PDMS微芯片吸附牛血清白蛋白的研究

材料表面的物理和化学性质对蛋白质的吸附具有很大的影响[1].对蛋白质吸附的研究是研制生物传感器、生物芯片和生物材料的基础.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Bovine serum albumin adsorption on nano-rough platinum surfaces studied by QCM-D

The adsorption of bovine serum albumin (BSA) on platinum surfaces with a root-mean-square roughness ranging from 1.49nm to 4.62nm was investigated using quartz crystal microbalance with dissipation (QCM-D). Two different BSA concentrations, 50microg/ml and 1mg/ml, were used, and the adsorption studies were complemented by monitoring the antibody interaction with the adsorbed BSA layer. The adsorption process was significantly influenced by the surface nano-roughness, and it was observed that the surface mass density of the adsorbed BSA layer is enhanced in a non-trivial way with the surface roughness. From a close examination of the energy dissipation vs. frequency shift plot obtained by the QCM-D technique, it was additionally observed that the BSA adsorption on the roughest surface is subject to several distinct adsorption phases revealing the presence of structural changes facilitated by the nano-rough surface morphology during the adsorption process. These changes were in particular noticeable for the adsorption at the low (50microg/ml) BSA concentration. The results confirm that the nano-rough surface morphology has a significant influence on both the BSA mass uptake and the functionality of the resulting protein layer.     

Investigating adsorption of bovine serum albumin on cellulosic substrates using magnetic resonance imaging

Cellulosic biomass is recalcitrant to enzymatic hydrolysis which greatly reduces the efficiency of biofuels production. Specifically, the lignin component of biomass is thought to provide non-productive binding sites for glycosyl hydrolases, effectively disabling the enzymes from completing further digestion. A thorough understanding of the adsorption rates of protein molecules on celluloses—especially lignocelluloses—is crucial to improving the cyclic steps of adsorption, diffusion, and reaction. We use magnetic resonance imaging (MRI) to detect concentrations of bovine serum albumin (BSA) in equilibrium with various cellulose substrates, including delignified and acid-treated lignocellulosic substrates. BSA is believed to be an effective adsorption blocker during enzymatic hydrolysis of lignocellulosics, and has been correlated with an increase in reaction yield. We found BSA to have little adsorption onto the chosen cellulose substrates in the low concentration range studied. Ultraviolet (UV) absorption measurements of reaction supernatants at 280 nm were used to confirm the MRI results for each of the substrate types. The advantages of the MRI technique are compared with that of the traditional UV measurement.     

Effects of anti-epileptic drugs on the L-tryptophan binding to human serum albumin.

Effects of four anti-epileptic drugs (AED; phenobarbital, phenytoin, carbamazepine and sodium valproate) on the L-tryptophan binding to human serum albumin were studied. Among these drugs examined, only sodium valproate inhibited the binding even within the concentrations of its therapeutic range, and the Klotz plotting analysis revealed that the inhibition was competitive. The results of examinations with sera from epileptic patients medicated with these AED and drug-free normal controls also suggested that the protein binding ratios of L-tryptophan were decreased in the blood plasma of some patients with the high valproate concentrations and the low albumin contents.     

固定化牛血清白蛋白吸附材料的制备及其对胆红素的吸附性能

降低高胆红素血症和重症肝炎患者血液中异常升高的胆红素浓度是血浆交换和血液灌流等疗法的目标之一。本文通过共价键合的方法将牛血清白蛋白(BSA)固定在甲基丙烯酸缩水甘油酯共聚三羟甲基丙烷三甲基丙烯酸酯[poly(GMA-co-TMPTMA)]大孔树脂微球上,制备得到对胆红素具良好吸附性能的固定化BSA吸附材料(BIA),吸附容量达48.7mg/g。由于血清白蛋白对胆红素的强烈相互作用,胆红素溶液中游离BSA的存在会显著降低BIA对胆红素的吸附量。BIA对胆红素的吸附量随吸附温度升高而增加。BIA在-80℃下储存31d后性能仍然稳定,对胆红素的吸附量几乎不变。上述结果表明所制备的BIA为以特异性吸附胆红素为目的的血液净化材料提供了新的选择。     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Human serum albumin adsorption on TiO2 from single protein solutions and from plasma

