Efficiency of blocking of non-specific interaction of different proteins by BSA adsorbed on hydrophobic and hydrophilic surfaces

The efficiency of a pre-absorbed bovine serum albumin (BSA) layer in blocking the non-specific adsorption of different proteins on hydrophobic and hydrophilic surfaces was evaluated qualitatively and quantitatively using infrared reflection spectroscopy supported by spectral simulations. A BSA layer with a surface coverage of 35% of a close-packed monolayer exhibited a blocking efficiency of 90–100% on a hydrophobic and 68–100% on a hydrophilic surface, with respect to the non-specific adsorption of concanavalin A (Con A), immunoglobulin G (IgG), and staphylococcal protein A (SpA). This BSA layer was produced using a solution concentration of 1 mg/mL and 30 min incubation time. BSA layers that were adsorbed at conditions commonly employed for blocking (a 12 h incubation time and a solution concentration of 10 mg/mL) exhibited a blocking activity that involved competitive adsorption–desorption. This activity resulted from the formation of BSA–phosphate surface complexes, which correlated with the conformation of adsorbed BSA molecules that was favourable for blocking. The importance of optimisation of the adsorbed BSA layer for different surfaces and proteins to achieve efficient blocking was addressed in this study.     

Influence of particle size on the binding activity of proteins adsorbed onto gold nanoparticles

We used optical extinction spectroscopy to study the structure of proteins adsorbed onto gold nanoparticles of sizes 5-60 nm and their resulting biological binding activity. For these studies, proteins differing in size and shape, with well-characterized and specific interactions-rabbit immunoglobulin G (IgG), goat anti-rabbit IgG (anti-IgG), Staphylococcal protein A, streptavidin, and biotin-were used as model systems. Protein interaction with gold nanoparticles was probed by optical extinction measurements of localized surface plasmon resonance (LSPR) of the gold nanoparticles. Binding of the ligands in solution to protein molecules already immobilized on the surface of gold causes a small but detectable shift in the LSPR peak of the gold nanoparticles. This shift can be used to probe the binding activity of the adsorbed protein. Within the context of Mie theory calculations, the thickness of the adsorbed protein layer as well as its apparent refractive index is shown to depend on the size of the gold nanoparticle. The results suggest that proteins can adopt different orientations that depend on the size of the gold nanospheres. These different orientations, in turn, can result in different levels of biological activity. For example, we find that IgG adsorbed on spheres with diameter ≥20 nm does not bind to protein A. This study illustrates the principle that the size of nanoparticles can strongly influence the binding activity of adsorbed proteins. In addition to the importance of this in cases of direct exposure of proteins to nanoparticles, the results have implications for proteins adsorbed to materials with nanometer scale surface roughness.     

A Piezoelectric Quartz Crystal Biosensor as a Direct Affinity Sensor

Abstract

A microprocessor controlled piezoelectric detector as sensor was employed to monitor in real time protein adsorption and immunoreactions using piezoelectric quartz crystals (AT-cut) with a basic resonant frequency of 10 MHz. The adsorbed protein was an immunoglobulin (h-lg G); in the immunosensing a covalent immobilized molecule (the pesticide 2,4-D) formed the receptor for the immobilized ligand sample (Mab anti-2,4-D) in a competitive assay.     

Investigation of protein adsorption and electrochemical behavior at a gold electrode

The adsorption of two model proteins, human serum albumin and immunoglobulin G, on a gold electrode surface was investigated using 125I radiolabeling and cyclic voltammetry (CV). 125I radiolabeling was used to determine the extent of protein adsorption, while CV was used to ascertain the effect of the adsorbed protein layer on the electron transfer between the gold electrode and an electroactive moiety in solution, namely, K3Fe(CN)6. The adsorbed amounts of HSA and IgG agreed well with previous results and showed approximately monolayer coverage. The amount of adsorbed protein increased when a positive potential (700 mV) was applied to the electrode, while the application of a negative potential (-800 mV) resulted in a decrease. When the solution pH was varied to alter the charge on the protein, the adsorption trends appeared to follow electrostatic interaction, namely, greater adsorption when the electrode and the protein possessed opposite charge and vice versa. The adsorbed protein layer had the effect of blocking the electron transfer. It was possible to correlate the degree of electron blocking with the amount of adsorbed protein to show that the greater the adsorption, the larger the blocking effect. Of the two proteins used, HSA proved to be more efficient at blocking the electron transfer.     

