Metal Nanoparticles on Polymer Surfaces: 5. Catalytic Activity of Colloidal Platinum Films Incorporated in Polystyrene Surface Layer

The fundamental possibility to enlarge Pt nanoparticles in monolayer ensembles formed on polystyrene surfaces by the adsorption from hydrosol in solution of isopropanol and K₂PtCl₄ is demonstrated for the first time. The enlargement of “seeding” nanoparticles is performed after their preliminary incorporation (partial embedding) into the polymer surface layer by the annealing of a system within the range between “surface” and “bulk” glass transition temperatures of polystyrene. It is shown that a colloidal film of metallic platinum with a thickness up to 200 nm is formed in the course of enlargement and it is mechanically fixed in the polymer surface layer. Such a system exhibits, over a long time, high catalytic activity in the model reaction of methyl viologen reduction with hydrogen.__________Translated from Kolloidnyi Zhurnal, Vol. 67, No. 3, 2005, pp. 398–403.Original Russian Text Copyright © 2005 by Rudoy, Sukhov, Dement’eva, Abkhalimov, Vereshchagina, Kartseva, Ershov.     

Prediction of binding constants of protein ligands: A fast method for the prioritization of hits obtained from de novo design or 3D database search programs

A dataset of 82 protein–ligand complexes of known 3D structure and binding constant K_i was analysed to elucidate the important factors that determine the strength of protein–ligand interactions. The following parameters were investigated: the number and geometry of hydrogen bonds and ionic interactions between the protein and the ligand, the size of the lipophilic contact surface, the flexibility of the ligand, the electrostatic potential in the binding site, water molecules in the binding site, cavities along the protein–ligand interface and specific interactions between aromatic rings. Based on these parameters, a new empirical scoring function is presented that estimates the free energy of binding for a protein–ligand complex of known 3D structure. The function distinguishes between buried and solvent accessible hydrogen bonds. It tolerates deviations in the hydrogen bond geometry of up to 0.25 Å in the length and up to 30 °Cs in the hydrogen bond angle without penalizing the score. The new energy function reproduces the binding constants (ranging from 3.7 × 10^-2 M to 1 × 10^-14 M, corresponding to binding energies between -8 and -80 kJ/mol) of the dataset with a standard deviation of 7.3 kJ/mol corresponding to 1.3 orders of magnitude in binding affinity. The function can be evaluated very fast and is therefore also suitable for the application in a 3D database search or de novo ligand design program such as LUDI. The physical significance of the individual contributions is discussed.     

Protein–protein interaction dynamics by amide H/H exchange mass spectrometry

Amide H/²H exchange detected by mass spectrometry provides a powerful tool for observing changes that occur upon protein–protein interactions. In general, it is possible to observe protection of surface amides, they become less solvent exposed when they are buried at the interface. The information thus obtained about the location of the protein–protein interface is useful for building a correct docked structure of the protein–protein complex. Examples of protein–protein interfaces that were correctly identified by such methods include the thrombin–thrombomodulin interaction and the interaction between the regulatory and catalytic subunit of protein kinase A. Amide exchange also affords a view into the subtle changes in the ensemble of states that occur upon protein modification or protein–protein binding. Examples of proteins in which amide exchange has been used to observe phosphorylation-induced changes include ERK2 and CheB. Amide exchange showed the pathway of communication between the cAMP-binding site and the catalytic subunit site within the regulatory subunit of protein kinase A. Clues as to how thrombomodulin regulates the catalytic activity of thrombin were also obtained.     

Determination of Proteins in a Mesofluidic Lab-on-Valve System

The application of a mesofluidic lab-on-valve system to the spectrophotometric determination of protein was investigated. Protein species in the sample solution reacts rapidly with Congo red at pH 4.1, which forms a complex with a maximum absorption at 496 nm. A univariant approach was used for the optimization of the experimental parameters. A sample volume of 20 μl was used along with 4.0 μl of Congo red solution of 0.9 g l⁻¹, and a flow rate for the detection process of 20 μl s⁻¹ was used. Under optimal conditions, a linear calibration curve was obtained in the range of 12.5–200 μg ml⁻¹ of bovine serum album, along with a detection limit (3σ) of 5.6 μg ml⁻¹ and a sampling frequency of 60 per hour. Protein concentrations in human serums, urine, milk, and yoghourt were determined using this procedure, and satisfactory agreements were obtained with that achieved using the Coomassie brilliant blue method.     

