Immobilization Of Albumin Research Articles

Adsorption of human serum albumin onto gold: a combined electrochemical and ellipsometric study

Human serum albumin adsorption onto gold surfaces was investigated by electrochemical and ellipsometric methods. Albumin adsorption onto gold was confirmed by the change of the open circuit potential of gold and by the ellipsometric parameter variation during albumin immobilization. In both experiments the parameters reached stable values within 10-15 min. The albumin adsorption layer thickness measured with the ellipsometer was about 1.5 nm. The adsorption of albumin under applied potential was also investigated and it was found that both positive and negative applied potential promote albumin adsorption. Changes in the optical parameters of bare gold and albumin adsorbed onto gold surface under applied potential were investigated with in situ ellipsometry. The similarity and reversibility of the optical changes showed that adsorbed albumin was stable on the gold surface under the applied potential range (-200-600 mV). The cyclic voltammograms of K3Fe(CN)6 on the modified gold surface showed that albumin could partly block the oxidation and reduction reaction.

Neural differentiation of midbrain cells on various protein-immobilized polyethylene films.

The effect of surface modification of polyethylene (PE) film on differentiation of midbrain (MB) cells obtained from rat embryos was determined by their micromass culture system. When cultured on untreated PE film, cell differentiation was suppressed to approximately two-thirds of that observed in a control culture dish. On the contrary, type I collagen-immobilized PE film increased differentiated foci of the MB cells more than did the untreated PE film. RGDS (Arg-Gly-Asp-Ser) peptide immobilization onto PE film resulted in almost the same differentiation activity as the collagen immobilized PE film. Bovine serum albumin (BSA) immobilization onto PE film enhance the differentiation activity more than did the untreated PE film, but not up to the levels of collagen- and RGDS-immobilized PE. The number of differentiated foci of the MB cells on untreated PE film were increased by the addition of the condition medium prepared from the collagen-immobilized PE film. However, the number of foci was not increased by the addition of other condition media obtained from control dish, untreated, BSA-, and RGDS-immobilized PE. On the other hand, none of these condition media enhanced a differentiation of the neuronal cell line of PC12 cells, suggesting that some factors effectively differentiate midbrain cells, composed of neuronal epithelial and mesenchymal cells, but not the PC12 cells secreted in the condition media prepared from collagen-immobilized PE. In addition, it is probable that neural growth factor was not secreted in these condition media, which could not induce the differentiation of PC12 cells.

The inhibitory mechanism of gap junctional intercellular communication induced by polyethylene and the restorative effects by surface modification with various proteins.

Gap junctional intercellular communication (GJIC) is a function that plays an important role in maintaining cell and tissue homeostasis and in regulating cell growth, development, and differentiation. Change in this function of V79 fibroblasts cultured on polyethylene films modified with albumin or collagen was estimated using fluorescence redistribution after photobleaching (FRAP) analysis. The GJIC function of V79 cells on nontreated polyethylene was strongly inhibited in comparison with those on a glass coverslip. When the cells were culture on collagen-immobilized polyethylene film, this function was recovered to about 70% of the cells cultured on the coverslip. However, albumin immobilization did not recover the function as much as collagen immobilization. Western blotting analysis and immunostaining of connexin 43, which is a major protein constituting gap junctional channel of these cells, revealed its abnormal expression and distribution in the cells on nontreated polyethylene, whereas its almost normal distribution was observed in the cells on collagen-immobilized polyethylene. This abnormal expression and distribution of connexin 43 induced by the surface of polyethylene may be ascribed to a strong inhibition of GJIC of V79 fibroblasts.

Capacitive monitoring of protein immobilization and antigen–antibody reactions on monomolecular alkylthiol films on gold electrodes

Self-assembled monolayers of ω-mercaptohexadecanoic acid and ω-mercaptohexadecylamine on gold electrodes are stable at neutral pH and display pure capacitive behavior at frequencies around 20 Hz. Different methods of covalent immobilization of proteins on these monolayers are compared. Various reagents including succinimides, thionylchloride, p-nitrophenol and carbodiimides were used to activate the carboxy groups of the adsorbed monolayer of ω-mercaptohexadecanoic acid. Glutaraldehyde, cyanuric chloride and phenylene diisocyanate were used to activate the amino groups of the monolayer of ω-mercaptohexadecylamine. The immobilization of albumin on the activated surface was studied by capacitive measurements. The N-hydroxysuccinimide and carbodiimide methods were identified as most suitable for protein immobilization in that they did not compromise the insulating properties of the alkylthiol layer and led to maximal increase of its dielectric thickness. These approaches were used for a layer-by-layer preparation of a capacitive immunosensor. Specifically, antibodies to human serum albumin were immobilized on the alkylthiol monolayer. Binding of the antigen led to a decrease of the electrode capacitance. The detection limit of the immunosensor is as low as 15 nM (1 mg/l).

