The adsorbed conformation of globular proteins at the air/water interface

External reflection FTIR spectroscopy and surface pressure measurements were used to compare conformational changes in the adsorbed structures of three globular proteins at the air/water interface. Of the three proteins studied, lysozyme, bovine serum albumin and beta-lactoglobulin, lysozyme was unique in its behaviour. Lysozyme adsorption was slow, taking approximately 2.5 h to reach a surface pressure plateau (from a 0.07 mM solution), and led to significant structural change. The FTIR spectra revealed that lysozyme formed a highly networked adsorbed layer of unfolded protein with high antiparallel beta-sheet content and that these changes occurred rapidly (within 10 min). This non-native secondary structure is analogous to that of a 3D heat-set protein gel, suggesting that the adsorbed protein formed a highly networked interfacial layer. Albumin and beta-lactoglobulin adsorbed rapidly (reaching a plateau within 10 min) and with little change to their native secondary structure.     

Preparation for Optimal Conformation of Lysozyme with Nanodiamond and Nanosilica as Carriers

Solutions of suspended particles of nanodiamond and nanosilica in deionized water 25 mg/10 mL were prepared for the purpose of achieving saturation adsorption. The respective saturation thresholds and adsorption reaction constants of lysozyme of extremely diluted concentrations 0–1000 nmol/L in 7 mmol/L (potassium phosphate buffer solution) PPBS on the surfaces of nanodiamond and nanosilica of 100 nm diameter have been measured. The adsorbed quantities of lysozyme on a unit surface area of both nanoparticles can be derived. The possible influences by the adsorption surface profile and adsorption surface area, adsorption ability of the nanoparticles, strength and activity of lysozyme have been discussed.     

Adsorption-induced conformational changes of proteins onto ceramic particles: differential scanning calorimetry and FTIR analysis

Three model proteins, bovine serum albumin, hen's egg lysozyme and bovine serum fibrinogen, were adsorbed from aqueous solution onto finely dispersed ceramic particles, namely different kinds of alumina and hydroxyapatite particles. The influence of adsorption on protein secondary structure was investigated. The FTIR spectroscopic findings were compared with the results of DSC measurements. In almost all cases it was found that adsorption results in destabilisation and structural loss of the bound protein. A decrease in transition enthalpy is correlated with a loss in alpha-helical structure, which seems to be the most sensitive structure on adsorption-induced rearrangements. A total collapse of structure in the adsorbed proteins was not determined on any ceramic surface. Some residual structure is always retained. Structural changes in the D- or E-domains of fibrinogen could be independently observed by two different calorimetric signals. The two techniques applied in the present study -- micro-DSC and FTIR spectroscopy -- can be concluded to provide complementary information on adsorption-induced structural changes on both the molecular (thermal stability, overall structure) and the sub-molecular level (secondary structure).     

Direct functionalization of nanodiamond particles using dopamine derivatives

The article reports on the strong linking of dopamine derivatives as a simple and a versatile strategy for the surface functionalization of hydroxyl-terminated nanodiamond (ND-OH) particles. Azide- (ND-N(3)) or poly-N-isopropylacrylamide-terminated (ND-PNIPAM) particles were obtained from ND-OH particles through the reaction with the corresponding dopamine derivatives. The azide-terminated ND particles were further derivatized with a fluorescent probe, alkynyl-pyrene, via copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. The modified ND particles were characterized using transmission Fourier transform infrared (FTIR) spectroscopy, UV-vis spectroscopy, electrochemical measurements, thermogravimetric analysis (TGA), and particle size measurements. The surface loading of ND particles with dopamine was estimated from TGA and UV-vis spectroscopy and was found to be around 0.27 mmol g(-1). Because of its simple, gentle nature and versatility, the chemistry developed in this work can be used as an avenue for the preparation of functional nanodiamond particles for various applications.     

