Degree Of Fluorescence Polarization Research Articles

Spectroscopic features of Pheophorbide-a binding to poly-L-lysine

Binding of Pheophorbide-a of to poly-L-lysine (PLL) have been studied in neutral aqueous buffered solutions of low and near-physiological ionic strengths with low ethanol content (2.4–5.9%) in a wide range of molar polymer-to-dye ratios (P/D) using absorption and polarized fluorescence spectroscopy, fluorimetric titration, fluorescence melting. The binding has highly cooperative character (cooperativity parameter q ≈ 1000) and results in 40-fold quenching of the dye fluorescence as well as in increase in the fluorescence polarization degree up to 0.16, that evidences formation of π-stacked dye aggregates on the polypeptide exterior. The spectroscopic properties of these aggregates were established. The cooperative biding constants were estimated by Schwarz's method.

Two-dimensional fluid viscosity measurement in microchannel flow using fluorescence polarization imaging

This study describes the development of a noncontact and two-dimensional fluid viscosity measurement technique based on fluorescence polarization microscopy. This technique exploits fluorescence depolarization due to rotational Brownian motion of fluorophores and determines fluid viscosity in microchannel flow by measuring steady-state fluorescence polarization. The main advantage of the technique is that planar distributions of fluid viscosity can be visualized by noncontact optical measurement, while commonly-used mechanical viscometers measure the viscosity of bulk liquids. Moreover, steady-state polarization measurements are realized using a simpler experimental setup compared to other noncontact techniques such as time-resolved fluorescence lifetime/polarization measurements. The relationship between the fluid viscosity (μ) and the fluorescence polarization degree (P) was experimentally obtained using casein molecules labeled with fluorescein isothiocyanate as a fluorescent probe. The fluid viscosity was controlled within the range of 0.7–3.0 mPa s, which is the range often encountered in biological materials, by mixing sucrose or glucose with the solution. The fluid temperature was maintained uniform at 30 °C during the measurement. The calibration result showed that 1/P linearly increased with 1/μ which qualitatively agreed well with the theoretical prediction. The measurement uncertainty was 7.5%–9.5% based on the slope of the calibration curve. The viscosity gradient generated by the mass diffusion between the two solutions co-flowing in the Y-shaped microchannel was clearly visualized under uniform temperature conditions by applying the calibration curve. Finally, the influence of the temperature change on P was experimentally evaluated. The results supported the applicability of the present technique for visualization of the viscosity distribution induced by temperature change. These results confirmed the feasibility of the present technique for analyzing microscale viscosity fields associated with mass transport or temperature change.

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03193-y.

Spectroscopic study of binding of a cationic Pheophorbide-a to an antiparallel quadruplex Tel22

Binding of water-soluble cationic Pheophorbide- a derivative (CatPheo- a ) to Na + -stabilized antiparallel quadruplex formed by 22-mer oligonucleotide d[AG 3 (T 2 AG 3 ) 3 ], a fragment of human telomeric DNA (Tel22, PDB ID: 143D), has been examined using experimental techniques of absorption and polarized fluorescent spectroscopy as well as absorption melting. The binding affinity of CatPheo- a to Tel22 was studied in titration experiments registering the dependence of the dye fluorescence intensity and polarization degree on molar phosphate-to-dye ratio ( P/D ). CatPheo- a was found to bind effectively to the quadruplex, and two competitive binding modes were detected. The first one predominates at the dye excess and results in the fluorescence quenching, whereas the second one is preferential at the biopolymer excess and results in the enhancement of pheophorbide emission. The effect of CatPheo- a on thermodynamic parameters of Tel22 quadruplex unfolding was estimated using a two-state model. It was found that CatPheo- a destabilizes the quadruplex structure of Tel22 slightly decreasing its 4→1 transition midpoint temperature, gives destabilizing increment into Gibbs standard free energy and 2-fold decrease in the equilibrium quadruplex folding constant at 37°C. In ethanol CatPheo- a exhibits 15% higher efficiency of singlet oxygen generation as compared to the parent Pheo- a compound that makes it a promising photosensitizer for photodynamic therapy.

