength, which was assigned due to the tautomerism by H-transfer within the porphyrin core. Quantum yields of fluorescence for 1 and 2 in methanolic solutions are Φf = 0.072 and 0.051, respectively. Although, the steady-state fluorescence spectra do not indicate that 1 and 2 form aggregates in methanolic and aqueous solutions (at <10-4 M), singlet decays measured by time-correlated single photon counting for both compounds were fit to multiexponential functions, which was explained by formation of aggregates in more concentrated solutions. Furthermore, at very high concentrations of 1 in CH3OH (>10-4 M), the dynamic formation of aggregates was evidenced by new bathochromically shifted band in the emission spectrum and the growth kinetics in the time-resolved fluorescence measurement. Triplet excited states of 1 and 2 were probed by laser flash photolysis. The triplets are populated with quantum yields of intersystem crossing, ΦISC = 0.34 and 0.41, respectively and lifetimes of the triplet excited states are in millisecond time-scale. Both triplets are quenched by O2 with the rate constants approaching diffusion limits. Relative quantum yields for the singlet oxygen formation are 0.86 and 0.92 respectively, rendering porphyrins 1 and 2 applicable for photodynamic therapy.

Relationship between non-photochemical quenching efficiency and the energy transfer rate from phycobilisomes to photosystem II.

The chromophorylated PBLcm domain of the ApcE linker protein in the cyanobacterial phycobilisome (PBS) serves as a bottleneck for Förster resonance energy transfer (FRET)from the PBS to the antennal chlorophyll of photosystem II (PS II) and as a redirection point for energy distribution to the orange protein ketocarotenoid (OCP), which is excitonically coupled to the PBLcm chromophore in the process of non-photochemical quenching (NPQ) under high light conditions. The involvement of PBLcm in the quenching process was first directly demonstrated by measuring steady-state fluorescence spectra of cyanobacterial cells at different stages of NPQ development. The time required to transfer energy from the PBLcm to the OCP is several times shorter than the time it takes to transfer energy from the PBLcm to the PS II, ensuring quenching efficiency. The data obtained provide an explanation for the different rates of PBS quenching in vivo and in vitro according to the half ratio of OCP/PBS in the cyanobacterial cell, which is tens of times lower than that realized for an effective NPQ process in solution.

Reconsideration of the ESIPT off mechanism for fluorescent probe MNC in aqueous solution

Fluorescent probes with excited state intramolecular proton transfer (ESIPT) properties play a significant role in the research of life science and material science. Guo et al. designed 3-hydroxy-2-(6-Methoxynaphthalen-2-yl)-4H-chromen-4-one (MNC) as a control to achieve the dual-color fluorescence imaging of lipid droplets and endoplasmic reticulum (ER). They deemed that the ESIPT process would be turned off in ER with high water content [J. Am. Chem. Soc. 2021, 143, 3169–3179]. However, contrary to the conventional ESIPT off case, the enol* state fluorescence intensity that should have been enhanced was severely quenched in water. Here, combined with ultrafast spectrum, steady-state fluorescence spectrum and potential energy surface, the mechanism of ESIPT process of MNC turned off in water is revised. Furthermore, the formation of aggregated states in water is responsible for the quenching of MNC fluorescence. This work is expected to provide broader ideas for the design of hydrophobic fluorescent probes.

A Comparative Study of Nanobio Interaction of Zn-Doped CdTe Quantum Dots with Lactoferrin Using Different Spectroscopic Methods.

In this paper, glutathione (GSH)-coated Zn-doped CdTe quantum dots (QDs) with different particle sizes were synthesized using the "reflow method", and the interaction mechanism between the two QDs and lactoferrin (LF) was investigated systemically with different spectroscopic methods. The steady-state fluorescence spectra showed that the LF formed a tight complex with the two QDs through static bursting and that the electrostatic force was the main driving force between the two LF-QDs systems. The complex generation process was found to be spontaneous (ΔG < 0) and accompanied by exothermic and increasing degrees of freedom (ΔH < 0, ΔS > 0) by using the temperature-dependent fluorescence spectroscopy. The critical transfer distance (R0) and donor-acceptor distance (r) of the two LF-QDs systems were obtained based on the fluorescence resonance energy transfer theory. In addition, it was observed that the QDs changed the secondary and tertiary structures of LF, leading to an increase in the hydrophobicity of LF. Further, the nano-effect of orange QDs on LF is much larger than that of green QDs. The above results provide a basis for metal-doped QDs with LF in safe nano-bio applications.