Adsorption Reaction Dynamics of Systems Lysozyme and Nanodiamond/Nanosilica at pH=7-13

Adsorption reactions between surfaces of nanodiamond and nanosilica with diameter of 100 nm prepared as suspension solutions of 0.25 μg/μL and lysozyme molecule with different concentrations of 7 mmol/L PPBS at pH=7, 9, 11, and 13 have been investigated by fluores-cence spectroscopy. Adsorption reaction constants and coverages of lysozyme with different concentrations of 0-1000 nmol/L under the influences of different pH values have been ob-tained. Helicities and conformations of the adsorbed lysozyme molecules, free spaces of every adsorbed lysozyme molecule on the surfaces of nanoparticles at different concentrations and pH values have been deduced and discussed. The highest adsorption capabilities for both sys-tems and conformational efficiency of the adsorbed lysozyme molecule at pH=13 have been obtained. Lysozyme molecules can be prepared, adsorbed and carried with optimal activity and helicity, with 2 and 10 mg/m2 on unit nanosurface, 130 and 150 mg/g with respect to the weight of nanoparticle, within the linear regions of the coverages at around 150-250 nmol/L and four pH values for nanodiamond and nanosilica, respectively. They can be prepared inthe tightest packed form, with 20 and 55 mg/m2, 810-1680 and 580-1100 mg/g at threshold concentrations and four pH values for nanodiamond and nanosilica, respectively.     

Spectroscopic Investigation for Purity Evaluation of Detonation Nanodiamonds: Experimental Approach in Absorbance and Scattering

Nanodiamond powders have excellent mechanical, chemical, physical, and optical properties. In this research, two non-pure groups of nanodiamond particles labeled as ND1 and ND2 have been selected for purity evaluation by means of absorbance and scattering analysis. The nanodiamond powders have been used for Raman and dynamic light scattering (DLS), Fourier transform infrared (FTIR) and UV–Vis absorbance approaches. The Raman spectra show a weak diamond signal in ND2 as purity and 4 basic set of bands. FTIR absorbance spectroscopy was used in the spectral range of 400–3600 cm^?1. The results show that ND2 is almost non-absorbance with wavelength. It means that the diamond phase purity of ND2 is greater than ND1, and ND2 shows a better structure of diamond. UV–Vis absorption spectra of the sample have been recorded in the spectral range of 200–800 nm by means of nanodiamond suspended in methanol (NDM). The UV–Vis absorbance of NDM1 is stronger than NDM2 at the same concentration. Therefore the diamond phase purity of ND2 is greater than ND1. Particle size distribution and zeta potential of DNDs were investigated by DLS method. Finally the structure and phase of samples have been evaluated by X-ray diffraction (XRD) for confirmation.     

SANS and UV-vis spectroscopy studies of resultant structure from lysozyme adsorption on silica nanoparticles

The interaction of lysozyme protein (M.W. 14.7 kD) with two sizes of silica nanoparticles (16 and 25 nm) has been examined in aqueous solution using UV-vis spectroscopy and small-angle neutron scattering (SANS). The measurements were performed on fixed concentration (1 wt %) of nanoparticles and varying concentration of protein in the range 0 to 2 wt %. The adsorption isotherm as obtained using UV-vis spectroscopy suggests strong interaction of the two components and shows an exponential behavior. The saturation values of adsorption are found to be around 90 and 270 protein molecules per particle for 16 and 25 nm sized nanoparticles, respectively. The adsorption of protein on nanoparticles leads to the aggregation of particles and these structures have been studied by SANS. The aggregates are characterized by fractal structure coexisting with unaggregated particles at low protein concentrations and free proteins at higher protein concentrations. Further, contrast variation SANS measurements have been carried out to differentiate the adsorbed and free protein in these systems.     

Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme

Adsorption of chicken egg lysozyme on silica nanoparticles of various diameters has been studied. Special attention has been paid to the effect of nanoparticle size on the structure and function of the adsorbed protein molecules. Both adsorption patterns and protein structure and function are strongly dependent on the size of the nanoparticles. Formation of molecular complexes is observed for adsorption onto 4-nm silica. True adsorptive behavior is evident on 20- and 100-nm particles, with the former resulting in monolayer adsorption and the latter yielding multilayer adsorption. A decrease in the solution pH results in a decrease in lysozyme adsorption. A change of protein structure upon adsorption is observed, as characterized by a loss in alpha-helix content, and this is strongly dependent on the size of the nanoparticle and the solution pH. Generally, greater loss of alpha helicity was observed for the lysozyme adsorbed onto larger nanoparticles under otherwise similar conditions. The activity of lysozyme adsorbed onto silica nanoparticles is lower than that of the free protein, and the fraction of activity lost correlates well with the decrease in alpha-helix content. These results indicate that the size of the nanoparticle, perhaps because of the contributions of surface curvature, influences adsorbed protein structure and function.     