Broadband polarized emission from P(NDI2OD-T2) polymer

We investigate the P(NDI2OD-T2) photophysical properties via absorbance and fluorescence spectroscopy, in association with the experimental approach baptized Stokes Spectroscopy, which provides valuable material information through the acquisition and analysis of the fluorescence polarization degree. By changing solvents and using different samples such as solutions, thick, and thin films, it is possible to control the polarization degree spectrum associated to the fluorescence emitted by the polymer’s isolated chains and aggregates. We show that the polarization degree could become a powerful tool to obtain information related to the samples morphology, which is connected to their microscopic structure. Moreover, the polarization degree spectra suggest that depolarization effects linked to energy and charge transfer mechanisms are likely taking place. Our findings indicate that P(NDI2OD-T2) polymers are excellent candidates for the advancement of organic technologies that rely on the emission and detection of polarized lights.

Liquid temperature measurement method in microchannels by using fluorescence polarization

A novel optical method that can measure fluid temperature at the microscopic scale by measuring fluorescence polarization is described in this paper. The measurement is much less influenced by fluorescence quenching effects, which is a significant issue in conventional laser-induced fluorescence methods. Therefore, the effects of the other properties of the fluid can be reduced considerably in the proposed method, thus has the potential of leading to greater reliability in measuring the temperature. An experiment was performed in a microchannel flow by using fluorescent molecule probes. The relationship between the fluid temperature and the measured fluorescence polarization degree is evaluated to derive the correlation curve. In addition, the effects of the fluid viscosity and fluid pH on the fluorescence polarization degree are discussed to evaluate the influence of the quenching effects. The results showed that the fluorescence polarization is considerably less sensitive to the quenching factors as compared with the fluorescence intensity measurements. Furthermore, a strong linear correlation between the polarization degree and the fluid temperature was obtained. This relationship agreed well with the theoretical one qualitatively and confirmed the validity of the measurements and feasibility of the proposed method.

Fluorescence polarization immunoassay of ractopamine

A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopamine–fluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.3–50 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.

Micro-scale temperature measurement method using fluorescence polarization

A novel method that can measure the fluid temperature in microscopic scale by measuring the fluorescence polarization is described in this paper. The measurement technique is not influenced by the quenching effects which appears in conventional LIF methods and is believed to show a higher reliability in temperature measurements. Experiment was performed using a microchannel flow and fluorescent molecule probes, and the effects of the fluid temperature, fluid viscosity, measurement time, and pH of the solution on the measured fluorescence polarization degree are discussed to understand the basic characteristics of the present method. The results showed that fluorescence polarization is considerably less sensible to these quenching factors. A good correlation with the fluid temperature, on the other hand, was obtained and agreed well with the theoretical values confirming the feasibility of the method.

Binding of Metallated Porphyrin-Imidazophenazine Conjugate to Tetramolecular Quadruplex Formed by Poly(G): a Spectroscopic Investigation.

The binding of telomerase inhibitor ZnTMPyP(3+)-ImPzn, Zn(II) derivative of tricationic porphyrin-imidazophenazine conjugate, to tetramolecular quadruplex structure formed by poly(G) was studied in aqueous solutions at neutral pH and near physiological ionic strength using absorption and polarized fluorescent spectroscopy techniques. Three binding modes were determined from the dependences of the fluorescence intensity and polarization degree for the porphyrin and phenazine moieties of the conjugate on molar polymer-to-dye ratio (P/D). The first one is outside electrostatic binding of positively charged porphyrin fragments to anionic phosphate groups of the polymer which prevails only at very low P/D values and manifests itself by substantial fluorescence quenching. It is suggested that the formation of externally bound porphyrin dimers occurs. The other two binding modes observed at high P/D are embedding of the ZnTMPyP(3+) moiety into the groove of poly(G) quadruplex accompanied by more than 3-fold enhancement of the conjugate emission, and simultaneous intercalation of the phenazine fragment between the guanine bases accompanied by the increase of its fluorescence polarization degree up to 0.25. Thus Zn(II) conjugate seems to be promising ligand for the stabilization of G-quadruplex structures since porphyrin binding to poly(G) is strengthened by additional intercalation of phenazine moiety.

G143 蛍光偏光法によるマイクロ流路内の流体温度計測手法の開発(OS-12:先端計測による熱工学の学際的展開(1))

A method that can measure the fluid temperature in microscopic scale by measuring the fluorescence polarization is described in this study. To understand the basic characteristics and advantage of the method, the effects of the fluid temperature, fluid viscosity, measurement time, and pH of the solution on the measured fluorescence polarization degree are discussed. The results showed that fluorescence polarization is considerably less sensible to the measurement time and pH compared to the fluorescence intensity demonstrating the stability of the temperature measurement. The correlations between the fluid temperature and the inverse value of P measured by two photo detectors (photomultiplier and CCD camera) both showed a linear relationship. This agreed with the theoretical characteristic, and confirmed the feasibility of this method.