Fluorescence Resonance Energy Transfer Properties and Auger Recombination Suppression in Supraparticles Self-Assembled from Colloidal Quantum Dots

Colloid quantum dots (CQDs) are recognized as an ideal material for applications in next-generation optoelectronic devices, owing to their unique structures, outstanding optical properties, and low-cost preparation processes. However, monodisperse CQDs cannot meet the requirements of stability and collective properties for device applications. Therefore, it is urgent to build stable 3D multiparticle systems with collective physical and optical properties, which is still a great challenge for nanoscience. Herein, we developed a modified microemulsion template method to synthesize quantum dot supraparticles (QD-SPs) with regular shapes and a high packing density, which is an excellent research platform for ultrafast optical properties of composite systems. The redshift of the steady-state fluorescence spectra of QD-SPs compared to CQD solutions indicates that fluorescence resonance energy transfer (FRET) occurred between the CQDs. Moreover, we investigated the dynamic processes of energy transfer in QD-SPs by time-resolved ultrafast fluorescence spectroscopy. The dynamic redshift and lifetime changes of the spectra further verified the existence of rapid energy transfer between CQDs with different exciton energies. In addition, compared with CQD solutions, the steady-state fluorescence lifetime of SPs increased and the fluorescence intensity decreased slowly with increasing temperature, which indicates that the SP structure suppressed the Auger recombination of CQDs. Our results provide a practical approach to enhance the coupling and luminescence stability of CQDs, which may enable new physical phenomena and improve the performance of optoelectronic devices.

Enhancement on Tb3+ luminescence in CaScBO4-based ceramic phosphors by Ce3+-induced resonance energy transfer and the generation of white light emission collaborated with Mn2+

A series of Tb3+-doped, Ce3+,Tb3+-co-doped and Ce3+,Tb3+,Mn2+-tri-doped CaScBO4 (CSB) ceramic phosphors were prepared by solid-state reaction. X-ray diffraction (XRD) was taken to confirm the phase purity of all as-prepared phosphors powder. Steady-state and transient-state fluorescence spectra were applied to evaluate the luminescence properties of the phosphors. The results indicated that significant enhancement on Tb3+ luminescence was successfully realized by the Ce3+-induced resonance energy transfer and confirmed that the energy transfer from Ce3+ to Tb3+ was governed by an electronic dipole-dipole interaction. Compared to the CSB:Tb3+ phosphors whose optimal excitation located at 378 nm, the occurrence of Ce3+-Tb3+ energy transfer not only allowed the optimal excitation to peak at around 390 nm but also gifted the CSB:Ce3+,Tb3+ phosphors color tunability from blue, cyan to green. Then, two different designs of white light by the introduction of Mn2+ luminescence were performed. One was the combination of CSB:Ce3+,Tb3+ and CSB:Ce3+,Mn2+ phosphors, based on the co-excitation of the above two phosphors at the same wavelength. Another was the direct use of the tri-doped CSB:Ce3+,Tb3+,Mn2+ phosphors. Both designs succeeded in the generation of white light emission. The performance of these two designs in real devices were also studied. The direct use of the tri-doped CSB:Ce3+,Tb3+,Mn2+ phosphor in white light-emitting diode (WLED) device can produce white light with a color rendering index (CRI) up to 85.3 while the use of the CSB:Ce3+,Tb3+ and CSB:Ce3+,Mn2+ phosphors combination can just produce a value of 76.6 in CRI.

Highly Coupled Heterobicycle-Fused Porphyrin Dimers: Excitonic Coupling and Charge Separation with Coordinated Fullerene, C60.

Porphyrin dimers have been widely explored and studied owing to their importance in photosynthetic systems. A vast variety of dimers linked by different groups and at different angles have been synthesized and studied; however, the means by which to synthesize rigidly fused porphyrins with direct conjugation of the chromophores remains limited. Such a class of porphyrins may possess interesting properties that unconjugated or stacked dimers may not exhibit. In this study, bisbenzimidazole-fused porphyrin dimers and their mono- and bis-zinc derivatives are synthesized and characterized. As a consequence of excitonic coupling, these dimers exhibit a split Soret band irrespective of the metal ion in the porphyrin cavity. Steady-state fluorescence and excitation spectra followed by femtosecond transient absorption spectral studies of the heterometallated dimer, (free-base and zinc porphyrin) reveals the occurrence of efficient singlet-singlet energy transfer (>95 % efficiency and rate constant >1012 s-1 ) within the dyad. Further, donor-acceptor conjugates were formed by metal-ligand axial coordination of phenyl imidazole functionalized C60 and were characterized by a variety of physicochemical techniques. Excited state charge separation from both singlet and triplet excited states of ZnP in the conjugates has been established. The lifetime of the final charge-separated state was in the 30-40 μs range revealing charge stabilization. Interestingly, no charge separation in the conjugate derived from the heterometallated dimer was observed wherein excitation transfer dominated the process. The present study brings out the importance of the rigid π-spacer connecting porphyrin dimers in governing the energy and electron transfer events when coupled with an electron acceptor.

The Ubiquity of High-Energy Nanosecond Fluorescence in DNA Duplexes.

