Fluorescence Polarization Spectroscopy Research Articles

Ultrasmall superparamagnetic iron oxide (USPIO)-based liposomes as magnetic resonance imaging probes

BackgroundMagnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.MethodsRegarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the signal enhancement on T1- and T2-weighted sequences of sMLs. To monitor the biodistribution and the clearance of the particles over time in vivo, sMLs were injected in wild type mice.ResultsDLS revealed a mean particle diameter of sMLs in the range between 100 and 200 nm, as confirmed by TEM. An effective iron oxide loading was achieved just for one type of USPIO, with an EE% between 74% and 92%, depending on the initial Fe concentration (being higher for lower amounts of Fe). MRI measurements demonstrated the applicability of these nanostructures as MRI probes.ConclusionOur results show that the development of sMLs is strictly dependent on the physicochemical characteristics of the nanocores. Once established, sMLs can be further modified to enable noninvasive targeted molecular imaging.

Structural basis of γH2AX recognition by human PTIP BRCT5-BRCT6 domains in the DNA damage response pathway

Human Pax2 transactivation domain-interacting protein (hPTIP), containing six BRCT domains, is an essential protein required for the IR induced DDR process with an unclear role. Here we report that the tandem BRCT5–BRCT6 domain of hPTIP recognizes the γH2AX tail, and this interaction depends on the phosphorylation of H2AX Ser139 and binding with the carboxyl ending peptide to the aminoacyl ending peptide. The 2.15Å crystal structure of hPTIP BRCT5/6–γH2AX complex and mutation analysis provide molecular evidence for direct interactions between PTIP and γH2AX. This interaction proffers a new clue to identify the role of PTIP in DDR pathways. Structured summary of protein interactionsPTIP and gamma H2AXbind by fluorescence polarization spectroscopy (View Interaction: 1, 2, 3, 4, 5, 6).PTIP and gamma H2AXbind by X-ray crystallography (View interaction).

Polarization single complex imaging of circular photosynthetic antenna

Single complex fluorescence polarization spectroscopy is applied to study the peripheral light harvesting antenna (LH2) from photosynthetic purple bacterium Rhodopseudomonas (Rps.) acidophila. The measured two-dimensional excitation-emission polarization plots are used to construct geometric representation for the absorbing B800 and emitting B850 as ellipses. The shape and orientation of the ellipses is discussed in terms of tilted LH2 complexes where emission occurs from energetically disordered B850 excitons.

Comprehensive studies on the interactions between chitosan nanoparticles and some live cells

As more and more oral formulations of nanoparticles are used in clinical contexts, a comprehensive study on the mechanisms of interaction between polymer nanoparticles and live cells seems merited. Such a study was conducted and the results were compared to the polymer itself in order to demonstrate different kinds of effects that are brought into the cell by polymer and its nanoparticles, especially the effects on the biomembrane. Several techniques, including surface plasmon resonance (SPR), Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, fluorescence polarization spectroscopy (FP), flow cytometry (FCM) with quantitative analysis, and confocal images with antibody staining were employed toward this end. The cytotoxicity in vitro was also evaluated. Chitosan (CS), a polycationic polymer, was used to prepare the nanoparticles. We demonstrate that chitosan nanoparticles (CS-NP) induce strong alterations in the distribution of membrane proteins, fluidity of membrane lipids, and general membrane structure. Furthermore, the uptake of CS-NP into Caco-2 cells was found to have a similar mechanism to that of CS molecules, but the differences in degree were noted. These results indicate that positive charge and nanoscale size were the factors that most significantly affected the interactions between the nanoparticles of polycationic polymers and live cells. However, no difference in cytotoxicity toward the Caco-2 cells was found between CS and CS-NP. This supports the idea that CS-NP is an effective and safe carrier for oral drug delivery.

