Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS) we measure the translational diffusion coefficient of asphaltene molecules in toluene at extremely low concentrations (0.03-3.0 mg/L): where aggregation does not occur. We find that the translational diffusion coefficient of asphaltene molecules in toluene is about 0.35 x 10(-5) cm(2)/s at room temperature. This diffusion coefficient corresponds to a hydrodynamic radius of approximately 1 nm. These data confirm previously estimated size from rotational diffusion studied using fluorescence depolarization. The implication of this concurrence is that asphaltene molecular structures are monomeric, not polymeric.     

Fluorescence Correlation Spectroscopy Study of Probe Diffusion in Poly(vinyl alcohol) Solutions and Gels

In this study, we demonstrate how the diffusion of probe particles in aqueous poly(vinyl alcohol) (PVA) solutions and gels is affected by: (i) the presence of cross-links, (ii) the cross-link density, (iii) the polymer concentration. We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion time of a rhodamine-based fluorescent particle (TAMRA) and TAMRA-labeled dextran in PVA solutions and gels prepared at various polymer concentrations (1% to 8.6% w/v) and cross-link densities (1/400 to 1/50 cross-link monomers per PVA monomers). The measurements indicate that the probe particles are slowed down with increasing polymer concentration and with increasing cross-link density. Also, FCS can detect differences in the diffusion times measured in “fresh” and “aged” PVA solutions. We find that FCS provides a quantitative measure of network inhomogeneities.     

Lateral diffusion coefficients of an eicosanyl-based bisglycerophosphocholine determined by PFG-NMR and FRAP

We report the lateral diffusion properties of 2,2'-di-O-decyl-3,3'-di-O-(eicosanyl)-bis-(rac-glycero)-1,1'-diphosphocholine (C20BAS) using pulsed-field gradient NMR (PFG-NMR) and fluorescence recovery after photobleaching (FRAP). C20BAS membranes display a melting transition at Tm = 15.7 degrees C, as determined by differential scanning calorimetry and 31P NMR chemical shift anisotropy. The lateral diffusion coefficient of C20BAS, as determined by PFG-NMR and FRAP, at 25 degrees C, were DPFG-NMR = 1.9 +/- 0.6 x 10(-8) cm2/s and DFRAP C20BAS = 1.2 +/- 0.1 x 10(-8) cm2/s, respectively. In comparison, the lateral diffusion coefficient of the monopolar phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was 1.8 +/- 0.9 x 10(-8) and 2.5 +/- 0.9 x 10(-8) cm2/s using PFG-NMR and FRAP, respectively.     

Characterization of Diblock Copolymer Order–Order Transitions in Semidilute Aqueous Solution Using Fluorescence Correlation Spectroscopy

The temperature and pH‐dependent diffusion of poly(glycerol monomethacrylate)‐block‐poly(2‐hydroxypropyl methacrylate) nanoparticles prepared via polymerization‐induced self‐assembly in water is characterized using fluorescence correlation spectroscopy (FCS). Lowering the solution temperature or raising the solution pH induces a worm‐to‐sphere transition and hence an increase in diffusion coefficient by a factor of between four and eight. FCS enables morphological transitions to be monitored at relatively high copolymer concentrations (10% w/w) compared to those required for dynamic light scattering (0.1% w/w). This is important because such transitions are reversible at the former concentration, whereas they are irreversible at the latter. Furthermore, the FCS data suggest that the thermal transition takes place over a very narrow temperature range (less than 2 °C). These results demon­strate the application of FCS to characterize order–order transitions, as opposed to order–disorder transitions.