Annealing of silica to reduce the concentration of isolated silanols and peak tailing in reverse phase liquid chromatography

Non-porous, colloidal silica particles were annealed at three different temperatures, 800, 900 and 1050 °C. The adsorption of lysozyme, a probe of surface roughness, was consistent with progressively reduced surface roughness as temperature increased. The heat treated silica particles were rehydroxylated and then used to pack UHPLC columns. The cationic protein lysozyme was used to probe silanol activity, which exhibited progressively less tailing as the annealing temperature increased. FTIR spectroscopy confirmed that the abundance of isolated silanols on the surface was reduced by annealing at 900 °C or 1050 °C. FTIR also revealed that there was markedly increased hydrogen bonding of the isolated silanols to neighbors after rehydroxylation. These results combine to support the hypothesis that (a) isolated silanols on silica cause tailing in RP-LC and (b) nonplanar topography gives rise to isolated silanols.     

Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores

The interactions of proteins with the surface of cylindrical nanopores are systematically investigated to elucidate how surface curvature and surface chemistry affect the conformation and activity of confined proteins in an aqueous, buffered environment. Two globular proteins, lysozyme and myoglobin, with different catalytic functions, were used as model proteins to analyze structural changes in proteins after adsorption on ordered mesoporous silica SBA-15 and propyl-functionalized SBA-15 (C(3)SBA-15) with carefully controlled pore size. Liquid phase ATR-FTIR spectroscopy was used to study the amide I and II bands of the adsorbed proteins. The amide I bands showed that the secondary structures of free and adsorbed protein molecules differ, and that the secondary structure of the adsorbed protein is influenced by the local geometry as well as by the surface chemistry of the nanopores. The conformation of the adsorbed proteins inside the nanopores of SBA-15 and C(3)SBA-15 is strongly correlated with the local geometry and the surface properties of the nanoporous materials, which results in different catalytic activities. Adsorption by electrostatic interaction of proteins in nanopores of an optimal size provides a favorably confining and protecting environment, which may lead to considerably enhanced structural stability and catalytic activity.     

Adsorption of 2-aminobenzothiazole on colloidal silver particles: an experimental and theoretical surface-enhanced Raman scattering study

Surface-enhanced Raman scattering (SERS) spectra of the biologically important 2-aminobenzothiazole (2-ABT) molecule adsorbed on silver hydrosols are compared with its FTIR spectrum and normal Raman spectroscopy (NRS) spectrum in the bulk and in solution. The optimized structural parameters and the computed vibrational wavenumbers of the compound have been estimated from ab initio (Hatree-Fock) and density functional calculations. Some vibrational modes of the molecule have been reassigned. Concentration-dependent SERS spectra of the molecule reveal the existence of two types of vertically adsorbed species on colloidal silver particles, whose relative population varies with the adsorbate concentrations. The adsorption geometry and structural parameters of one type of adsorbed species are related to the NRS spectrum of the chemically prepared and theoretically modeled 2-ABT-Ag(I) coordination compound.     

Electrosurface properties of single-crystalline detonation nanodiamond particles obtained by air annealing of their agglomerates

A complex study of electrosurface properties has been performed for single-crystalline detonation nanodiamond particles with sizes of 4–5 nm obtained by air annealing of their agglomerates. FTIR spectroscopy and X-ray photoelectron spectroscopy data indicate that the investigated properties result from the presence of two types of ionogenic functional groups on the particle surface, i.e., acidic carboxyl and amphoteric hydroxyl groups. Acid-base potentiometric titration, laser Doppler electrophoresis, and conductometry have been employed to measure the Γ_H⁺(pH) and Γ_OH^-(pH) adsorption isotherms of potential-determining ions, as well as the pH dependences (in a pH range of 3.5–10.5) of the surface charge density, electrophoretic mobility, and specific surface conductivity of detonation nanodiamond particles in aqueous 0.0001–0.01 M KCl solutions.     