In the present work, the adsorption of human serum albumin (HSA) on commercially pure titanium with a titanium oxide layer formed in a H(2)O(2) solution (TiO(2) cp) and on TiO(2) sputtered on Si (TiO(2) sp) was analyzed. Adsorption isotherms, kinetic studies, and work of adhesion determinations were carried out. HSA exchangeability was also evaluated. Surface characterization was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and wettability studies. The two TiO(2) surfaces have very distinct roughnesses, the TiO(2) sp having a mean R(a) value 14 times smaller than the one of TiO(2) cp. XPS analysis revealed consistent peaks representative of TiO(2) on sputtered samples as well as on Ti cp substrate after 48 h of H(2)O(2) immersion. Nitrogen was observed as soon as protein was present, while sulfur, present in disulfide bonds in HSA, was observed for concentrations of protein higher than 0.30 mg/mL. The work of adhesion was determined from contact angle measurements. As expected from the surface free energy values, the work of adhesion of HSA solution is higher for the TiO(2) cp substrate, the more hydrophilic one, and lower for the TiO(2) sp substrate, the more hydrophobic one. The work of adhesion between plasma and the substrates assumed even higher values for the TiO(2) cp surface, indicating a greater interaction between the surface and the complex protein solutions. Adsorption studies by radiolabeling of albumin ((125)I-HSA) suggest that rapid HSA adsorption takes place on both surfaces, reaching a maximum value after approximately 60 min of incubation. For the higher HSA concentrations in solution, a multilayer coverage was observed on both substrates. After the adsorption step from single HSA solutions, the exchangeability of adsorbed HSA molecules by HSA in solution was evaluated. The HSA molecules adsorbed on TiO(2) sp seem to be more easily exchanged by HSA itself than those adsorbed on TiO(2) cp after 24 h. In contrast, after 72 h, nearly all the adsorbed albumin molecules effectively exchange with other albumin molecules.     

Viscoelastic study of the adsorption of bovine serum albumin on gold and its dependence on pH

Using a quartz crystal resonator system operating at 5 MHz the shear wave propagating properties of bovine serum albumin (BSA) have been monitored as it is adsorbed on a gold surface from a phosphate buffered saline (PBS) solution. Employing a 2-layer model for the combined BSA layer and PBS solution, the viscoelasticity of the BSA layer may be determined in real time as the adsorption on gold proceeds. The viscoelasticity is found to depend on the pH of the PBS solution and changes gradually over long times. It is suggested that at the low frequency of the measurement, large-scale molecular motions are being monitored which are a consequence of the structural changes in the protein molecules undergoing adsorption. Such low-frequency molecular motions are difficult to examine by any other technique. The results and their interpretation in viscoelastic terms demonstrate the considerable potential of the quartz crystal resonator system for assessing the stability of proteins on surfaces and their suitability as coatings for prosthetic materials.     

Patterns of the adsorption of bovine serum albumin on carboxymethyl dextran and carboxymethyl cellulose films

Patterns of the adsorption of bovine serum albumin on carboxymethyl dextran and carboxymethyl cellulose films are studied by means of microcontact printing, atomic force microscopy, and quartz crystal microbalance. It is shown that both the charge of polysaccharide macromolecules and the technique for deposition of their films onto the surface (via adsorption from a solution or covalent cross-linking) are factors that determine the degree of nonspecific adsorption of the protein on such films.     