During the past few years, several studies reported that a significant part of the intrinsic fluorescence of DNA duplexes decays with surprisingly long lifetimes (1-3 ns) at wavelengths shorter than the ππ* emission of their monomeric constituents. This high-energy nanosecond emission (HENE), hardly discernible in the steady-state fluorescence spectra of most duplexes, was investigated by time-correlated single-photon counting. The ubiquity of HENE contrasts with the paradigm that the longest-lived excited states correspond to low-energy excimers/exciplexes. Interestingly, the latter were found to decay faster than the HENE. So far, the excited states responsible for HENE remain elusive. In order to foster future studies for their characterization, this Perspective presents a critical summary of the experimental observations and the first theoretical approaches. Moreover, some new directions for further work are outlined. Finally, the obvious need for computations of the fluorescence anisotropy considering the dynamic conformational landscape of duplexes is stressed.

Distinguishing Healthy and Carcinoma Cell Cultures Using Fluorescence Spectra Decomposition with a Genetic-Algorithm-Based Code.

In this paper, we analysed the steady state fluorescence spectra of cell suspensions containing healthy and carcinoma fibroblast mouse cells, using a genetic-algorithm-spectra-decomposition software (GASpeD). In contrast to other deconvolution algorithms, such as polynomial or linear unmixing software, GASpeD takes into account light scatter. In cell suspensions, light scatter plays an important role as it depends on the number of cells, their size, shape, and coagulation. The measured fluorescence spectra were normalized, smoothed and deconvoluted into four peaks and background. The wavelengths of intensities' maxima of lipopigments (LR), FAD, and free/bound NAD(P)H (AF/AB) of the deconvoluted spectra matched published data. In deconvoluted spectra at pH = 7, the fluorescence intensities of the AF/AB ratio in healthy cells was always higher in comparison to carcinoma cells. In addition, the AF/AB ratio in healthy and carcinoma cells were influenced differently by changes in pH. In mixtures of healthy and carcinoma cells, AF/AB decreases when more than 13% of carcinoma cells are present. Expensive instrumentation is not required, and the software is user friendly. Due to these attributes, we hope that this study will be a first step in the development of new cancer biosensors and treatments with the use of optical fibers.

Optimized RED spectral band Fluorescence of edible plants leaves extracts

Chlorophyll pigments in Ethanol solvent were extracted from 20 edible plants leaves to allocate optimized fluorescence domains for bio-spectral applications. Steady state optical absorption and fluorescence spectra of many samples were acquired at room temperature and compared for a wide range of spectral parameters. The goal was to find the plants with maximum fluorescence and least spectral bandwidth. Diluting the extracts of Chl-a concentrations starting at about 15 μg/cc showed an increase of the leading Gaussian fluorescence peak in the RED spectral band. Typical optimized maximum fluorescence of most plants was obtained for diluted concentrations near 0.5 μg/cc. Further dilutions beyond the optimal concentrations values decrease the fluorescence and changes also the bandwidth of the Gaussian fluorescence peak. They also shift the peak wavelength to 674 nm. The enhanced fluorescence obtained by dilution and peak shifting towards the blue zone, concomitantly with the Gaussian bandwidth reduction is explained. Clusters of plants in domains with optimum fluorescing properties were mapped.

Kinetics of Ca2+ Dissociation from Cod Parvalbumin Studied by Fluorescent Stopped-flow Method.

Small Ca2+-binding protein parvalbumin possesses two strong Ca2+/Mg2+- binding sites located within two EF-hand domains. Most parvalbumins have no tryptophan residues, while cod protein contains a single tryptophan residue, which fluorescence (spectrum maximum position and fluorescence quantum yield) is highly sensitive to the Ca2+ association/dissociation. Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca2+ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca2+-induced changes in the protein, both in steady-state and kinetic mode. Steady-state fluorescence spectra of cod parvalbumin were measured using Cary Eclipse spectrofluorimeter. Stopped-flow accessories in combination with a novel high-speed spectrofluorimeter were used for measurements of kinetics of Ca2+ dissociation from cod parvalbumin after fast mixing of Ca2+-loaded protein with a chelator of divalent metal cations ethylenediaminetetraacetic acid (EDTA). The fluorescent phase plots (fluorescence intensity at a fixed wavelength plotted against a fluorescence intensity at another fixed wavelength), constructed from steady state and kinetical data, shows a break at [Ca2+]/[parvalbumin] ratio close to 1. This means that the transition passes through an intermediate state, which is a protein with one bound calcium ion. These observations indicate that the binding of Ca2+ to cod parvalbumin is sequential. The results of the present spectral study showed that the binding of Ca2+ to cod parvalbumin is a sequential process. Calcium dissociation rate constants for the two binding sites of cod parvalbumin evaluated from the kinetic data are koff1 = 1.0 s-1 and koff2 = 1.5 s-1.