Catalytic conversion of C<Subscript>2</Subscript>-C<Subscript>3</Subscript> alcohols on detonation nanodiamond and its modifications

The catalytic activity of detonation nanodiamond and its modifications obtained through treatment with hydrogen or air at elevated temperatures is studied in the conversion of C₂-C₃ alcohols. The catalysts were characterized by means of electron microscopy, optical (FTIR) spectroscopy, elemental analysis and pulse microcatalytic method. It has been established that nanodiamond exhibits high catalytic activity in the conversion of alcohols. The oxidizing and reducing treatment of nanodiamond changes its activity and selectivity, and the activity of oxidized nanodiamond is considerably higher than that of reduced nanodiamond.     

Chemical imaging of protein adsorption and crystallization on a wettability gradient surface

The use of self-assembled monolayers is an established method to study the effect of surface properties on proteins and other biological materials. The generation of a monolayer with a gradient of chemical properties allows for the study of multiple surface properties simultaneously in a high throughput manner. Typically, in order to detect the presence of proteins or biological material on a surface, the use of additional dyes or tags is required. Here we present a novel method of studying the effect of gradient surface properties on protein adsorption and crystallization in situ through the use of ATR-FTIR spectroscopic imaging, which removes the need for additional labeling. We describe the successful application of this technique to the measurement of the growth of a gradient monolayer of octyltrichlorosilane across the surface of a silicon ATR element. ATR-FTIR imaging was also used to study the adsorption of lysozyme, as a model protein, onto the modified surface. The sensitivity of measurements obtained with a focal plane array (FPA) detector were improved though the use of pixel averaging which allowed small absorption bands to be detected with minimal effect on the spatial resolution along the gradient. Study of the effect of surface hydrophobicity on both adsorption of lysozyme to the element and lysozyme crystallization revealed that more lysozyme adsorbed to the hydrophobic side of the ATR element and more lysozyme crystals formed in the same region. These findings strongly suggest a correlation exists between surface protein adsorption and protein crystallization. This method could be applied to the study of other proteins and whole cells.     

Thermodynamic analysis of lysozyme adsorbed to silica

The structural stability of hen egg white lysozyme in solution and adsorbed to small colloidal silica particles at various surface concentrations was investigated using hydrogen-deuterium (H/D) exchange in combination with mass spectrometry (HDX-MS) and differential scanning calorimetry (DSC). The combination of HDX-MS and DSC allows a full thermodynamic analysis of the lysozyme structure as both the enthalpy and the Gibbs free energy can be derived from the various measurements. Moreover, both HDX-MS and DSC provide information on the relative structural heterogeneity of lysozyme in the adsorbed state compared to that in solution. Results demonstrated that at high surface coverage, the structural stability of lysozyme was only marginally affected by adsorption to silica particles whereas the unfolding enthalpy decreased by more than 10%, meaning that the entropy of lysozyme increased with a similar value upon adsorption. Furthermore, the structural heterogeneity increased considerably. At lower surface concentrations, the structural heterogeneity increased further whereas the enthalpy of unfolding decreased. Further analyses of the HDX-MS experiments clearly indicated that folding/unfolding of lysozyme occurs through a two-domain process. These two domains had a similar amount of structural elements and a difference in stabilization energy of 8 kJ/mol, regardless if lysozyme was in solution or adsorbed to silica.     

Adsorption behavior of methylene blue and its congeners on a stainless steel surface

Methylene blue and its congeners as model dyes were adsorbed onto stainless steel particles at different ionic strengths, pH values, and ethanol contents, and the adsorption mechanism was investigated. A Fourier transform infrared spectroscopy (FTIR) analysis of the dyes adsorbed on the stainless steel plate was carried out to determine the orientations of the adsorbed dyes on stainless steel surface. The adsorption isotherms for all the dyes tested were approximated by a Langmuir equation (Q=Kq(m)C/(1+KC)) in most cases except under strongly basic conditions. From the ionic strength and ethanol content dependencies of the K value in the Langmuir equations, both the electrostatic and hydrophobic interactions were indicated to contribute to the adsorption of the dyes at neutral pH. By comparing the K and q(m) values for the methylene blue congeners and with the aid of the FTIR analyses, it was found that the kind of substituent groups at most positions of the polyheterocycles of methylene blue strongly affects the adsorption behavior, particularly the area occupied by an adsorbed dye molecule, the affinity for the stainless steel surface, and the orientation of the adsorbed dye molecule on the stainless steel surface.     