Porous Protein‐Based Nanoparticle Hydrogel for Protein Chips with Improved Sensitivity

We have discovered a novel method to prepare a protein‐based hydrogel, that is, a ‘three‐dimensional nanostructured protein hydrogel’ (3D NPH), which is composed of loosely inter‐connected protein–polymer hybrid nanoparticles. The 3D NPH can be easily prepared by spotting a protein/polymer mixture on a substrate. Surprisingly, gold nanoparticles carrying protein molecules easily diffuse into the 3D NPH through pores and spaces. We have shown that the protein chip made by our 3D NPH method has tremendously improved sensitivity in detecting protein–protein interactions compared with that by direct protein immobilization methods.

     

Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein–ligand complex structure analysis

Room-temperature (RT) protein crystallography provides significant information to elucidate protein function under physiological conditions. In particular, contrary to typical binding assays, X-ray crystal structure analysis of a protein–ligand complex can determine the three-dimensional (3D) configuration of its binding site. This allows the development of effective drugs by structure-based and fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein–ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein–ligand complex analysis, the simultaneous preparation of protein–ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein–ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein–ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin–ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.

Room temperature protein crystallography and its application to protein–ligand complex structure analysis was demonstrated using a microfluidic protein crystal array device.     

Determination of ultra trace amounts of protein by 4-chlorosulfo-(2′-hyaroxylphenylazo)-rhodanine-Ti(IV) complex [ClSARP-Ti(IV)] as the fluorescence spectral probe in AOT microemulsion

Experiments indicated that protein can enhance the fluorescence of the 4-chlorosulfo-(2′-hydroxylophenylazo)-rhodanine-Ti(IV) complex [ClSARP-Ti(IV)] in the presence of bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) microemulsion. Based on this, a sensitive and reproducible fluorometric method for the determination of micro amount protein was developed. The calibration curves of four proteins were given. Under the optimum experimental conditions, the enhanced fluorescence intensity of the system was in proportional to the concentration of protein in the range of 0.1–11 μg mL⁻¹ for bovine serum albumin (BSA), 1.0–10 μg mL⁻¹ for human serum albumin (HSA), 1.0–50 μg mL⁻¹ for ovalbumin (Ova) and 2.5–18 μg mL⁻¹ for γ-globulins (γ-G). Their detection limits were 0.070, 0.071, 0.33 and 0.22 μg mL⁻¹, respectively. The ClSARP-Ti(IV) complex as a spectral probe can be used to the determination of protein in milk powder and oatmeal yielding with satisfactory results. Therefore, the proposed method is one of the most sensitive methods available. In addition, the interaction mechanism of this system is studied by multi-techniques.     

A comprehensive analysis in one run – in-depth conformation studies of protein–polymer chimeras by asymmetrical flow field-flow fractionation

Polymer-based protein engineering has enabled the synthesis of a variety of protein–polymer conjugates that are widely applicable in therapeutic, diagnostic and biotechnological industries. Accurate characterizations of physical–chemical properties, in particular, molar masses, sizes, composition and their dispersities are critical parameters that determine the functionality and conformation of protein–polymer conjugates and are important for creating reproducible manufacturing processes. Most of the current characterization techniques suffer from fundamental limitations and do not provide an accurate understanding of a sample''s true nature. In this paper, we demonstrate the advantage of asymmetrical flow field-flow fractionation (AF4) coupled with multiple detectors for the characterization of a library of complex, zwitterionic and neutral protein–polymer conjugates. This method allows for determination of intrinsic physical properties of protein–polymer chimeras from a single, rapid measurement.

Precise characterization of structural parameters and their polydispersities of protein–polymer conjugates is performed with rapid analysis using asymmetrical flow field-flow fractionation coupled with multiple detectors.     