Complex Kinetics of Apobec3g Interaction with Single-Stranded Nucleic Acids

Human APOBEC3G (A3G) is a host cell cytidine deaminase capable of restricting replication of retroviruses by deaminating single-stranded (ss) viral DNA, and also by directly inhibiting RT-catalyzed polymerization reactions. We have studied the kinetics of A3G interaction with model 10-, 20-, 40- and 60-nt ssDNAs as well as with long polymeric DNA by three independent approaches: (i) surface plasmon resonance (SPR) analysis, (ii) fluorescence polarization spectroscopy (FP), and (iii) single-molecule (SM) DNA stretching. Our results suggest the following: (1) Binding kinetics of A3G protein to all ssDNA oligonucleotides are multi-step with the faster, weaker binding gradually converting to more stable binding with much slower on/off rates; (2) The typical A3G-ssDNA on/off times measured in our experiments range between ∼10 and 10,000 sec, and the corresponding Kd values range between 0.1 and 10 μM; (3) Higher concentrations of A3G, longer A3G/ssDNA incubation times, as well as ssDNA length were found to correlate with slower ssDNA on/off kinetics, suggesting that A3G/A3G interactions in the bound state are responsible for slow protein kinetics.Taken together, these observations suggest that multimerization of A3G on ssDNA leads to a gradual decrease in its on/off kinetics, which in turn enhances this protein's ability to stall reverse transcription via formation of a “roadblock” for reverse transcriptase.

Enhanced Optical and Nonlinear Optical Responses in a Polyelectrolyte Templated Langmuir−Blodgett Film

Optical and nonlinear optical properties like fluorescence and second harmonic generation (SHG) of molecular materials can be strongly influenced by the mode of assembly of the molecules. The Langmuir-Blodgett (LB) technique is an elegant route to the controlled assembly of molecules in ultrathin films, and complexation of ionic amphiphiles in the Langmuir film by polyions introduced in the aqueous subphase provides a simple and efficient access to further control, stabilization, and optimization. The monolayer LB film of the hemicyanine-based amphiphile, N-n-octadecyl-4-[2-(4-(N,N-ethyloctadecylamino)phenyl)ethenyl]pyridinium possessing a "tail-head-tail" structure, shows fluorescence as well as SHG response. The concomitant enhancement of both of these linear and nonlinear optical attributes is achieved through templating with the polyanion of carboxymethylcellulose. Brewster angle and atomic force microscopy reveal the influence of polyelectrolyte templating on the morphology of the Langmuir and LB films. Polarized absorption and fluorescence spectroscopy provide insight into the impact of complexation with the polyelectrolyte on the orientation and deaggregation of the hemicyanine headgroup leading to fluorescence and SHG enhancement in the LB film.

Two-photon-excitation fluorescence depolarization in solutions and nano-scale-organized (macro)molecular media: Application of the wide-angular detection-aperture technique

We here discuss a problem of time-resolved fluorescence polarization spectroscopy at two-photon excitations of macroscopically isotropic molecular media, that can be organized locally on a nano-scale. The simplifying assumptions on the hydrodynamical shape of fluorophores and on the form of the two-photon absorption tensor, are considered and displayed on the corresponding examples of the emission anisotropy synthetic decays. These assumptions reduce the number of the model parameters making the theoretical description of the discussed spectroscopic technique applicable in the experimental practice. Because the two-photon excitations of fluorescence require the application of the exciting light of appropriately high intensity, and that may have a negative influence on the quality of the experimental data collected, we discuss the application of the wide-angular detection-aperture technique in such experiments. Furthermore, we consider the application of the symmetry adapted callibration (SAC) method enabling one to accurately analyze the experimental data even if the collecting optics employed is not ideal and is affected by several unexpected technical imperfections that are hard to be accounted for analytically.

ChemInform Abstract: Electronic States of Benzo(a)pyrene. Linear and Magnetic Circular Dichroism, Polarized Fluorescence, and Quantum Chemical Calculations.

The carcinogen benzo[a]pyrene is investigated by UV and IR linear dichroism (LD) spectroscopy in stretched polyethylene, by UV magnetic circular dichroism (MCD) spectroscopy, and by fluorescence polarization spectroscopy. The combined results lead to detailed experimental characterization of transitions in the near-UV region, with determination of polarization directions for five electronic transitions. The experimental results are compared with theoretical predictions using the PPP, CNDO/S, CNDO-SDCI, and LCOAO quantum chemical models. Excellent agreement with observed values in the low-energy region, including transition transition moment directions and MCD signs, is obtained within the LCOAO model. The theoretical analysis reveals a strong breakdown of the alternant pairing symmetry for benzo[a]pyrene, compared with those observed in the related hydrocarbons benz[a]anthracene and chrysene. 44 refs., 7 figs., 4 tabs.