Reflective interferometric fourier transform spectroscopy: a self-compensating label-free immunosensor using double-layers of porous SiO2

An interferometric biosensor comprised of two layers of porous Si, stacked one on top of the other, is described. A fast Fourier transform (FFT) of the reflectivity spectrum reveals three peaks that correspond to the optical thickness of the top layer, the bottom layer, and both layers together. Binding of immunoglobulin G to a protein A capture probe adsorbed to the surface of the top layer induces changes in reflectivity at the top layer/solution interface. The FFT method allows discrimination of target analyte binding from matrix effects due to nonspecific changes in the analyte solution. The sensor response is shown to be insensitive to the addition of 4000-fold excess sucrose or 80-fold excess bovine serum albumin interferents.     

Sandwich-type amperometric immunosensor for human immunoglobulin G using antibody-adsorbed Au/SiO2 nanoparticles

A novel sandwich-type electrochemical immunosensor for human immunoglobulin G (hIgG) was developed using Au/SiO₂ nanoparticles (NPs) with adsorbed horseradish peroxidase-anti-hIgG as the secondary antibody layer. The signal readout is based on the amperometric response to the catalytic reduction of hydrogen peroxide at an AuNPs-polythionine modified glassy carbon electrode. Under optimized conditions, the linear range is from 0.1 to 200 ng·mL^?1, with a detection limit of 0.035 ng·mL^?1 (at an S/N of 3). The immunosensor exhibited a performance that is better than that based on Au/SiO₂NPs-excluded secondary antibody.     

Adsorption of albumin and IgG to porous and smooth titanium

The possibility to load submicrometer porous titanium surfaces with relatively small proteins, albumin and immunoglobulin G (IgG) was investigated. The loading ability is of interest due to the possibility of slow release of molecules from biomaterial surfaces, and may be important for the manipulation of wound healing around prostheses. Iodine-125 (125I) labeled albumin and IgG were adsorbed onto smooth and to porous titanium with a pore diameter of 200-300 nm. The smooth and porous surfaces were divided into three groups: hydrophilic, hydrophobic, or to amine-terminated silane (3-aminopropyltriethoxysilane) that bound proteins via glutaraldehyde. The protein solution pH and protein concentrations were varied, and the adsorption experiments made without or in the presence of calcium and magnesium ions. The adsorbed amounts were quantified with a gamma counter. Two to eleven times more proteins adsorbed onto porous than smooth surfaces and the adsorbed amounts increased with increasing protein concentration (0.1-10 mg/ml) during a constant incubation time. The elutability by sodium dodecyl sulphate (SDS) was incomplete on porous surfaces.     

Simultaneous monitoring of protein adsorption at the solid–liquid interface from sessile solution droplets by ellipsometry and axisymmetric drop shape analysis by profile

In this paper two in situ techniques are combined to simultaneously examine protein adsorption at the solid–liquid interface from sessile solution droplets. With axisymmetric drop shape analysis by profile (ADSA-P) the change in solid–liquid interfacial tension is determined, while ellipsometry is employed to measure the amount of protein adsorbed from the same solution droplet at the solid–liquid interface. Three proteins (human serum albumin (HSA), immunoglobulin G (IgG) and fibrinogen (Fb)) were dissolved to a concentration of 0.05 mg ml⁻¹ in PBS (pH 7) and sessile droplets were placed for 2 h on a 47.8 nm thick gold coating on glass. The gold coated glass was positioned onto a quartz prism with immersion oil. The prism was aligned in a rotating analyser ellipsometer and the optical beam was thus allowed to be reflected at the hydrophobic gold surface. The ADSA-P set-up was built in 90° cross-beamed set-up around the prism. By combining the results for the adsorbed amounts and the interfacial tension changes over the two hour adsorption period, two stages in the adsorption process could be distinguished. In the first stage, the adsorbed amounts increase in correspondence with the interfacial tension changes, indicating that the interfacial tension changes are caused by adsorption, whereas in the second stage interfacial tension changes continue despite the adsorbed amounts being constant. Consequently, the second stage must be associated with conformational changes of the adsorbed proteins. For HSA and Fb, the conformational contribution to the interfacial tension changes (7.8 and 5.3 mJ m⁻², respectively) were approximately 2-fold the adsorption contribution, while for IgG both were equal around 3 mJ m⁻².     