     

NMR diffusion spectroscopy as a measure of host-guest complex association constants and as a probe of complex size

The complexes of cyclohexylacetic acid and cholic acid with beta-cyclodextrin were studied by NMR diffusion coefficient measurements. The diffusion coefficient of the 1:1 cyclohexylacetic acid/beta-cyclodextrin complex, K(a) = 1800 +/- 100 M(-1), is slightly slower (3.23 +/- 0.07 x 10(-6) cm(2) s(-1)) than that of beta-cyclodextrin (3.29 +/- 0.07 x 10(-6) cm(2) s(-1)). The diffusion coefficient of the 1:1 cholic acid/beta-cyclodextrin complex, K(a) = 5900 +/- 800 M(-1), is significantly slower (2.93 +/- 0.07 x 10(-6) cm(2) s(-1)) than that of beta-cyclodextrin. The results indicate that caution should be exercised when studying host-guest complexation by the so-called 'single point' technique. A novel data treatment is introduced which takes into account the diffusion behavior of all of the species when determining K(a). Experimentally determined diffusion coefficients of complexes are also a useful probe of the size of host-guest complexes.     

Probing the molecular interaction of chymotrypsin with organophosphorus compounds by 31P diffusion NMR in aqueous solutions

In the present study, we applied for the first time (31)P diffusion NMR to resolve different species obtained by the addition of organophosphorus compounds (OP) such as diisopropyl phosphorofluoridate (DFP) or 1-pyrenebutyl phosphorodichloridate (PBPDC) to alpha-chymotrypsin (Cht). (31)P diffusion NMR was used since the products of these reactions constitute a mixture of OP-covalent conjugates of the enzyme and OP-containing hydrolysis products that have noninformative (1)H NMR spectra. It was shown that the peak, attributed to the covalent native diisopropylphosphoryl-Cht (DIP-Cht) conjugate by chemical shift considerations, has a greater diffusion coefficient (D = (0.65 +/- 0.01) x 10(-5) cm(2) s(-1)) than expected from its molecular mass (approximately 25 kDa). This peak was therefore suggested to consist of at least two superimposed signals of diisopropyl phosphoryl (DIP) pools of high and low molecular weights that happen to have the same chemical shift. This conclusion was substantiated by the use of DMSO-d(6) that separated the overlapping signals. Diffusion measurements performed on the extensively dialyzed and unfolded DIP-Cht conjugate still resulted in a high diffusion coefficient ((0.30 +/- 0.05) x 10(-5) cm(2) s(-1)) relative to the assumed molecular mass. This observation was attributed to a dynamic dealkylation at the OP moiety (i.e., aging) that occurred during the relatively long diffusion measurements, where DIP-Cht was converted to the corresponding monoisopropyl phosphoryl Cht (MIP-Cht) conjugate. Homogeneous aged forms of OP-Cht were obtained by use of DFP and heat-induced dealkylation of DIP-Cht, and by PBPDC that provided the aged form via the hydrolysis of a P-Cl bond (PBP-Cht). The thermally stable aged conjugates enabled a reliable determination of the diffusion coefficients over several days of data acquisition, and the values found were (0.052 +/- 0.002) x 10(-5) cm(2) s(-1) and (0.054 +/-0.004) x 10(-5) cm(2) s(-1) for the MIP-Cht and the PBP-Cht adducts, respectively, values in the range expected for a species with a molecular weight of 25 kDa. The advantages and limitations of (31)P diffusion NMR in corroborating the type of species that prevail in such systems are briefly discussed.     

Calibration and Limits of Camera‐Based Fluorescence Correlation Spectroscopy: A Supported Lipid Bilayer Study