Removal of Fluorescein using different iron oxides as adsorbents: effect of pH

In this work the adsorption process of Fluorescein (dye with aril-methane group) as a function of pH on three different adsorbents: goethite, Co-goethite, and magnetite has been studied experimentally and theoretically. FTIR and Raman spectroscopy have been performed in an attempt to confirm the structure of surface complexes formed by sorption of the Fluorescein to different iron oxides. Typical anionic adsorption behaviour was observed for this dye onto goethite and Co-goethite whereas the adsorption level was practically constant in the range of pH studied when the adsorbent was magnetite. The diffuse layer model was employed to fit the experimental results. The surface complexes proposed from the adsorption data were in agreement with the patterns obtained from FTIR and Raman spectroscopy. The surface structure of the oxides affects the adsorption process and the final adsorbed amount at the equilibrium. Our model of diffuse double layer with the addendum of the effect of hydrophobic forces fits well the adsorption data of Fluorescein on iron oxides at different pH in the studied range. At lower pH electrostatic forces by ligand-exchange are predominant. In the range of pH 9-11 hydrophobic forces are managing the Fluorescein adsorption on the iron oxides, with the formation of outer-sphere complexes through van der Waals/hydrophobic forces. It is interesting that in the three iron oxides studied, the adsorbed amount in this range is similar.     

In situ conformational analysis of fibrinogen adsorbed on Si surfaces

Fibrinogen is a major plasma protein. Previous investigations of structural changes of fibrinogen due to adsorption are mostly based on indirect evidence after its desorption, whereas our measurements were performed on fibrinogen in its adsorbed state. Specific enzyme-linked immunosorption experiments showed that the amount of adsorbed fibrinogen increased as the surface became more hydrophobic. Atomic force microscopy (AFM) investigations revealed the trinodular shape of fibrinogen molecules adsorbed on hydrophilic surfaces, whereas all of the molecules appeared globular on hydrophobic surfaces. The distribution of secondary structures in adsorbed fibrinogen was quantified by in situ Fourier-transform infrared (FTIR) analysis. Substrates of identical chemical bulk composition but different surface hydrophobicity permit direct comparison among them. Adsorption properties of fibrinogen are different for each degree of hydrophobicity. Although there is some increase of turn structure and decrease of β-sheet structure, the secondary structure of adsorbed fibrinogen on hydrophilic surface turned out to be rather similar to that of the protein in solution phase with a major -helix content. Hydrophilic surfaces exhibit superior blood compatibility as required for medical applications.     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

Concentration-dependent surface-enhanced resonance Raman scattering of a porphyrin derivative adsorbed on colloidal silver particles

Surface-enhanced Raman spectra (SERS) of 5,10,15,20-tetrakis(1-decylpyridium-4-yl)-21H,23H-porphintetrabromide or Por 10 (H(2)Tdpyp) adsorbed on silver hydrosols are compared with the FTIR and resonance Raman spectrum (RRS) in the bulk and in solution. Comparative analysis of the RR and the FTIR spectra indicate that the molecule, in its free state, has D(2h) symmetry rather than C(2v). The SERS spectra, obtained on adsorption of this molecule on borohydride-reduced silver sol, indicate the formation of silver porphyrin. With the change in the adsorbate concentration, the SERS shows that the molecule changes its orientation on the colloidal silver surface. The appearance of longer wavelength band in the electronic absorption spectra of the sol has been attributed to the coagulation of colloidal silver particles in the sol. The long wavelength band is found to be red-shifted with the decrease in adsorbate concentration. The excitation profile study indicates that the resonance of the Raman excitation radiation with the original sol band is more important than that with the new aggregation band for the SERS activity. This indicates a large contribution of electromagnetic effect to surface enhancement.