Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250–310 nm), two-photon (570–620 nm) and three-photon (750–930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pK_a 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λ_max 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565–580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at ?60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos⁶ θ to cos² θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.     

Characterization of photoluminescence spectra from poly allyl diglycol carbonate (CR-39) upon excitation with the ultraviolet radiation of various wavelengths

The induced photoluminescence (PL) from the π-conjugated polymer poly allyl diglycol carbonate (PADC) (CR-39) upon excitation with the ultraviolet radiation of different wavelengths was investigated. The absorption and attenuation coefficients of PADC (CR-39) were recorded using a UV–visible spectrometer. It was found that the absorption and attenuation coefficients of the PADC (CR-39) exhibit a strong dependence on the wavelength of ultraviolet radiation. The PL spectra were measured with a Flormax-4 spectrofluorometer (Horiba). PADC (CR-39) samples were excited by ultraviolet radiation with wavelengths in the range from 260 to 420 nm and the corresponding PL emission bands were recorded. The obtained results show a strong correlation between the PL and the excitation wavelength of ultraviolet radiation. The position of the fluorescence emission band peak was red shifted starting from 300 nm, which was increased with the increase in the excitation wavelength. The PL yield and its band peak height were increased with the increase in the excitation wavelength till 290 nm, thereafter they decreased exponentially with the increase in the ultraviolet radiation wavelength. These new findings should be considered carefully during the use of the PADC (CR-39) in the scientific applications and in using PADC (CR-39) in eyeglasses.     

Optical Properties of Carbazole-Based Photorefractive Polymers

The optical properties of several carbazole-based photorefractive polymers which we prepared were determined. Through analysis of the UV-vis spectrum, the ratio of the azo nitrobenzene was adjusted to control the content of azo chromophore functional components. The solid transient fluorescence emission spectra of the photorefractive polymers were tested with a 325 nm emission wavelength. The results showed that the J-aggregates and several large closed-loop structures formed in the photorefractive polymers resulted in a long lifetime of their transient fluorescence. Spin-orbit coupling among the molecules increased the intersystem crossing rate, and induced delayed fluorescence.     

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

The emission behaviour of Aequorea green fluorescent protein (A-GFP) chromophore, in both neutral (N) and anionic (A) form, was studied in the temperature range from 20 °C to 75 °C and at pH = 7. Excitation wavelengths of 399 nm and 476 nm were applied to probe the N and A forms environment, respectively. Both forms exhibit distinct fluorescence patterns at high temperature values. The emission quenching rate, following a temperature increase, is higher for the chromophore N form as a result of the hydrogen bond network weakening. The chromophore anionic form emission maximum is red shifted, upon temperature increase, due to a charge transfer process occurring after A form excitation.     

Combined Quenching Mechanism of Anthracene Fluorescence by Cetylpyridinium Chloride in Sodium Dodecyl Sulfate Micelles

The Stern-Volmer quenching constant (K_SV) for quenching of anthracene fluorescence in sodium dodecyl sulfate (SDS) micelles by pyridinium chloride has been reported previously to be 520 M^?1 based on steady state fluorescence measurements. However, such measurements cannot distinguish static versus dynamic contributions to the overall quenching. In the work reported here, the quenching dynamics of anthracene in SDS micelles by cetylpyridinium chloride (CPC), an analogue of pyridinium chloride, were investigated using both steady state and time resolved fluorescence quenching. Concurrent measurement of the decrease in fluorescence intensity and lifetime of anthracene provide a quantitative evaluation of collision induced (i.e. dynamic) versus complex formation (i.e. static) quenching of the anthracene fluorophore. The results reveal that a combined quenching mechanism is operative with approximately equal constants of 249?±?6 M^?1 and 225?±?12 M^?1 for dynamic and static quenching, respectively.     

Fluorescence Properties of Intercalating Neutral Chromophores in Complexes with Polynucleotides of Various Base Compositions and Secondary Structures

Changes in the relative quantum yield and polarization degree of steady-state fluorescence of daunomycin (DM) on its binding to six synthetic single- and double-stranded polynucleotides of various nucleotide compositions were measured over a wide range of molar polymer-to-dye ratios, in solutions of low ionic strength. Guanine base was found to be an effective quencher of DM fluorescence [in the DM–poly(G) complex the intensity of residual emission was ~0.5% of the free dye intensity]. The quenching of DM fluorescence by another purine base, i.e., adenine, was also revealed. But, unlike guanine, adenine exhibits quenching activity when it is in close contact with the DM chromophore, as realized in the complex with single-stranded poly(A). In intercalative complexes with double-stranded nucleic acids, where such contact is lacking, the quenching ability of adenine does not manifest itself, which has been demonstrated with the DM–poly(A) · poly(U) complex. It was found that the interaction with pyrimidine bases does not substantially change the DM quantum yield. The quenching feature of DM fluorescence is identical to that observed earlier by us for a glycoside phenazine dye containing, like DM, a neutral chromophore.     