Camera‐based fluorescence correlation spectroscopy (FCS) approaches allow the measurement of thousands of contiguous points yielding excellent statistics and details of sample structure. Imaging total internal reflection FCS (ITIR‐FCS) provides these measurements on lipid membranes. Herein, we determine the influence of the point spread function (PSF) of the optical system, the laser power used, and the time resolution of the camera on the accuracy of diffusion coefficient and concentration measurements. We demonstrate that the PSF can be accurately determined by ITIR‐FCS and that the laser power and time resolution can be varied over a wide range with limited influence on the measurement of the diffusion coefficient whereas the concentration measurements are sensitive to changes in the measurement parameters. One advantage of ITIR‐FCS is that the measurement of the PSF has to be performed only once for a given optical setup, in contrast to confocal FCS in which calibrations have to be performed at least once per measurement day. Using optimized experimental conditions we provide diffusion coefficients for over ten different lipid membranes consisting of one, two and three constituents, measured in over 200000 individual correlation functions. Using software binning and thus the inherent advantage of ITIR‐FCS of providing multiple observation areas in a single measurement we test the FCS diffusion law and show how they can be complemented by the local information provided by the difference in cross‐correlation functions (ΔCCF). With the determination of the PSF by ITIR‐FCS and the optimization of measurement conditions ITIR‐FCS becomes a calibration‐free method. This allows us to provide measurements of absolute diffusion coefficients for bilayers with different compositions, which were stable over many different bilayer preparations over a time of at least one year, using a single PSF calibration.     

Molecular basis of carbon dioxide transport in polycarbonate membranes

Gas transport of carbon dioxide in poly[bisphenol A carbonate-co-4,4'-(3,3,5-trimethylcyclohexylidene)diphenol carbonate] films over a wide range of pressure is described. The interpretation of the experimental results in terms of the dual mode model allowed the evaluation of the parameters of the model that govern the gas permeation process. The value of the diffusion coefficient obtained for carbon dioxide at zero concentration was 2.4 x 10(-8) cm(2) s(-1), at 303 K. This parameter was also measured by using pulsed field gradient NMR finding that its value reaches a nearly constant value of (2.7 +/- 0.9) x 10(-8) cm(2) s(-1), at 298 K, for diffusion times greater than 20 ms. Both the diffusion and solubility coefficients were also computed by using simulation methods based on the transition states theory and the Widom method, respectively. The value obtained for the diffusion coefficient was 1.8 x 10(-8) cm(2) s(-1), at 303 K, which compares very favorably with the experimental measurements. The drop of the simulated solubility coefficient with increasing pressure is sharper than that of the experimental one, at low pressures, and similar, at high pressures.     

Proton dynamics in lithium-ammonia solutions and expanded metals

Quasielastic neutron scattering has been used to study proton dynamics in the system lithium-ammonia at concentrations of 0, 4, 12, and 20 mole percent metal (MPM) in both the liquid and solid (expanded metal) phases. At 230 K, in the homogenous liquid state, we find that the proton self-diffusion coefficient first increases with metal concentration, from 5.6x10(-5) cm2 s(-1) in pure ammonia to 7.8x10(-5) cm2 s(-1) at 12 MPM. At higher concentrations we note a small decrease to a value of 7.0x10(-5) cm2 s(-1) at 20 MPM (saturation). These results are consistent with NMR data, and can be explained in terms of the competing influences of the electron and ion solvation. At saturation, the solution freezes to form a series of expanded metal compounds of composition Li(NH3)4. Above the melting point, at 100 K, we are able to fit our data to a jump-diffusion model, with a mean jump length (l) of 2.1 A and residence time (tau) of 3.1 ps. This model gives a diffusion coefficient of 2.3x10(-5) cm2 s(-1). In solid phase I (cubic, stable from 88.8 to 82.2 K) we find that the protons are still undergoing this jump diffusion, with l=2.0 A and tau=3.9 ps giving a diffusion coefficient of 1.8x10(-5) cm2 s(-1). Such motion gives way to purely localized rotation in solid phases IIa (from 82.2 to 69 K) and IIb (stable from 69 to 25 K). We find rotational correlation times (tau(rot)) of the order of 2.0 and 7.3 ps in phases IIa and IIb, respectively. These values can be compared with a rotational mode in solid ammonia with tau(rot) approximately 2.4 ps at 150 K.     