基于碳纳米管修饰玻碳电极的唾液sIgA可再生免疫传感器

将壳聚糖(CHI)分散的羧基化多壁碳纳米管(MWCNT)吸附到玻碳电极(GCE)表面,形成负电荷界面,利用静电吸附和金-氮(Au-N)、金-硫(Au-S)共价键作用将阳离子电子媒介体硫堇(THI)和纳米金颗粒(GNPs)层层自组装到电极表面,通过纳米金单层吸附唾液分泌性免疫球蛋白A(sIgA),最后用辣根过氧化物酶(H...     

The effective and specific isolation of antibodies from human serum by using thiophilic paramagnetic polymer beads

A rapid and effective method to specifically isolate immunoglobulin G (IgG) from human serum by thiophilic paramagnetic polymer beads was developed. The thiophilic paramagnetic beads were synthesized with vinyl acetate and divinylbenzene by microsuspension polymerization in the presence of magnetite nanoparticles. Divinylsulfone and 2-mercapto-benzothiazole were subsequently used to modify the surface of these beads, resulting in thiophilic particles that exhibited a high specificity to the antibodies in serum at low salt concentration. The adsorbed IgG was eluted by 0.8 M KCl and 72% of the IgG in the serum was recovered. The purity of the isolated IgG reached 98.4% and the bioactivity was fully maintained (>99%). The high efficiency, mild conditions and simplicity make this technology suitable for the economic purification of antibodies in a large scale.     

The research on physiological property of functionalized hyaluronan: interaction between sulfated hyaluronan and plasma proteins

Hyaluronan (HA) is a natural polysaccharides which has no sulfated group but high molecular weight in comparison with other glycosaminoglycans (GAGs). Previously it has been cleared up that the cell function of human keratinocytes is affected by S‐HA (HA substituted with sulfated groups). Most biomedical materials contact with blood components, proteins, cells, etc. In this study, the interaction between S‐HA and blood components is discussed, that is, plasma proteins. And the application of S‐HA for new analytical and separation method of some proteins is pointed out. None of the proteins were adsorbed to HA. Fibronectin and fibinogen were adsorbed to S‐HA, but immunoglobulin‐G and insulin were not adsorbed to it as well as heparin. However, albumin could interact only with heparin, and it did not interact with S‐HA. Furthermore S‐HA adsorbed the plasma proteins that did not adsorb to heparin. It is clear that S‐HA showed different interaction with the plasma proteins in comparison with natural sulfated polysaccharides. Copyright © 2004 John Wiley & Sons, Ltd.     

Influence of bovine immunoglobulin G on faradaic reactions at electrodes

The effect of bovine immunoglobulin G (IgG), adsorbed on different electrode materials, on faradaic reactions of hexacyanoferrate(III)-hexacyanoferrate(II), hydroquinone-benzoquinone, nicotinamide adenine dinucleotide, reduced from (NADH), and phenol is described and discussed. The reactions are partially inhibited by IgG, but the current and peak potentials reach protein-independent values for higher concentrations of IgG. The hydroquinone-benzoquinone couple can be used for detection purposes at IgG-covered electrodes in electrochemical immunoassays.     

Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates

Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins behaved similarly on addition of the alkaline-surfactant cleaning solution, in that platinum and gold exhibited an increase, while tungsten, titanium, and stainless steel showed a decrease in weight. According to dissipation measurements with the QCM-D, the adsorbed layer for platinum and gold was rigid, while that for the tungsten, titanium, and stainless steel was much more flexible. The removal efficiency of adsorbed-protein by alkaline solution of SDS depended on the water content of the adsorbed layers for W, Ti, and SS, while for Pt and Au, it depended on secondary structural content. When protein adsorption was high (Pt, Au), protein-protein interactions and protein-surface interactions were dominant and the removal of protein layers was limited. Water content of the adsorbed protein layer was the determining factor for how efficiently the layer was removed by alkaline SDS when protein adsorption was low. Hence, protein-protein and protein-surface interactions were minimal and protein structure was less perturbed in comparison with those for high protein adsorption. Secondary structural content determined the efficient removal of adsorbed protein for high adsorbed amount.     