Study of Anisotropic Photoluminescence and Energy Transfer in Oriented Dye-incorporating Zeolite-L Monolayer

Development of the methods to organize zeolite microcrystals into closely packed and uniformly aligned monolayers on various substrates have been pursued viewing microparticles as a novel class of building blocks. We now report that the vertically aligned zeolite monolayer can be applied as novel supramolecularly organized systems for anisotropic photoluminescence in high dichroic ratio, to study energy transfer dynamics between the internal and external fluorophores, and to develop zeolite-based advanced materials. Study of polarized fluorescence spectroscopy and angle-dependent intensity change with dye molecules in different surroundings further provides insight into molecular interactions that can be used for the future design of optoelectronic device in nanometer size. In addition, this report shows that isolating of organic dye through surface treatment is crucial for preventing the egress of the incorporated dye molecules from the channels of zeolite to the solution and to enhance the anisotropic luminescence.

Diphenylacetylene Polymer Nanofiber Mats Fabricated by Freeze Drying: Preparation and Application for Explosive Sensors

AbstractFinely structured diphenylacetylene polymer nanofibers were successfully prepared by a freeze drying method. SEM revealed that the morphology and structure of the fibers were significantly dependent on the concentration of the polymer solution, the ‘frozen in’ quenching temperature and the chemical structure of the polymer derivatives. Polarized fluorescence spectroscopy revealed that the polymer chains within the nanofiber were uniaxially oriented and highly elongated. The polymer nanofiber was remarkably sensitive to explosive nitroaromatic compounds when compared to the corresponding thin films. The fluorescence of polymer nanofiber obtained from 0.003 wt.‐% cryogenic benzene solution rapidly decreased to 50% of its initial value in ≈35 s by exposing to 2,4‐dinitrotoluene.magnified image

Fluorescence Amplification in Self-Assembled Organic Nanoparticles by Excitation Energy Migration and Transfer

The self-assembly of a low molecular weight organic chromophore occurs upon reprecipitation in water and yields 120 nm wide disk-like nanoparticles (NPs), as shown by fluorescence correlation spectroscopy (FCS) and atomic force microscopy (AFM). The NPs are able to incorporate perylene molecules previously present in water at nanomolar concentrations, thus switching ON and sensitizing their fluorescence. The doped NPs display a very high brightness as a result of their significant fluorescence quantum yield (up to 48%), the cumulated molecular absorbance, and the light-harvesting process. Fluorescence polarization spectroscopy also reveals that the efficiency of the donor-to-acceptor energy transfer process is amplified by a donor-to-donor excitation energy migration.

Pre‐Nucleation Structuring of TAG Melts Revealed by Fluorescence Polarization Spectroscopy and Molecular Mechanics Simulations

AbstractWe have studied the pre‐nucleation behavior of tripalmitin (TP) and tristearin (TS) in blends with triolein (TO), high oleic safflower oil (HOSfO) and soybean oil (SBO) by means of fluorescence polarization spectroscopy (FPS) and molecular mechanics simulations (MM). The FPS measurements at different temperatures showed that there is an increase in the anisotropy of the TP:HOSfO and TP:SBO blends as opposed to the TP:TO sample. This increase is directly related to an increase in the microviscosity of the blend which is interpreted as a structuring step prior to the nucleation and growth of the crystals. A similar but less pronounced effect was also observed in the TS:SBO blends. We performed MM simulations in an attempt to understand the molecular interactions responsible for this behavior. The simulation results have shown that short range van der Waals (vdW) interactions are the ones responsible for the increase in the microviscosity of the blends prior to crystallization. Our results also indicate that the presence of molecules that contain at least one chain of palmitic acid in their triglyceride (TAG) composition will induce a pre‐nucleation increase in the microviscosity of the blend in both TP and TS containing systems. Lastly, we studied the applicability of these conclusions to longer chain TAG analogues. Our MM results show that hypothetical blends of TS and TAGs containing stearic acid in their structure, will not have a low enough vdW energy to account for an increase in the microviscosity. Hence, there seems to be a specific interaction particularly favorable when the oil contains TAGs with at least one palmitic acid chain.