End-to-end diffusion on the microsecond timescale measured with resonance energy transfer from a long-lifetime rhenium metal-ligand complex

We measured the end-to-end diffusion coefficient of an alkyl chain-linked donor-acceptor pair using the time-resolved frequency-domain decay of the donor. The donor was a rhenium metal-ligand complex with a mean decay time ranging from 2.1 to 7.9 microseconds in the absence of the Texas red acceptor. The decay time was used to measure the donor-to-acceptor distance distribution and the mutual diffusion coefficient. Using this long lifetime donor, it was easily possible to determine a diffusion coefficient near 2 x 10(-8) cm2/s and diffusion coefficients as low as 1.3 x 10(-9) cm2/s were measurable. Such long lifetime donors should be valuable for measuring the flexing of peptides on the microsecond timescale, domain motions of proteins and lateral diffusion in membranes. The availability of microsecond decay time luminophores now allows luminescence spectroscopy to be useful generally for studies of microsecond dynamics of biological macromolecules.     

Fluorescence correlation spectroscopy of water-in-oil microemulsions: an application in specific characterisation of droplets containing biomolecules

Fluorescence correlation spectroscopy (FCS) has been successfully used to characterise water-in-oil (w/o) microemulsions. The investigated systems were stabilised by sodium bis-2-ethylhexyl sulphosuccinate (AOT) and the measured diffusion times have been related to the radii of the aggregated species, which for some systems, were separately determined by small-angle neutron scattering (SANS). We demonstrate that FCS is capable of measuring hydrodynamic radii of microemulsions rapidly and at surfactant concentrations lower than previously reported for other techniques. FCS was also used to specifically interrogate microemulsion droplets containing a fluorescently-labelled biomolecule, specifically phalloidin, a peptide fungal toxin from Amanita phalloides, and the enzyme -chymotrypsin (-CT). The microemulsion droplets are only marginally increased in size if a small peptide (phalloidin) is included in the water phase, whereas the droplet size is significantly increased when a larger protein (-CT) is included.     

Slow diffusion of macromolecular assemblies by a new pulsed field gradient NMR method

The translational diffusion coefficient of an integral membrane protein/surfactant complex has been measured using a novel pulsed field gradient NMR method. In this new approach, the information about the localization of the molecules is temporarily stored in the form of longitudinal magnetization of isotopes with long spin-lattice relaxation times. This allows one to increase the duration of the diffusion interval by about 1 order of magnitude. Unlike standard proton NMR methods using pulsed field gradients and stimulated echoes, the new method can be applied to macromolecular assemblies with diffusion coefficients well below 10(-10) m(2) s(-1), corresponding to masses in excess of 25 kDa in aqueous solution at room temperature. The method was illustrated by application to a water-soluble complex of tOmpA, the hydrophobic transmembrane domain of bacterial outer membrane protein A, with the detergent octyl-tetraoxyethylene (C(8)E(4); overall mass of complex approximately 45 kDa). The diffusion coefficient was found to be D = (4.99 +/- 0.07) x 10(-11) m(2) s(-1), consistent with measurements by size exclusion chromatography and by ultracentrifugation. The method has also been applied to a solution of recombinant human tRNA(3)(Lys), which has a molecular mass of 24 kDa, and the diffusion coefficient D = (1.05 +/- 0.015) x 10(-10) m(2) s(-1).     

Approaching complete peroxisome characterization by gas-phase fractionation

We examined the utility of gas-phase fractionation (GPF) in the m/z dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) analysis of the peptides produced by proteolytic digestion of unfractionated proteins from a yeast whole-cell lysate and in a peroxisomal membrane protein fraction derived from isolated yeast peroxisomes. We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPF(m/z) and GPF(RI). Comparison of results of direct nuLC/ESI-MS/MS analysis of the unfractionated mixture of peptides from proteolysis of a yeast whole cell lysate by DD ion selection from 400-1800 m/z in triplicate and GPF(m/z) from 400-800, 800-1200 and 1200-1800 produced the following results: (i) 1.3 x more proteins were identified by GPF(m/z) for an equal amount of effort (i.e., 3 microLC/ESI-MS/MS) and (ii) proteins identified by GPF(m/z) had a lower average codon bias value. Use of GPF(RI) identified more proteins per m/z unit scanned than GPF(m/z) or triplicate analysis over a wide m/z range. After tryptic digestion of all the proteins from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins we detected 93% (38/41) of known peroxisomal proteins using GPF(m/z), but only 73% using a standard wide m/z range survey scan.     