A Ratiometric and near-Infrared Fluorescent Probe for Imaging Cu<Superscript>2+</Superscript> in Living Cells and Animals

We have rationally constructed a novel ratiometric and near-infrared Cu²⁺ fluorescent probe based on a tricarbocyanine chromophore. The new probe NIR-Cu showed a ratiometric fluorescent response to Cu²⁺ with a large emission wavelength shift (up to 142 nm) in the far-red to near-infrared region. The probe also displayed a large variation in the fluorescence ratio (I₆₃₆/I₇₇₈) to Cu²⁺ species with high sensitivity and selectivity. Additionally, the developed probe NIR-Cu was suitable for fluorescence imaging of Cu²⁺ in living cells and mice.     

头孢呋辛酯与牛血清白蛋白相互作用特征研究

采用荧光光谱、三维荧光光谱、同步荧光光谱和紫外吸收光谱法,研究了不同温度下头孢呋辛酯（CFA）的浓度在1.959×10-6至13.71×10-6 mol·L-1范围内,牛血清白蛋白(BSA)的浓度为2.0×10-6 mol·L-1时两者之间的相互作用,按照Sterm-Volmer方程、Lineweaver-Burk方程和热力学方程分析和处理实验数据,计算了表观作用常数（K_LB: 3.907×106 L·mol-1）,热力学参数的平均值（焓变ΔH：-13.43 kJ·mol-1,熵变ΔS：81.90 J·K-1和标准吉布斯自由能变化ΔGθ：-38.34 kJ·mol-1）,测定了作用位点数（n: 1.042）,讨论了CFA对BSA的荧光猝灭作用机理。BSA和CFA作用可能形成了一种新的复合物,猝灭作用主要属于静态猝灭。认可热力学参数ΔH≈0, ΔS＞0和ΔGθ＜0,反应力主要是熵驱动力和静电作用力。在同步荧光光谱中色氨酸和酪氨酸的峰波长明显红移;在三维荧光光谱中,在加入CFA后二峰的发射波长蓝移;在紫外-可见吸收光谱图中,三体系的最大吸收明显不同,这些均显现色氨酸和酪氨酸所处的微环境的变化,同时说明了加入CFA后BSA的构象发生了变化。这为讨论BSA的构象变化,阐明CFA的药理作用和在生物体内的生物学效应等提供重要信息。     

Tryptophan Environment and Functional Characterization of a Kinetically Stable Serine Protease Containing a Polyproline II Fold

The single tryptophan residue from Nocardiopsis sp. serine protease (NprotI) was studied for its microenvironment using steady state and time-resolved fluorescence. The emission maximum was observed at 353 nm with excitation at 295 nm indicating tryptophan to be solvent exposed. Upon denaturation with 6 M guanidinum thiocyanate (GuSCN) the emission maxima was shifted to 360 nm. Solute quenching studies were performed with neutral (acrylamide) and ionic (I^- and Cs⁺) quenchers to probe the exposure and accessibility of tryptophan residue of the protein. Maximum quenching was observed with acrylamide. In the native state, quenching was not observed with Cs⁺ indicating presence of only positively charged environment surrounding tryptophan. However; in denatured protein, quenching was observed with Cs⁺, indicating charge reorientation after denaturation. No quenching was observed with Cs⁺ even at pH 1.0 or 10.0; while at acidic pH, a higher rate of quenching was observed with KI. This indicated presence of more positive charge surrounding tryptophan at acidic pH. In time resolved fluorescence measurements, the fluorescence decay curves could be best fitted to monoexponential pattern with lifetimes of 5.13 ns for NprotI indicating one conformer of the trp. Chemical modification studies with phenyl glyoxal suggested presence of Arg near the active site of the enzyme. No inhibition was seen with soyabean trypsin and limabean inhibitors, while, CanPI uncompetitively inhibited NprotI. Various salts from Hofmeister series were shown to decrease the activity and PPII content of NprotI.     