Spectroscopic observations, spin-orbit functions, and coupled-channel deperturbation analysis of data on theA Σ1u+andb Π3ustates ofRb2

We present experimentally derived potential curves and spin-orbit interaction functions for the strongly perturbed $A\text{ }{^{1}\ensuremath{\Sigma}}_{u}^{+}$ and $b\text{ }{^{3}\ensuremath{\Pi}}_{u}$ states of the rubidium dimer. The results are based on laser-induced fluorescence and optical-optical double-resonance polarization spectroscopy measurements combined with earlier laser-induced fluorescence data. We used an analytic potential (Hannover form) incorporated in a discrete variable representation of the Hamiltonian matrix for numerical energy-level calculation. A previous vibrational assignment of the $A$ levels is confirmed, and very probable vibrational assignment for the $b$ levels is also obtained. Currently, the rms residual of our fit is $0.053\text{ }{\text{cm}}^{\ensuremath{-}1}$ as compared to the typical experimental uncertainties that are estimated to be $0.005\text{ }{\text{cm}}^{\ensuremath{-}1}$. Fitted diagonal and off-diagonal spin-orbit functions are obtained and compared with ab initio calculations by all electrons and effective core pseudopotential methods. Analysis of the computational results yields an explanation for the ubiquitous single minima in these spin-orbit functions, which can be represented approximately by Morse-type functions.

Singlet/Triplet Reversal in Strongly-Coupled GaSe Nanoparticle Aggregates

GaSe nanoparticles have been synthesized in the absence of tightly binding edge ligands. These particles are ligated primarily with TOP and TOPO ligands that are easily displaced by far less bulky alkyl aldehydes. Ligand replacement results in nanoparticle aggregates in which the lowest energy optical transitions are strongly coupled, resulting in a large red shift of the absorption spectrum (about 2600 cm−1) and a reversal of the singlet and triplet states. The reversal of the spin states results in changes in the polarization spectroscopy and a dramatic decrease in the radiative lifetime. Specifically, the exciton singlet states are linear oscillators, and time-resolved fluorescence polarization spectroscopy gives an initial anisotropy very close to the linear oscillator limit of 0.4. The radiative lifetime of this fluorescence is about 12 ns, compared to about 82 ns for the monomers. Upon aggregation, the fluorescence quantum yield increases from 4.7 to 61%.

Determination Of The Lipid Membrane Composition Of J774 Macrophages Cells Surexpressing Mrp Protein (resistant To Ciprofloxacin)

Ciprofloxacin (CIP) is a fluoroquinolone antibiotic with an activity towards both extracellular and intracellular bacteria (Seral et al., 2005). Diffusion and efflux processes modulate accumulation of this drug within eukaryotic cells. When J774 macrophages were grown in presence of ciprofloxacin, the antibiotic is subject to constitutive efflux through the activity of an MRP-related transporter (Michot et al., 2004).In view of the critical role of lipids for both drug uptake and activity of MRP proteins (Hinrichs et al, 2004), together with the ability of fluoroquinolones to interact with lipids (Bensikaddour et al., 2008 (a,b)), we investigated the composition of lipids in resistant and sensitive J 774 macrophages to ciprofloxacin.Firstly, we characterized by thin layer chromatography the phospholipids composition of J774 macrophages cells sensitive (WT) and resistant to ciprofloxacin (CIP). Results showed that sphingomyelin (SM) decreased 2 times whereas phosphatidylinositol increased 1.5 fold in resistant cells. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and cholesterol didn't show any significant change. Secondly, we studied membrane fluidity of liposomes mimicking WT and CIP resistant J774 cells, by fluorescence polarization spectroscopy. In this respect, we observed a decrease in the melting temperature in vesicles mimicking the membrane composition of CIP resistant cells, indicating that changes in the fluidity of these membranes may be due to the decrease of SM. Studies are currently performed to investigate potential changes in other components of rafts like glucosylceramides. These data might have important relevance to relate the interaction of fluoroquinolones with lipids and change in the processes involved in cellular accumulation of fluoroquinolones.