Amine groups functionalized gel-type resin supported Pd catalysts: Physicochemical and catalytic properties in hydrogenation of alkynes

Palladium catalysts (0.125–0.5 wt.% Pd) supported by amine groups—functionalized gel-type resin (FCN) were studied in the hydrogenation of alkynes reagents, 2-butyne-1,4-diol and phenylacetylene. The catalysts were prepared by two routes. The first, “OAc” is based on the immobilization of Pd-precursor in the pre-swollen resin from THF solution of Pd(OAc)₂, followed by chemical reduction of the Pd-centers. This method produces Pd particles of size in nano-scale. The second procedure, “aq” implies the deposition of Pd-species on dry resin beads using aqueous solution of PdCl₂. Reduction of these Pd-species gives relatively large Pd particles, dominating are 30–50 nm in size. The SEM studies performed over the cross-section of catalysts grains showed location of Pd in outer shell of polymer beads in both “OAc” and “aq” catalysts; however, thinner layer of Pd appears in “aq” series catalysts. In the presence of all catalysts, prepared by “OAc” and “aq” methods the selectivity towards alkenes is high, above 90%. The catalysts of “aq’ series are much more active and more selective than “OAc” analogues giving selectivity to alkene ca. 94% at almost complete conversion of alkynes. Moreover, catalytic performance of “aq’ series catalyst is unchanged under recycling use. The catalyst was recovered and reused 4 times, maintaining its catalytic efficiency.     

Ferrocene clicked poly(3,4-ethylenedioxythiophene) conducting polymer: Characterization, electrochemical and electrochromic properties

The “click” chemistry, Cu(I)-catalyzed azide–alkyne cycloaddition reaction, was applied to covalently functionalize the poly(3,4-ethylenedioxythiophene) (PEDOT) conducting polymer film with an excellent electron transfer mediator (ferrocene). Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy were used to characterize the ferrocene-grafted PEDOT conducting polymer film, and it was proved that the grafting procedure via click reaction had a high efficiency. The ferrocene groups covalently grafted in the polymer films turned out to own a relatively fast electron transfer rate and show multi-color states via adjusting applied potential.     

Preparation of Porous Protein-Based Hydogel for Highly Sensitive Protein Chips

Summary: Protein chips are important tools for high-throughput analysis of biological events. We have developed a novel method to prepare a protein-based hydrogel, that is, a “Three-Dimensional Nano-structured Protein Hydrogel” (3-D NPH), which is composed of protein and polymer nano-particles. The 3-D NPH could be easily prepared by dispensing a protein and polymer mixture on a substrate. Surprisingly, gold particles conjugated with protein A diffused into the 3-D NPH which was made of mouse IgG through the pores. We have shown that the protein chips made with our 3-D NPH method has tremendously improved sensitivity in detecting protein-protein interactions compared with that of direct protein immobilization methods.     

Temperature-dependent interfacial properties of hydrophobically end-modified poly(2-isopropyl-2-oxazoline)s assemblies at the air/water interface and on solid substrates

We describe herein the properties at the air/water (A/W) interface of hydrophobically end-modified (HM) poly(2-isopropyl-2-oxazoline)s (PiPrOx) bearing an n-octadecyl chain on both termini (telechelic HM-PiPrOx) or on one chain end (semitelechelic HM-PiPrOx) for different subphase temperatures and spreading solvents using the Langmuir film balance technique. The polymer interfacial properties revealed by the π–A isotherms depend markedly on the architecture and molecular weight of the polymer. On cold water subphases (14 °C), diffusion of PiPrOx chains onto water takes place for all polymers in the intermediate compressibility region (5 mN m⁻¹). At higher subphase temperatures (36 and 48 °C), the HM-PiPrOx film exhibited remarkable stability with time. Brewster angle microscopy (BAM) imaging of the A/W interface showed that the polymer assembly was not uniform and that large domains formed, either isolated grains or pearl necklaces, depending on the polymer structure, the concentration of the spreading solution and the subphase temperature. The Langmuir films were transferred onto hydrophilic substrates (silica) by the Langmuir–Blodgett (LB) technique and onto hydrophobic substrates (gold) by Langmuir–Schaefer (LS) film deposition, resulting in the formation of adsorbed particles ranging in size from 200 to 500 nm, depending on the polymer architecture and the substrate temperature. The particles presented “Janus”-like hydrophilic/hydrophobic characteristics.     