Study on the Fluorescence Quenching Reaction of Amitriptyline and Clomipramine Hydrochlorides with Eosin Y and its Analytical Application

Amitriptyline.HCl (AMI) and clomipramine.HCl (CMI) react with eosin Y (EY) in pH 3.8 NaAc-AcH buffer solution to form ion association complex which results in quenching of fluorescence of EY and appearance of a new resonance Rayleigh scattering (RSS) spectrum at 620 nm. The spectral characteristics of absorption, fluorescence and RSS spectra have been investigated. The factors influencing the reaction were studied and optimum conditions for the reaction have been determined. Based on fluorescence quenching, a simple and sensitive spectrofluorimetric method for determination of AMI and CMI has been developed. The fluorescence quenching intensity was measured at 550 nm using an excitation wavelength of 310 nm. The calibration graph was found to be rectilinear in the range 0.08–2.0 μg?mL^?1 with detection limit of 0.017 μg?mL^?1 for AMI and 0.06–2.0 μg?mL^?1 with detection limit of 0.015 μg?mL^?1 for CMI. The method can be satisfactorily applied to the determination of AMI and CMI in tablets without interference from commonly occurring exicipients. The recovery and RSD values obtained indicate good accuracy and precision of the method. The mechanism of the reaction and fluorescence quenching has also been discussed.     

Red Emissive Biocompatible Nanoparticles from Tetraphenylethene‐Decorated BODIPY Luminogens for Two‐Photon Excited Fluorescence Cellular Imaging and Mouse Brain Blood Vascular Visualization

BODIPY (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene) is an emissive chromophore in solutions but suffers from fluorescence quenching when aggregated due to its flat molecular conformation and small Stokes shift. To create aggregate‐state emissive BODIPY luminogens, tetraphenylethene (TPE), which is a popular luminogen with intriguing aggregation‐induced emission (AIE) characteristic, is introduced as periphery to a methylated BODIPY core. Three TPE‐BODIPY adducts are synthesized and characterized, and their photophysical properties and electronic structures are investigated. The incorporation of AIE‐active TPE units alleviates aggregation‐caused quenching of BODIPY core, furnishing emissive nanoparticles based on TPE‐BODIPY adducts. Significantly, the two‐photon absorption (TPA) and two‐photon excited fluorescence (TPEF) properties are improved as more TPE units are attached. The luminogens with 3TPE units (3TPE‐BODIPY) shows the strongest TPA and TPEF in the wavelength range of 750–830 nm, with cross‐section values of 264 and 116 GM at 810 nm, respectively. Red emissive nanoparticles with a Stokes shift of 60 nm and a fluorescence quantum yield of 16% are attained by encapsulating 3TPE‐BODIPY with 1,2‐sistearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol)‐2000]. The nanoparticles are biocompatible and function well in TPEF cellular imaging and mouse brain blood vascular visualization.     

Photochemical Properties of a Novel Cyanobacteriochrome from Anabaena sp. PCC7120

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as photoreceptor sensors. We report the photochemistry of All4261 GAF2, a novel cyanobacteriochrome from the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. All4261 contains four tandemly arranged GAF domains. The respective chromophore domains were co-expressed in Escherichia coli with the genes for phycocyanobilin biosynthesis enzymes, HO1, and PcyA. The resulting proteins were analyzed by zinc-induced fluorescence, UV-Vis absorption and emission fluorescence spectra. Only All4261 GAF2 binded chromophore covalently, having zinc-induced fluorescence band in SDS-PAGE gel with the zinc acetate solution existed. Absorption spectra analysis showed that All4261 GAF2 had absorption peaks at 340 nm and 590 nm, failing to show photoreversibility. Fluorescence spectra revealed that All4261 GAF2 had a fluorescence emission peak at 645 nm, with high fluorescence quantum yield and molar extinction coefficient.     

Laser-induced synthesis of silver nanoparticles and their conjugation with protein

Partially oxidized spherical silver nanoparticles (AgNPs) of different size are prepared by pulsed laser ablation in water and directly conjugated to protein S-ovalbumin for the first time and characterized by various optical techniques. UV–Visible spectrum of AgNPs showed localized surface plasmon resonance (LSPR) peak at 396 nm which red shift after protein addition. Further the increased concentration of AgNPs resulted a decrease in intensity and broadening of S-ovalbumin peak (278 nm), which can be related to the formation of protein NPs complex caused by the partial adsorption of S-ovalbumin on the surface of AgNPs. The red shift in LSPR peak of AgNPs after mixing with S-ovalbumin and decrease in protein-characteristic peak with increased silver loading confirmed the formation of protein–AgNPs bioconjugates. The effect of laser fluence on the size of AgNPs and nanoparticle–protein conjugation in the size range 5–38 nm is systematically studied. Raman spectra reveal broken disulphide bonds in the conjugated protein and formation of Ag–S bonds on the nanoparticle surface. Fluorescence spectroscopy showed quenching in fluorescence emission intensity of tryptophan residue of S-ovalbumin due to energy transfer from tryptophan moieties of albumin to AgNPs. Besides this, small blue shift in emission peak is also noticed in presence of AgNPs, which might be due to complex formation between protein and nanoparticles. The binding constant (K) and the number of binding sites (n) between AgNPs and S-ovalbumin have been found to be 0.006 M^?1 and 7.11, respectively.     