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single‐particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 μm^?2) and very low surface concentrations (single‐molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well‐suited for the intermediate concentration range of about 0.1–100 μm^?2. However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z‐scan FCS, is calibration‐free for membrane measurements, but requires several experiments at different well‐controlled focusing positions. A recently established FCS method, electron‐multiplying charge‐coupled‐device‐based total internal reflection FCS (TIR‐FCS), referred to here as imaging TIR‐FCS (ITIR–FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR–FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.     

Enhanced translational diffusion of rubrene in sucrose benzoate

The translational diffusion of rubrene in the fragile molecular glass former, sucrose benzoate (SB) (fragility index m approximately 94), has been studied from T(g)+6 K to T(g)+71 K(T(g)=337 K) by using the technique of holographic fluorescence recovery after photobleaching. In the temperature range of the measurements, the translational relaxation functions were observed to decay exponentially, indicating that Fick's law of diffusion governs the translational motion of rubrene in sucrose benzoate. The value of the translational diffusion coefficient D(T) obtained from the 1e time of the translational relaxation function varied from 5.3 x 10(-15) cm2 s(-1) at 343 K to 5.0x10(-9) cm2 s(-1) at 408 K. The temperature dependence of D(T) for diffusion of rubrene in SB is compared with that of the viscosity and the dielectric relaxation time tau(D) of SB. The temperature dependence of D(T) is weaker than that of Teta for T<1.2T(g) but tracks the reciprocal of the dielectric relaxation time 1tau(D) for 1.05T(g)相似文献   

Rhodamine B diffusion in hair as a probe for structural integrity

The aim of this work was to investigate the diffusion of Rhodamine B into bleached, photo bleached and abraded hair, treated or not with an emulsion of ceramide using two different techniques: spectrophotometry and fluorescence optical microscopy with image analysis. This comparison, combined with the Einstein-Smoluchowski equation, allowed validating a methodology that uses the apparent diffusion coefficient of a dye as an index for hair damage. Distinct behaviors of the dye were observed in the cuticle and in the cortex. For a bleached hair sample the apparent diffusion coefficient in the cuticle ranges from 8.2 x 10(-11) cm2 s(-1) to 10 x 10(-11)cm2 s(-1), while for the cortex this value drops to 4.0 x 10(-11) cm2 s(-1) to 4.2 x 10(-11) cm2 s(-1). The diffusion is always faster in the cuticle than in the cortex and the apparent diffusion coefficient shows up to a seven-fold decrease when the dye penetrates the cortex. The chemical, photochemical and physical treatments applied to hair significantly change the values of the apparent diffusion coefficients in the cuticle. The data also proved that the penetration of Rhodamine B into hair occurs via an intercellular path.     