Excitonic nature of the dual fluorescence of 2,3-diazabicyclo[2.2.2]oct-2-ene and 1,4-dimethyl-(2,3-diazabicyclo[2.2.2]oct-2-ene)

Absorption, fluorescence excitation, and fluorescence spectra and the dependence of the degree of fluorescence polarization on the emission wavelength are measured for glass-like ethanol solutions of 2,3-diazabicyclo[2.2.2]oct-2-ene and 1,4-dimethyl-(2,3-diazabicyclo[2.2.2]oct-2-ene) at a temperature of 77 K. The analysis of the spectral polarization data shows that two excited electronic states S1 and S2 that contribute to the emission of the compounds are related to the exciton splitting and correspond to the symmetric (S2) and antisymmetric (S1) wave functions.

Analysis of Pigment Orientation in Photosystem II at Different Temperatures by Polarization Fluorescence and Molecular Exciton Theory

The effect of temperature on pigment orientation in photosystem II (PSII) was studied by fl uorescence excitation and polarization fl uorescence spectra of spinach thylakoid solution and molecular exciton theory. Experimental results showed that at 15 to 45 °C, the absorption band of chlorophyll a at 436 nm at room tempe rature red-shifted with increased temperature. The excitation spectra intensity reached the maximum at 35 °C but signifi cantly dropped at 65 and 78 °C. The polarization fl uorescence spectra revealed that the fl uorescence peak of PSII did not change at 15 and 45 °C, and the calculated degree of fl uorescence polarization increased with increased temperature. Spectral and molecular exciton theory analyses indicated that temperature affected pigment orientation in PSII, as well as the coupling strength between pigments or pigment and protein, thereby changing photosynthetic effi ciency. These results can serve as a reference for studies on energy absorption, energy transmission, regulation mechanism, and prospective applications in solar cell materials.

Polarization of the fluorescence excited in erbium-doped fibers in 1490–1570 nm spectral range

We present data on the polarization degree of the fluorescence, excited in erbium-doped fibers (EDF) by linearly polarized light at three essentially different wavelengths in the spectral range of the fundamental absorption of Er3+ (1492, 1526, and 1568nm). Experiments were performed in otherwise similar single-mode fibers with a broad range of erbium concentration (≈640–5600wtppm). The polarization degree of the fluorescence demonstrated expected decay with the incident light power and proved to be significantly weaker for the central excitation wavelength of 1526nm. The maximum value of the experimentally observed effect (≤0.3%) is significantly lower than it is predicted by model of the randomly oriented non-interacting dipoles. This discrepancy is explained by the reduced anisotropy (to the value 0.7–0.8) of the erbium ions absorption/emission, the effect which was utilized to explain the experimentally observed weak polarization hole burning in EDF earlier. Additional reduction in the fluorescence polarization degree and accelerated depolarization relaxation which were experimentally observed in fibers with high erbium concentration are attributed to the spatial migration of the excited state.

Denaturation of bovine serum albumin under the action of cetyltrimethylammonium bromide, according to data from fluorescence analysis

The tryptophan fluorescence of bovine serum albumin (BSA) in solutions with different concentrations of cationic detergent cetyltrimethylammonium bromide (CTAB) at different pH is investigated, providing information on BSA denaturation under the action of CTAB. It is found that BSA denaturation under the action of CTAB at all of the investigated pH values (3.5–8.0) is a single-stage process, as determined by BSA tryptophan fluorescence quenching, by an increased degree of the BSA tryptophan fluorescence polarization, and by the values of the parameters for the rotational diffusion of BSA molecules in CTAB solutions. It is shown that the cationic detergent CTAB is more efficient for BSA denaturation at pH values higher than the BSA isoelectric point (4.9).

Investigation of the rotational diffusion of the Rose Bengal fluorescent nanomarker in human serum albumin solutions

The polarized fluorescence of the Rose Bengal fluorescent nanomarker in HSA solutions was investigated and parameters of its rotational diffusion were calculated. The increase in the degree of fluorescence polarization, rotational relaxation time, and the effective hydrodynamic radius of Rose Bengal, as well as the decrease of the rotational diffusion coefficient in HSA solutions, were found. The effects of the electronegativity of atoms in the structure of the nanomarker on the parameters of its rotational diffusion were established based on comparison of Rose Bengal with other nanomarkers of the homologous family.