Fluorescence,Photophysical Behaviour and DFT Investigation of E,E-2,5-bis[2-(3-pyridyl)ethenyl]pyrazine (BPEP)

E,E-2,5-bis[2-(3-pyridyl)ethenyl]pyrazine (BPEP) has been prepared by aldol condensation between 2,5-dimethylpyrazine and pyridine-3-carboxaldehyde. It is characterized by IR, ¹H NMR, and ¹³C NMR. The electronic absorption and emission properties of BPEP were studied in different solvents. BPEP displays a slight solvatochromic effect of the absorption and emission spectrum, indicating a small change in dipole moment of BPEP upon excitation. The dye solutions (1 × 10^?4 M) in CHCl₃, EtOH and dioxane give laser emission in blue region upon excitation by a 337.1 nm nitrogen pulse (λ = 337 nm). The tuning range, gain coefficient (α) and emission cross – section (σ_e) have been determined. Ground and excited states electronic geometric optimizations were performed using density functional theory (DFT) and time-dependent density functional theory (TD-DFT), respectively. A DFT natural bond analysis complemented the ICT. The simulated maximum absorption and emission wavelengths are in line the observed ones in trend, and are proportionally red-shifted with the increase of the solvent polarity. The stability, hardness and electrophilicity of BPEP in different solvents were correlated with the polarity of the elected solvents. BPEP dye displays fluorescence quenching by colloidal silver nanoparticles (AgNPs). The fluorescence data reveal that radiative and non-radiative energy transfer play a major role in the fluorescence quenching mechanism.     

Hydrophobic Interactions in Donor-Disulphide-Acceptor (DSSA) Probes Looking Beyond Fluorescence Resonance Energy Transfer Theory

Donor –linker –acceptor (DSSA) is a concept in fluorescence chemistry with acceptor being a fluorescent compound (FRET) or quencher. The DSSA probes used to measure thiol levels in vitro and in vivo. The reduction potential of these dyes are in the range of ?0.60 V, much lower than the best thiol reductant reported in literature, the DTT (?0.33 V). DSSA disulphide having an unusually low reduction potential compared to the typical thiol reductants is a puzzle. Secondly, DSSA probes have a cyclized rhodamine ring as acceptor which does not have any spectral overlap with fluorescein, but quenches its absorbance and fluorescence. To understand the structural features of DSSA probes, we have synthesized DSSANa and DSSAOr. The calculated reduction potential of these dyes suggest that DSSA probes have an alternate mechanism from the FRET based quenching, namely hydrophobic interaction or dye to dye quenching. The standard reduction potential change with increasing complexity and steric hindrance of the molecule is small, suggesting that ultra- low Eo’ has no contribution from the disulphide linker and is based on structural interactions between fluorescein and cyclized rhodamine. Our results help to understand the DSSA probe quenching mechanism and provide ways to design fluorescent probes.     

Excitation-wavelength Dependent Fluorescence of Ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate

The excitation wavelength dependence of the steady-state and time-resolved emission spectra of ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate (EAADCy) in tetrahydrofuran (THF) at room temperature has been examined. It is found that the ratio of the fluorescence intensity of the long-wavelength and short-wavelength fluorescence bands strongly depends on the excitation wavelength, whereas the wavelengths of the fluorescence excitation and fluorescence bands maxima are independent on the observation/excitation wavelengths. The dynamic Stokes shift of fluorophore in locally excited (LE) and intramolecular charge transfer (ICT) states has been studied with a time resolution about 30 ps. The difference between Stokes shift in the LE and ICT states was attributed to the solvent response to the large photoinduced dipole moment of EAADCy in the fluorescent charge transfer state. On this base we can state that, the relaxation of the polar solvent molecules around the fluorophore was observed.     