Effects of acetonitrile on adsorption behavior of bovine serum albumin onto synthetic calcium hydroxyapatite particles

The objective of this study was to examine the effects of acetonitrile (AN) on the adsorption behavior of bovine serum albumin (BSA) onto calcium hydroxyapatite [Ca10(PO4)6(OH)2 Ca10, Hap] materials by combining the ultraviolet (UV) and circular dichroism (CD) measurements of BSA solution. The structural change of BSA molecules with addition of AN was investigated by UV and CD spectroscopy measurements prior to studying adsorption behavior of BSA onto Hap. The CD spectra revealed that the fraction of alpha-helical content of BSA is remarkably decreased at AN concentrations above 30 vol.%, while beta-sheet content is increased. On the other hand, the percentages of random coil and turn contents were decreased only slightly. In addition to this secondary structural change of BSA, the UV spectra suggested that the tertiary structure of protein molecules was also changed by the addition of large amounts of AN; BSA molecules associate to form molecular aggregates at [AN]> or =40 vol.%. From the adsorption of BSA onto Hap particles (ca. 30 nm in the particle length) from a water-AN mixed solution, it was revealed that the adsorption behavior of BSA strongly depends on the change of secondary and tertiary structures of BSA by addition of AN. The contraction of BSA molecules at low AN concentrations (10-20 vol.%) gave their small cross-sectional area, providing a large amount of adsorption (n(BSA)), although n(BSA) was decreased above 30 vol.% AN by enlargement of BSA molecules with solvation and unfolding some alpha-helix domains. The n(BSA) values of the systems with AN exhibited a maximum; n(BSA) was increased at a lower BSA concentration region, although it was decreased at a higher BSA concentration due to self-association. Accompanying the change of n(BSA) with AN addition, the maxima of electrophoretic mobility (em) of the Hap particles were observed for the systems with AN, although the em of Hap particles was normally increased and saturated with increase in protein coverage for the native structure on the system without AN. On the other hand, because the aggregated BSA molecules could be cooperatively bound, the adsorption of BSA onto the Hap particles with large size (108 nm in the particle length) was enhanced in the presence of AN.     

Effect of phosphorylated organic compound on the adsorption of bovine serum albumin by hydroxyapatite.

The amount of adsorption of bovine serum albumin (BSA) by hydroxyapatite (HAP) increased with a concentration of CaCl2 due to the bridging effect of Ca2+ between adsorbate BSA and adsorbent HAP. On the other hand, it decreased remarkably with a concentration of K2HPO4. This was explained in terms of the effects of ionic strength and competitive adsorption between inorganic phosphate anion (Pi) and BSA, because BSA is in negatively charged over the examined pHs. A similar effect was observed in the presence of phosphorylated compounds such as phosphoserine, phytate, and phosphorylated polyvinylalcohol. The inhibiting effect of these compounds was stronger than that of their mother compounds (serine, inositol, and polyvinylalcohol). This result shows that phosphate groups bound to the mother compounds interfere with the adsorption of BSA by HAP in the same manner that Pi does. Although the adsorption of BSA was almost irreversible with respect to dilution with water, desorption was performed when these organic phosphorylated compounds were added after the accomplishment of the adsorption of BSA. However, the effective concentration of the phosphorylated compounds for the desorption of BSA was fairly higher than that for the competitive inhibition against the BSA adsorption.     

Spectroscopic studies on in vitro binding of cefixime and tolcapone drugs with serum albumin

The interaction between cefixime (antibacterial) and tolcapone (Parkinson’s disease) drugs with bovine serum albumin (BSA) was investigated using several spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism. The thermodynamic parameters of the interactions were calculated, which indicated that the binding processes are spontaneous and H-bonding and van der Waals forces play a major role in BSA–cefixime interaction and hydrophobic interactions dominate BSA–tolcapone complexation. Cefixime quenches the intrinsic fluorescence of BSA by dynamic process while tolcapone through static process. The binding constant of the BSA–tolcapone complex (10⁷ L mol^?1) is found to be relatively higher than that of BSA–cefixime complex (10⁴ L mol^?1). The binding distance between BSA and cefixime and tolcapone is calculated to be 3.3 and 4.2 nm, respectively. Both fluorescence and circular dichrosim spectral studies confirmed conformational changes in BSA upon binding with these drugs. Molecular docking studies suggest the possible binding sites in the protein molecule.'     