Effect of nanobubbles on friction forces between hydrophobic surfaces in water

Micro- and nanoscale combined hierarchical polymer structures were fabricated by UV-assisted capillary force lithography. The method is based on the sequential application of engraved polymer molds with a UV-curable resin of polyurethane acrylate (PUA) followed by surface treatment with a trichloro(1H,1H,2H,2H-perfluorooctyl) silane in vapor phase. Two distinct wetting states were observed on these dual-roughness structures. One is “Cassie–Wenzel state” where a water droplet forms heterogeneous contact with microstructures and homogeneous contact with nanostructures. The other is “Cassie–Cassie state” where a droplet makes heterogeneous contact both with micro- and nanostructures. A simple thermodynamic model was developed to explain static contact angle, hysteresis, and wetting transition on dual-roughness structures.     

PLIMSTEX: a novel mass spectrometric method for the quantification of protein–ligand interactions in solution

Protein–ligand interactions by mass spectrometry, titration, and H/D exchange (PLIMSTEX) is a new mass spectrometric method for determining association constants and binding stoichiometry for interactions of proteins with various ligands, as well as for quantifying the conformational changes associated with ligand binding to proteins. The association constants determined with PLIMSTEX agree with literature values within a factor of six, establishing its validity for protein interactions involving metal ions, small organic molecules, peptides, and proteins. PLIMSTEX provides solution, not gas-phase, properties by taking advantage of ESI and MALDI mass spectrometry to measure accurately the mass of a protein as it undergoes amide H/D exchange. The approach sidesteps the problem of relating gas-phase abundances of the protein or protein–ligand complex ions to their solution concentrations. With on-column concentration and desalting, high picomole quantities of proteins are sufficient for reproducible mass detection, and the concentration of the protein can be as low as 10⁻⁸ M. It is amenable to different protein/ligand systems in physiologically relevant media. No specially labeled protein or ligand is needed. PLIMSTEX offers minimal perturbation of the binding equilibrium because it uses no denaturants, no additional spectroscopy or reaction probes, and no physical separation of ligand and protein during binding.     

Protein interactions and association: an open challenge for colloid science

The solution behaviour of globular proteins still presents many puzzling aspects, calling for a deeper understanding of the physical properties of lyophilic colloids. For instance, protein interactions in ‘salting-out’ conditions require to take explicitly into account ‘Donnan’ effects on the small ion distribution, while understanding of temperature and salt–specificity effects on protein solubility presumably calls for a careful analysis of hydrophobic contributions. Yet, very unexpected effects can be found even at low salt concentration. In particular, we discuss the very distinctive properties of β-lactoglobulin A (BLGA) solutions, where strong attractive interactions show up, displaying a marked non-monotonic trend as a function of the solution ionic strength. These ‘anomalous’ attractions drive very peculiar reversible association processes, resulting in the spontaneous formation of short-lived clusters with a well-defined small aggregation number. We suggest that other protein association processes of physiological interest may parallel BLGA clustering.     

Quantifying adsorbed protein on surfaces using confocal fluorescence microscopy

The quantification and analysis of protein adsorption on solid surfaces are of significant importance in many areas of biosensors, biomaterials, and biomedical devices research. The accurate, in situ, measurement of multiple physicochemical properties from the thin protein layers which adsorb on surfaces is critical to understanding biocompatibility, surface chemistry factors, and the performance of implanted medical devices. To implement such studies, new tools and simple protocols based on instrumentation available in typical bioscience laboratories are desirable. In this work, we have developed an approach using confocal fluorescence microscopy to quantify the amount of bovine serum albumin (BSA) adsorbed onto a flat hydrophilic glass surface, under different pH conditions. This approach which can be implemented using most confocal fluorescence microscopes is described in detail and its limitations are discussed. This quantitative method coupled with the Langmuir model allowed for the determination of adsorption parameters at pH 2.0, 4.0, 7.4, and 9.2. The adsorption parameters were validated by comparison with literature values obtained from different techniques for a similar protein–surface system. The Derjaguin–Landau–Verwey–Overbeek (DLVO) theory was then used for a detailed analysis of these parameters, to understand in general terms how pH affected the surface adsorption interactions.     