Conformational Flexibility of Cytokine-Like C-Module of Tyrosyl-tRNA Synthetase Monitored by Trp144 Intrinsic Fluorescence

The non-catalytic COOH-terminal module formed after proteolytic cleavage of full-length mammalian tyrosyl-tRNA synthetase displays dual function: tRNA binding ability and cytokine activity. With the aim to explore the intramolecular dynamics of C-module in solution we used fluorescence spectroscopy to study conformational changes of isolated protein. We used information from fluorescence spectra and computational model for characterization of a microenvironment of a single tryptophan residue (Trp144). Its fluorescence parameters and protection from quenching by Cs⁺ ions indicate the internal localization—buried into protein globule. The fluorescence quenching of Trp144 by acrylamide suggests rapid conformation dynamics of the C-module in nanosecond time scale. The temperature-induced conformational changes in the C-module were monitored by the fluorescence measurements of Trp144 emission and by red-edge excitation shift. An emission maximum shift up to ∼349 nm and significant decrease of the red-edge shift effect at 37–52 °C indicated a major conformational transition of Trp144 from buried native state into highly relaxing polar solvent environment.     

Photoactivated Green Fluorescence Emission by Femtosecond Oscillator from Indole Solutions

We reported a novel femtosecond-laser-activated fluorescence emission from indole solutions upon excitation by the second harmonic wavelength of a femtosecond oscillator. A new absorption band around 400 nm and corresponding fluorescent band in the green domain were produced after the irradiation of femtosecond laser. This femtosecond-laser-activated luminescence process that allows the use of visible wavelength as a substitute for UV light to excite fluorescence from indole would extend applications based on indole chromophore. Furthermore, the photoactived emission can act as a fluorescence lifetime probe to measure the polarity in complex biological systems since it is polarity-sensitive. High performance liquid chromatography with fluorescence detector (HPLC-FLU) and high performance liquid chromatography with mass spectrometer (HPLC-MS) analysis demonstrate that the origin of the photoactivated fluorescence is new molecular species that generated in indole solution upon femtosecond laser irradiation.     

Synthesis of Water Dispersible Fluorescent Carbon Nanocrystals from <Emphasis Type="Italic">Syzygium cumini</Emphasis> Fruits for the Detection of Fe<Superscript>3+</Superscript> Ion in Water and Biological Samples and Imaging of <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> Cells

In this work, water dispersible fluorescent carbon nanocrystals (NCs) were synthesized by a simple, green and low cost hydrothermal method using Syzygium cumini (jamun) as a carbon source at 180 °C for 6 h. The average size of carbon NCs was found to be 2.1 ± 0.5 nm and shown bright blue fluorescence when excited at 365 nm under UV lamp. The carbon NCs were characterized by spectroscopic (UV-visible and fluorescence, Fourier transform infrared and dynamic light scattering) and high resolution transmission electron microscopic techniques. The quantum yield of carbon NCs was found to be ~5.9 % at 438 nm emission wavelength when excited at 360 nm. It was noticed that none of the metal ions quenched the fluorescence intensity of carbon NCs at 438 nm except for Fe³⁺, indicating the formation of Fe³⁺ ion-carbon NCs complexes. The linear range was observed in the concentration range of 0.01–100 μM with the corresponding detection limits of 0.001 μM, respectively. Furthermore, the carbon NCs were used as probes for imaging of fungal (Fusarium avenaceum) cells.     

A Dual-Readout Method for Biothiols Detection Based on the NSET of Nitrogen-Doped Carbon Quantum Dots–Au Nanoparticles System

In this paper, a rapid, simple and highly sensitive method with dual-readout (colorimetric and fluorometric) based on the nanometal surface energy transfer (NSET) between nitrogen-doped carbon quantum dots (NCQDs) and gold nanoparticles (AuNPs) for detection of biothiols is described. Highly luminescent NCQDs were prepared via a simple one-step hydrothermal method by applying sucrose and glycine as carbon and nitrogen sources. The results showed the obtained NCQDs had an average particle diameter of 5 nm and highly luminescent. The maximum emission wavelength was 438 nm with an excitation wavelength of 360 nm. In this system, NCQDs and AuNPs were respectively treated as energy donors and energy acceptors, which enable the nanometal surface energy transfer (NSET) from the NCQDs to the AuNPs, quenching the fluorescence. However, biothiols was used as a competitor in the NSET by the strongly Au-S bonding to release NCQDs from the Au surface, which subsequently produces fluorescent signal recovery and the red-to-purple color change quickly. This probe showed rapid response, high selectivity and sensitivity for biothiols with dual colorimetric and fluorescent turn-on signal changes. The low detection limit was calculated as 20 nM by using L-cysteine acted as target melocules. The method was also successfully applied to the determination of biothiols in human serum samples, and the results were satisfying.