Qualitative study of the competition of drugs in binding to serum albumin

Summary The competitive binding of five drugs and a detergent to bovine serum albumin at pH 7.4 and room temperature was studied by Hummel-Dreyer method in high performance liquid chromatography. The five drugs are: warfarin, sulfinpyrazone, aspirin, quinidine gluconate and lidocaine and the detergent is the sodium dodecyl sulfate (SDS). While the quantitative techniques of Hummel-Dreyer method have been well developed during last twenty years, this paper reports, perhaps for the first time, the qualitative techniques of Hummel-Dreyer method as an analytic tool to ascertain whether a drug would bind to protein and how one drug would affect another drug in binding, if the binding does occur. The results on the basis of qualitative observation indicate the strength of binding in the following order: warfarin > aspriin > lidocaine > sulfinpyrazone > quinidine gluconate. The SDS has capacity to disturb the binding site on the surface of protein.     

Fibrinogen adsorption on blocked surface of albumin

We have investigated the adsorption of albumin and fibrinogen onto PET (polyethylene terephthalate) and glass surfaces and how pre-adsorption of albumin onto these surfaces can affect the adsorption of later added fibrinogen. For materials and devices being exposed to blood, adsorption of fibrinogen is often a non-wanted event, since fibrinogen is part of the clotting cascade and unspecific adsorption of fibrinogen can have an influence on the activation of platelets. Albumin is often used as blocking agent for avoiding unspecific protein adsorption onto surfaces in devices designed to handle biological samples, including protein solutions. It is based on the assumption that proteins adsorbs as a monolayer on surfaces and that proteins do not adsorb on top of each other. By labelling albumin and fibrinogen with two different radioactive iodine isotopes that emit gamma radiation with different energies, the adsorption of both albumin and fibrinogen has been monitored simultaneously on the same sample. Information about topography and coverage of adsorbed protein layers has been obtained using AFM (Atomic Force Microscopy) analysis in liquid. Our studies show that albumin adsorbs in a multilayer fashion on PET and that fibrinogen adsorbs on top of albumin when albumin is pre-adsorbed on the surfaces.     

Electrically polarized biphasic calcium phosphates: adsorption and release of bovine serum albumin

In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled as well as unpoled surfaces of the composites was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as a drug or growth factor delivery vehicle.     

Structural characteristics of activated carbons and ibuprofen adsorption affected by bovine serum albumin

Structural characteristics of a series of MAST carbons were studied using scanning electron microscopy images and the nitrogen adsorption isotherms analyzed with several models of pores and different adsorption equations. A developed model of pores as a mixture of gaps between spherical nanoparticles and slitlike pores was found appropriate for MAST carbons. Adsorption of ibuprofen [2-(4-isobutylphenyl)propionic acid] on activated carbons possessing different pore size distributions in protein-free and bovine serum albumin (BSA)-containing aqueous solutions reveals the importance of the contribution of mesopores to the total porosity of adsorbents. The influence of the mesoporosity increases when considering the removal of the drug from the protein-containing solution. Cellulose-coated microporous carbon Norit RBX adsorbs significantly smaller amounts of ibuprofen than uncoated micro/mesoporous MAST carbons whose adsorption capability increases with increasing mesoporosity and specific surface area, burnoff dependent variable. A similar effect of broad pores is observed on adsorption of fibrinogen on the same carbons. Analysis of the ibuprofen adsorption data using Langmuir and D'Arcy-Watt equations as the kernel of the Fredholm integral equation shows that the nonuniformity of ibuprofen adsorption complexes diminishes with the presence of BSA. This effect may be explained by a partial adsorption of ibuprofen onto protein molecules immobilized on carbon particles and blocking of a portion of narrow pores.