Albumin Immobilized Polyurethane and its Blood Compatibility

Surface pretreatment with albumin on a blood contacting material inhibits platelet adhesion, activation, and subsequent thrombus formation. Although adsorbed albumin improves blood compatibility, rapid desorption occurs when this surface is exposed to circulating blood. In this study, human serum albumin was immobilized on a polyurethane (PU) surface to investigate its blood compatibility and extended effects on a blood-material interface. The PU surface was treated with hexamethylene diisocyanate (HMDI), and the PU-HMDI was further grafted with albumin to produce an albumin immobilized PU surface (PU-albumin). The PU-albumin surface was characterized by attenuated total reflection infrared electron spectroscopy for chemical analysis, scanning electron microscopy, and dynamic contact angle. Blood compatibility was evaluated by in vitro protein adsorption, platelet adhesion, and occlusion time in an ex vivo rabbit arterio-arterial shunt. Immobilization of albumin was confirmed by the disappearance of the -NCO peak observed at 2,250 cm-1 on the PU-HMDI surface by infrared spectroscopy and the existence of sulfur atomic percent by electron spectroscopy for chemical analysis. The concentration of PU-albumin was approximately 5.8 micrograms/cm2. The PU-albumin also showed a slight increase in hydrophilicity on the Wilhelmy plate method, and there was less fibrinogen adsorption than a PU control. In addition, PU-albumin had less platelet adhesion, platelet activation, and thrombogenicity. The ex vivo occlusion time of untreated PU was 50 min, that of PU-albumin was extended to 150 min, indicating that a PU-albumin surface has better blood compatibility than PU alone.

Effect of albumin immobilization by plasma polymerization on platelet reactivity

Albumin was immobilized on polyethylene (PE) film by the plasma polymerization method. Immobilized albumin on formaldehyde polymerized PE film was hard to desorb even when the film was dipped in blood plasma. Amounts of platelet adhesion on the film and serotonin release decreased clearly. Aggregated platelets were observed on PE film but not observed on the albumin immobilized surface. These results seemed to be due to not only immobilized albumin, but also the surface charge determined by plasma protein adsorption. That is, the slightly negatively charged surface indicated good anti-thrombogenicity.

Stepwise immobilization of proteins via their glycosylation.

Glycosyl derivatives of bovine serum albumin in which the glycosyl residue is represented by mono- or disaccharide can be, after periodate oxidation, coupled to polyhydrazides having a macroporous matrix (cross-linked polyacrylamide, bead cellulose). The amount of the linked neoglycoprotein depends not only on the physical structure of the matrix but also on the degree of its substitution with hydrazide groups and on the type and concentration of glycosyl residue in the neoglycoprotein. A high degree of substitution as well as the presence of the D-galactosyl unit both play a positive role. Owing to the fact that the glucosyl unit in disaccharide residues (cellobiosyl, lactosyl) also contributes positively to spacer properties, in the monolactosyl derivative of albumin exhibits good binding properties towards macroporous polyhydrazides. While the high sugar-containing conjugates of glycosyl derivatives of albumin with polyhydrazides are stable for two weeks at pH 6-9, the conjugates of the monolactosyl derivative of albumin can only be stored at pH 7.5. The binding site of albumin immobilization is considered.

Study of Drug-Protein Binding by Affinity Chromatography: Interaction of Bovine Serum Albumin and Salicylic Acid

The affinity chromatographic technique was used to study the interaction of bovine serum albumin and salicylic acid at 3.3±1.1°. Beaded agarose gel, on which the albumin was immobilized by covalent linkage, was packed in a column as an affinity adsorbent. Frontal analysis was performed on this column to evaluate the binding parameters for the interaction. The effect of albumin immobilization on drug binding was investigated by comparing the binding parameters of two affinity adsorbents, directly coupled albumin and albumin coupled through a spacer arm. The latter mode of attachment gave binding characteristics comparable to those of the soluble albumin. The method is simple and precise. The affinity adsorbent can be used repeatedly for many months for various drugs, including those that do not diffuse through dialysis membranes.