Molecular dynamics simulations of DiI-C18(3) in a DPPC lipid bilayer

We performed a 40 ns simulation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18(3)) in a 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) bilayer in order to facilitate interpretation of lipid dynamics and membrane structure from fluorescence lifetime, anisotropy, and fluorescence correlations spectroscopy (FCS). Incorporation of DiI of 1.6 to 3.2 mol% induced negligible changes in area per lipid but detectable increases in bilayer thickness, each of which are indicators of membrane structural perturbation. The DiI chromophore angle was 77 +/- 17 degrees with respect to the bilayer normal, consistent with rotational diffusion inferred from polarization studies. The DiI headgroup was located 0.63 nm below the lipid head group-water interface, a novel result in contrast to some popular cartoon representations of DiI but consistent with DiI's increase in quantum yield when incorporated into lipid bilayers. Importantly, the fast component of rotational anisotropy matched published experimental results demonstrating that sufficient free volume exists at the sub-interfacial region to support fast rotations. Simulations with non-charged DiI head groups exhibited DiI flip-flop, demonstrating that the positively-charged chromophore stabilizes the orientation and location of DiI in a single monolayer. DiI induced detectable changes in interfacial properties of water ordering, electrostatic potential, and changes in P-N vector orientation of DPPC lipids. The diffusion coefficient of DiI (9.7 +/- 0.02 x 10(-8) cm2 s(-1)) was similar to the diffusion of DPPC molecules (10.7 +/- 0.04 x 10(-8) cm2 s(-1)), supporting the conclusion that DiI dynamics reflect lipid dynamics. These results provide the first atomistic level insight into DiI dynamics, results essential in elucidating lipid dynamics through single molecule fluorescence studies.     

Determination of critical micelle concentrations and aggregation numbers by fluorescence correlation spectroscopy: aggregation of a lipopolysaccharide

Fluorescence correlation spectroscopy (FCS) is often used to determine the mass or radius of a particle by using the dependence of the diffusion coefficient on the mass and shape. In this article we discuss how the particle size of aggregates can be measured by using the concentration dependence of the amplitude of the autocorrelation function (ACF) instead of the temporal decay. We titrate a solution of aggregates or micelles with a fluorescent label that possesses a high affinity for these structures and measure the changes in the amplitude of the ACF. We develop the theory describing the change of the ACF amplitude with increasing concentrations of labels and use it to fit experimental data. It is shown how this method can determine the aggregation number and critical micelle concentration of a standard detergent nonaethylene glycol monododecyl ether (C₁₂E₉) and a lipopolysaccharide (LPS: Escherichia coli 0111:B4).     

An NMR determination of CO diffusion on platinum electrocatalysts

We report the first direct measurement of CO diffusion on nanoparticle Pt electrocatalysts at the solid/liquid interface, carried out using 13C nuclear magnetic resonance (NMR) with a spin-labeling pulse sequence. Diffusion parameters were measured in the temperature range of 253-293 K for CO adsorbed on commercial Pt-black under saturation coverage. 2H NMR of the same system indicates that the electrolyte remains in the liquid state at temperatures where the CO diffusion experiments were performed. The CO diffusion parameters follow typical Arrhenius behavior with an activation energy of 6.0 +/- 0.4 kcal/mol and a pre-exponential factor of (1.1 +/- 0.6) x 10-8 cm2/s. Exchange between different CO populations, driven by a chemical potential gradient, is suggested to be the main mechanism for CO diffusion. The presence of the electrolyte medium considerably slows down the diffusion of CO as compared to that seen on surfaces of bulk metals under UHV conditions. This work opens up a new approach to the study of surface diffusion of adsorbed molecules on nanoparticle electrode catalysts, including the possibility of correlating diffusion parameters to catalytic activity in real world applications of broad general interest.     

Ion-exchange investigation of the arsenazo III-uranyl system: confirmation of the physical existence of a weak M(2)L-type new complex compound

Ion-exchange experiments have proved the physical existence in the arsenazo III uranyl system of an M(2)L complex species in addition to the well-known ML complex. The emerald-green M(2)L complex exhibits a very low stability which permits the preparation of pure stoichiometric bluish ML complex solutions by percolation of the former through a cation-exchanger in the sodium form. Both complexes exhibit a similar visible spectrum with maxima located at 605 and 655 nm, the maximum absorptivity of M(2)L being slightly greater (e(655) approximately 5.8 x 10(4) 1. mole(-1). cm(-1)) than that of ML (E(655) = 5.0 +/- 0.3 x 10(4)). The complexes have a net negative charge which depends strongly on the particular washing conditions used for the complexes sorbed on chloride-form anion-exchange resins.