Effect of Disalignment due to Electron Collision on Laser-Induced Fluorescence Polarization Spectroscopy for Measuring Electric Field in Plasmas

The effect of disalignment due to electron collisions on the determination of electric fields in laser-induced fluorescence (LIF) polarization spectroscopy was studied. The aligned He I n1D (n=3,4) atoms were produced by laser excitation of 21S state through electric quadrupole transition in Penning discharge plasma. The polarization components of the subsequent LIF (n1D→21P) were observed as a function of electron density in the range from 2×1010 to 2×1012 cm-3. From the dependences of disalignment rates for 31D and 41D states on electron density, their rate coefficients were estimated to be (1.5±0.3)×10-5 and (4.7±1.2)×10-5 cm3/s, respectively. Critical disalignment rates, where the polarization of LIF disappeared, were also estimated using a rate-equation model taking into account the disalignment effect. On the basis of experimental rate coefficients and critical disalignment rates, we deduced the limitation of measurable electric field for electron density in LIF polarization spectroscopy.

Optical response and surface morphology of crystalline Nd 3+-doped fluoride films grown on monocrystalline LiYF 4 substrates by pulsed laser deposition

The realization of crystalline films of Nd 3+ :YF 3 and Nd 3+ :LiYF 4 on a monocrystalline LiYF 4 substrate by pulsed laser deposition is reported. The films were obtained by laser ablation with 355 nm photons of a bulk LiYF 4 crystal doped with Nd 3+ ions at 1.5% atomic concentration in the presence of different ablation/deposition parameters. The films optical characteristics, analyzed via laser induced polarized fluorescence spectroscopy upon IR excitation, are presented. Lifetime measurements of the fundamental Nd 3+ ion transition in the film were also performed. All these results were compared with those obtained in the Nd 3+ :LiYF 4 bulk crystal. The surface morphology of the depositions was analyzed via a scanning electron microscope. When the production of the deposition took place in high vacuum (1 × 10 − 4 Pa) and the substrate temperature was 750 °C, the grown film was Nd 3+ :YF 3. A 1 Pa controlled atmosphere of He in the ablation chamber and a substrate temperature of 650 °C favoured the growth of a Nd 3+ :LiYF 4 film. In the latter case the film showed also a smoother surface.

Chain-Length Dependent Nematic Ordering of Conjugated Polymers in a Liquid Crystal Solvent

In this communication we demonstrate the dependence of the solute order parameter on the solute molecular weight for polymer solutes dissolved in liquid crystalline solvents. Using ensemble absorption polarization spectroscopy together with single molecule fluorescence polarization measurements, we have determined the order parameter of the conjugated polymer MEH-PPV (poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene]) in the liquid crystal 5CB (4-cyano-4-n-pentylbiphenyl) as a function of polymer chain length. Ensemble absorption polarization measurements agree well with results obtained by single molecule fluorescence polarization spectroscopy, indicating a large-scale ordering of the MEH-PPV solute in 5CB. These results demonstrate that the increasing number of defects for larger polymer weights inherently limits the alignment of the polymer solute.

Complex of dipeptidyl peptidase II with adenosine deaminase

Dipeptidyl peptidase II (DPPII) from bovine kidney cortex and lung was purified to the electrophoretically homogeneous state. The molecular and catalytic characteristics of the enzyme were determined. It was revealed that DPPII preparations possess adenosine deaminase (ADA) activity at all purification steps. For the first time, the ADA-binding ability of DPPII has been shown similar to the well-known ADA-binding enzyme, DPPIV. The dissociation constant of the DPPII-ADA complex was estimated using a resonant mirror biosensor (80 nM), fluorescence polarization (60 nM), and differential spectroscopy (36 nM) techniques. The data demonstrate that DPPII can form a complex with ADA, but with one order of magnitude higher dissociation constant than that of DPPIV (7.8 nM).

Fluorescent Studies on Cooperative Binding of Cationic Pheophorbide‐a Derivative to Polyphosphate

The cooperative binding of a novel water-soluble cationic derivative of pheophorbide-a (CatPheo-a) to inorganic polyphosphate (PPS) in buffered aqueous solutions was studied by means of polarized fluorescence spectroscopy in a wide range of molar phosphate-to-dye ratios (P/D). Under low P/D values, CatPheo-a forms extended stacking associates on the PPS matrix, while under high P/D the dye binds to PPS in the dimer form. The CatPheo-a self-association is accompanied by 40-fold dye fluorescence quenching and a substantial increase in the fluorescence polarization degree. The fluorescent titration data were used for determination of cooperative binding parameters by Schwarz's method.