An experimental study on the “sequential order” rules in generalized two-dimensional correlation spectroscopy

Following our theoretical analysis on the “sequential order” rules in generalized two-dimensional (2D) correlation spectroscopy (H. Huang, Anal. Chem. 79 (2007) 8281–8292), an experimental study was conducted to test the “sequential order” rules using the FT-NIR data of poly(3-hydroxybutyrate) (PHB)/poly(l-lactic acid) (PLA) blends under uniaxial elongation and parallel polarization. The local sequential order concept proposed for the generalized two-dimensional (2D) correlation spectroscopy is now more clearly stated; “the intensity change at ν1 occurs predominantly before ν2” means that the starting time of the intensity change at ν1 is prior to that at ν2. It is this local sequential order which reflects the real and intuitive sequential order between two events in generalized situations. It has been found that the integrated/overall sequential order results obtained from the 2D correlation analysis may be contradictory to the intuitive local sequential order. In addition, different integrated/overall sequential orders could be obtained by selection of different sampling intervals from a certain set of experimental data, or choosing different number of the contours for the same sampling interval. These new experimental findings are a perfect reinforcement to our previous theoretical study and have further demonstrated the uncertainty of applying the “sequential order” rules in generalized 2D correlation spectroscopy.     

Nucleation and growtn of polythiophene films on gold electrodes

The nucleation and growth of polythiophene films on gold electrodes has been studied using potentiostatic steps. The mechanism has been deduced and estimates made of the kinetic parameters. Dissolution of the gold substrate at potentials where thiophene polymerisation occurs is suppressed by the initial rapid formation of a monolayer of polymer. The data indicate that formation of bulk film occurs by the instantaneous nucleation and three-dimensional growth of polymer on top of this monolayer. Rate constants for growth parallel to the surface on the bare gold substrate and the covering polymer layer are surprisingly very similar. Growth perpendicular to the surface is slightly more rapid, typically by a factor of 1.5–3, although it is less dependent on potential. The high density of nuclei results in their overlap at an early stage, after which growth is only possible perpendicular to the surface. Within a narrow potential range, the observation of maxima and minima in current-time transients is interpreted in terms of the “death” and “rebirth” of growing centres.     

Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads

The immobilization of trypsin on porous glycidyl methacrylate (GMA–GDMA) beads has been investigated. In particular, the distribution within the beads of trypsin and of dextran used for hydrophilizing the bead surface prior to protein immobilization was investigated with confocal microscopy. For the system investigated, the fluorescence intensity profiles obtained when using borate buffer as an ambient solution displayed a distinct minimum at the center of the beads, irrespective of the observation depth. However, by reduction of the refractive index difference between the solution and the beads through the addition of glucose to the aqueous solution, artifacts relating to optical length differences could be reduced. For both low molecular weight fluorescein isothiocyanate (FITC), FITC-labeled trypsin, and FITC-labeled dextran, an essentially homogeneous distribution throughout the beads was observed. This simple “contrast matching” method seems therefore to be an interesting tool when investigating the distribution of immobilized protein in porous chromatography media.     

Adsorption mechanism of Cu from aqueous solution by chitosan-coated magnetic nanoparticles modified with α-ketoglutaric acid

In order to better understand the adsorption mechanism of chitosan-coated magnetic nanoparticles modified with α-ketoglutaric acid (α-KA-CCMNPs), the removal of Cu²⁺ by α-KA-CCMNPs from aqueous solution was investigated in a batch system at 18, 35 and 50 °C. Different experimental approaches were applied to show mechanistic aspects, such as adsorption isotherms, kinetics and thermodynamics studies. Adsorption equilibrium studies showed that Cu²⁺ adsorption followed Langmuir model. The kinetics of the interactions was best described by pseudo-second-order mechanism. The thermodynamic parameters (ΔG°, ΔH° and ΔS°) analysis predicted that the adsorption process was strongly dependent on temperature of medium, and spontaneous and endothermic process. The XPS combined with FT-IR spectra revealed that N atom of –NH– group and O atom of carboxyl group in α-KA-CCMNPs coordinated with Cu²⁺. Experimental results from this study provide data that would be required if this heavy metal adsorption system was to be “scaled up” for industrial application.