Doubly and Triply Coupled Nanowire Antennas

Nanoantenna is one of the most important optical components for light harvesting. In this study, we show experimental evidence of interactions between coupled nanowires by comparing the fluorescence properties of quantum dots on single nanowire as well as doubly and triply coupled nanowire arrays. Because of the localized surface plasmon mode, there are strong polarization dependences in this photon–plasmon–exciton conversion process. It is interesting that both the polarization-dependent enhancement and the degree of fluorescence polarization are more pronounced for triply coupled nanowires than that of doubly coupled nanowire, while the case of single nanowire is weakest. Our theoretical analysis indicates the above phenomena can be ascribed to the coupled plasmon from the nanowire antennas. Our investigations demonstrate a potential method to control the polarization of emitters using coupled nanowire arrays.

Fluorescent and photooptical properties of H-bonded LC composites based on stilbazole derivative

A novel series of H-bonded LC (smectic and nematic) composites and “model” mixtures without hydrogen bonding based on the stilbazole derivative were prepared. As the matrices of the composites following polymers were synthesized: smectic homopolymer containing p-oxybenzoic groups, nematic copolymer containing p-oxybenzoic and methoxyphenylbenzoic side groups, and nematic homopolymer with methoxyphenylbenzoate groups. The spectral properties of composite films were studied in detail. Significant differences in the fluorescence spectra of H-bonded LC composites and “model” mixtures were revealed. Mechanism of the processes occurring in the films of the LC composites under the action of the UV light was proposed. The mechanism includes a reversible proton transfer involved in the H-bonds formation. It was found that in the oriented films of the nematic composite degree of fluorescence polarization is higher than in the films of a similar mixture without H-bonding. The effect of linearly polarized light on the optical properties of the composite films was studied. It was shown that the highest values of the photoinduced dichroism are achieved in nematic liquid crystals composites. It was revealed that for the irradiated LC composites annealing leads to decease of dichroism values (degree of polarized emission drops to zero), whereas for “model” LC mixture without hydrogen bonding a significant increase in these parameters was found.

Rotational diffusion of fluorescein family markers in solutions of human serum albumin

Rotational diffusion of fluorescein family nanomarkers (initial fluorescein and its halogenated derivatives, eosin and erythrosine) in solutions of human serum albumin (HSA) was studied at various pH values. In solutions of HSA, the degree of fluorescence polarization, rotational relaxation time, and Einstein radius of nanomarkers are larger and the rotational diffusion coefficient of nanomarkers smaller than in solutions without the protein. An increase in the electronegativity of atoms in the structural formulas of nanomarkers increases the degree of polarization of their fluorescence, decreases the coefficient of their rotational diffusion, and increases rotational relaxation time and the effective Einstein radius.

Effect on optical anisotropy of intramolecular lability in various molecular rotational configurations

It is shown that the effect of intramolecular lability in complex molecules on the degree of fluorescence polarization of rarified vapors is mainly mediated through perturbations of rotational motion in the region of separatrixes of A- and C-forms of rotation stereo dynamics. It is most pronounced for the orientation of optical transition dipole moment components along the axis of the molecule average moment of inertia (B) and manifests itself in all types of molecular tops. Such lability for dipole orientations along the A and C axes usually causes the degree of anisotropy to decrease by an order of magnitude and more. So it is advisable to use optical transitions with dipole moments oriented along the A or C axes for optical polarization analysis of molecules in the gaseous state, for example in analytical high-temperature polarized fluorescence.

Effect of solvation on polarization characteristics of fluorescence for 1,3,4-oxadiazole derivatives in binary solvents

We have studied the degree of fluorescence polarization for 1,3,4-oxadiazole derivatives in the binary solvents hexane-ethanol, toluene-ethanol, toluene-acetonitrile, 2-propanol-water, dioxane-water, 2-propanol-glycerol, glycerol-water vs. the composition of the solvent. We have shown that in systems with predominant universal interactions, the characteristic changes in the degree of polarization reflect the change in the viscosity of the mixture, while in systems with pronounced specific interactions they reflect the characteristic structural features of the fluorescent centers.