Evaluation of endothelial cell adhesion onto different protein/gold electrodes by EIS

To study cell attachment to biomaterials, several proteins such as fibronectin, collagen IV, heparin, immunoglobulin G, and albumin have been deposited onto polystyrene adsorbed on a self-assembled monolayer (silane or thiol) on glass or gold, respectively. The different steps of this multilayer assembly have been characterized by electrochemical impedance spectroscopy (EIS). These data are compared to those of adhesion rate, viability percentage, and cytoskeleton labeling for a better understanding of the cell adhesion process to each protein. All the proteins are endothelial cell adhering biomolecules but not with the same features. A linear relationship has been established between adhesion rate and resistance of the endothelial cell/protein interface for all negatively charged proteins.     

Removal of detergent and solvent from solvent-detergent-treated immunoglobulins.

The solvent-detergent (S/D) method was applied for inactivation of lipid-enveloped viruses during the production of immunoglobulins. Amberlite XAD-7 resin was used for removal of solvent (tri-n-butyl phosphate, TnBP) and detergent (Triton X-100) after the performed S/D inactivation procedure. The S/D reagents from the immunoglobulin preparation were adsorbed on Amberlite XAD-7, while immunoglobulins passed through the column and retained their biological activity. Using the method developed here, the final immunoglobulin preparation contains less than 1 ppm of Triton X-100 and less than 2 ppm TnBP.     

Adsorption and Bioactivity of Protein A on Silicon Surfaces Studied by AFM and XPS

The adsorption of protein A on silicon surfaces was studied by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy. The deposition was made statically from various concentrations of protein A in water solution. The biological activity was checked by the immobilization of rabbit immunoglobulin G. The protein adsorption occurs in least two different phases and leads to a multilayer film. The first monolayer of proteins is rapidly adsorbed on the surface. The adsorption of the second layer of proteins occurs much more slowly (a thousand times slower) and also involves the third monolayer. The protein A of the first monolayer is denaturated and biologically inactive. On the contrary, the proteins of the second monolayer keep their natural diameter and remain biologically active. AFM artifacts such as the convolution with small objects and the resulting estimation of the coverage ratio are discussed. Copyright 2001 Academic Press.     

γ-Mo2N催化剂上H2及NO吸附性质的TPD-MS研究

采用TPD-MS方法研究了H2及NO在γ-Mo2N上的吸附状况．单独的H2-TPD结果表明,当H2在673K吸附时,在443K、573K及723K得到了三个H2脱附峰,表明γ-Mo2N上有三种不同能量的H2吸附位．NO－TPD结果表明,NO吸附后亦有三个脱附峰（383K、493K、543K）,对应着γ-Mo2N上三种不同能量的NO吸附位：低、中、高能吸附位．NO既可以以解离状态,又可以以一种NO三聚态（dimerordinitrosyl）的形式吸附在γ－Mo2N上,这些吸附物种在脱附过程中产生大量的N2及少量的N2O.对比NO吸附在不同处理条件的γ-Mo2N上的TPD结果可知,NO是吸附在γ-Mo2N上的MO的配位不饱和中心上,这些吸附中心既可通过还原催化剂,又可通过在773K抽空钝化态的γ－Mo2N而产生,H2和NO共吸附的结果表明,预吸附H2再吸附NO后,H2和NO的脱附量均大大减少,且只有两个脱附峰出现.NO只在363K及493K出现两个脱附峰,表明预吸附氢占据了NO的强吸附位,且NO很难取代它,从而使NO只能吸附在能量较低的吸附位上;而H2只在523K及723K出现两个脱附峰,且伴随着H2的脱出有N2和H2O的产生,表明在γ－Mo2N上NO可能与预吸附氢形成了一种复合相MoHx（NO）y,它在脱附时分解为H2、N2及H2O.     

Metal-enhanced fluoroimmunoassay on a silver film by vapor deposition

We studied a fluoroimmunoassay using metal-enhanced fluorescence (MEF) detection on silver film generated by vapor deposition method. The morphology of the silver film was controlled through the thickness of the film. A silica layer was coated on the silver film to protect the film and separate the fluorophore from the metal surface. Rabbit immunoglobulin G (IgG) was adsorbed on the silica by physiosorption and then dye-labeled anti-rabbit IgG was bound to the immobilized rabbit IgG. It was observed that the fluorophore was quenched on a thin silver film (2 nm), enhanced on a thick film (>5 nm), and reached saturation (ca. 10 times enhancement) at 20 nm. The MEF was also dependent on the thickness of the silica with a maximum at 10 nm. The lowest lifetime was observed on the 20 nm silver film, which was consistent with the saturation of MEF. These results showed the properties of a silver film needed for a maximum increase of fluorescence intensity in a fluoroimmunoassay. Dependence of the MEF on the emission wavelength was also studied using different dye-labeled anti-rabbit IgGs.     

Native and Hydrophobically Modified Human Immunoglobulin G at the Air/Water Interface

The adsorption of human immunoglobulin G (IgG) at the air/water interface was monitored both by the in situ radiotracer technique using [(14)C] labeled IgG and by surface tension measurements. The results reveal that adsorption of IgG from single protein systems displays bimodality due to molecular rearrangements at the interface. Above the threshold value of 1.5x10(-2) mg/ml solution concentration, adsorbed IgG molecules reoriented from the side-on to the end-on configuration. The existence of a lag time which did not appear in Gamma=f(t) curves, was observed in Pi=f(t) relationships at low protein concentrations and was due to the limits of the surface pressure technique to detect protein adsorption. The adsorption of native IgG was also carried out in the presence of a hydrophobized IgG obtained by grafting capryloyl residues to its lysine groups by reaction with N-hydroxysuccinimide ester of caprylic acid, which yielded 19 covalently bound alkyl chains to the IgG molecule (19C(8)-IgG). This modified IgG exhibited enhanced adsorption at the air/water interface, as manifested by its increased adsorption efficiency relative to the native protein. Sequential and competitive adsorption experiments from binary mixtures of native IgG and 19C(8)-IgG clearly demonstrate that the displacement of the native protein from the air/water interface strongly depended on the manner of how 19C(8)-IgG and native IgG competed with each other. When the two proteins competed simultaneously, 19C(8)-IgG predominantly occupied the available area but when native IgG was adsorbed first, for 2 h, the sequentially adsorbed 19C(8)-IgG was incapable of substantially displacing it from the interface. Copyright 2001 Academic Press.     

Structural and topographical characteristics of adsorbed WPI and monoglyceride mixed monolayers at the air-water interface

In this work we have analyzed the structural and topographical characteristics of mixed monolayers formed by an adsorbed whey protein isolate (WPI) and a spread monoglyceride monolayer (monopalmitin or monoolein) on the previously adsorbed protein film. Measurements of the surface pressure (pi)-area (A) isotherm were obtained at 20 degrees C and at pH 7 for protein-adsorbed films from water in a Wilhelmy-type film balance. Since the surface concentration (1/A) is actually unknown for the adsorbed monolayer, the values were derived by assuming that the A values for adsorbed and spread monolayers were equal at the collapse point of the mixed film. The pi-A isotherm deduced for adsorbed WPI monolayer in this work is practically the same as that obtained directly by spreading. For WPI-monoglyceride mixed films, the pi-A isotherms for adsorbed and spread monolayers at pi higher than the equilibrium surface pressure of WPI are practically coincident, a phenomenon which may be attributed to the protein displacement by the monoglyceride from the interface. At lower surface pressures, WPI and monoglyceride coexist at the interface and the adsorbed and spread pi-A isotherms (i.e., the monolayer structure of the mixed films) are different. Monopalmitin has a higher capacity than monoolein for the displacement of protein from the air-water interface. However, some degree of interactions exists between proteins and monoglycerides and these interactions are higher for adsorbed than for spread films. The topography of the monolayer corroborates these conclusions.