Effect of polysaccharide structure on protein adsorption

Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and γ-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces.     

Biofouling of dextran-derivative layers investigated by quartz crystal microbalance

This study reports the fouling of carboxymethyl dextran (CMD) layers in cell culture medium, fibronectin, and serum solutions. CMD layers were covalently immobilized onto amine groups available either on an n-heptylamine plasma polymer (HApp) layer or onto a polyethylenimine (PEI) coating grafted to an acetaldehyde plasma polymer (AApp) layer. The successful immobilization of the graft layers was demonstrated by X-ray photoelectron spectroscopy (XPS). Primary amines on HApp and AApp + PEI surfaces were quantified using a colorimetric assay. Quartz crystal microbalance (QCM) was used to investigate in real-time the fouling of the graft layers upon incubation in cell culture medium (RPMI), fibronectin, and foetal bovine serum (FBS) solutions. HApp, AApp and AApp + PEI layers exhibited large fouling in fibronectin and FBS solutions, while fouling in RPMI cell culture medium was not significant. Protein repellent properties of CMD layers in FBS and fibronectin have been demonstrated compared to the other tested surfaces. QCM has shown that both CMD layers were fouled to a small extent in RPMI medium.     

壳聚糖膜固定生物活性短肽RGDS的研究

为改善壳聚糖对细胞的特异性吸附,采用水溶性碳二亚胺将生物活性短肽精氨酸-甘氨酸-天冬氨酸-丝氨酸(RGDS)固定到壳聚糖膜的表面,采用X射线光电子能谱检测固定多肽前后的壳聚糖膜表面,发现反应后壳聚糖膜表面氮元素含量增大,Nls和Cls曲线拟合谱中酰胺键增多,表明RGDS短肽已固定到壳聚糖膜的表面;人角膜缘上皮细胞体外培养实验表明,固定RGDS后壳聚糖膜的细胞黏附率有了明显提高,固定RGDS后的壳聚糖膜在角膜组织工程支架等方面有更好的应用潜力。     

Development of dextran-derivative arrays to identify physicochemical properties involved in biofouling from serum

To control protein adsorption on surfaces, low-fouling polymer coatings such as poly(ethylene oxide) (PEG or PEO) and polysaccharides are used. Their ability to resist protein adsorption is related to the layer structure, hence the immobilization mode. A polymer array technology was developed to study the structural diversity of carboxymethyl dextran (CMD) layers, whose immobilization conditions were varied. CMD arrays were analyzed by X-ray photoelectron spectroscopy (XPS) and by atomic force microscopy (AFM) colloidal probe force measurements. Serum protein adsorption was studied directly on the CMD arrays using surface plasmon resonance (SPR) microscopy. Physicochemical characterization revealed that pinning density regulates surface coverage and the amount of adsorbed molecules, and that salt concentration influences the surface structure of the charged polymer, forming extended or short layers. Protein adsorption experiments from serum showed that repulsive CMD layers are dense, with extended flexible chains. The present study underlines the usefulness of polymer arrays to study structural diversity of thin graft layers and to relate their physicochemical properties to their resistance to nonspecific protein adsorption.     

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.     

DNA修饰电极的研究(Ⅸ)--DNA探针在金基底上的固定、表征及其表面分子杂交

采用疏基化合的自组装／共价键合反应的逐层固定方法将双链DNA固定到金表面得到DNA修饰电极,并对该电极表面进行了电化学和X射线光电子能谱表征。研究了电极表面固定化DNA的表面分子杂交。对开发电化学基因诊断芯片和基因传感器具有一定意义。     

DNA修饰电极的研究(ⅠΧ)——DNA探针在金基底上的固定、表征及其表面分子杂交

采用巯基化合物自组装 /共价键合反应的逐层固定方法将双链 DNA固定到金表面得到 DNA修饰电极 ,并对该电极表面进行了电化学和 X射线光电子能谱表征 .研究了电极表面固定化 DNA的表面分子杂交 .对开发电化学基因诊断芯片和基因传感器具有一定意义     

One-step photochemical attachment of NHS-terminated monolayers onto silicon surfaces and subsequent functionalization

N-Hydroxysuccinimide (NHS)-ester-terminated monolayers were covalently attached in one step onto silicon using visible light. This mild photochemical attachment, starting from omega-NHS-functionalized 1-alkenes, yields a clean and flat monolayer-modified silicon surface and allows a mild and rapid functionalization of the surface by substitution of the NHS-ester moieties with amines at room temperature. Using a combination of analytical techniques (infrared reflection absorption spectroscopy (IRRAS), extensive X-ray photoelectron spectroscopy (XPS) in combination with density functional theory calculations of the XPS chemical shifts of the carbon atoms, atomic force microscopy (AFM), and static contact angle measurements), it was shown that the NHS-ester groups were attached fully intact onto the surface. The surface reactivity of the NHS-ester moieties toward amines was qualitatively and quantitatively evaluated via the reaction with para-trifluoromethyl benzylamine and biotin hydrazide.     

GaN nanowire functionalized with atomic layer deposition techniques for enhanced immobilization of biomolecules

We report the use of atomic layer deposition (ALD) coating as a nanobiosensor functionalization strategy for enhanced surface immobilization that may enable higher detection sensitivity. Three kinds of ALD coating films, Al(2)O(3), TiO(2), and SiO(2), were grown on the gallium nitride nanowire (GaN NW) surfaces and characterized with high-resolution transmission electron microscopy (HRTEM) and vacuum Fourier transform infrared spectroscopy (FTIR). Results from HRTEM showed that the thicknesses of ALD-Al(2)O(3), ALD-TiO(2) and ALD-SiO(2) coatings were 4-5 nm, 5-6 nm, and 12-14 nm, respectively. Results from FTIR showed that the OH contents of these coatings were, respectively, ～6.9, ～7.4, and ～9.3 times that of piranha-treated GaN NW. Furthermore, to compare protein attachments on the different surfaces, poly(ethylene glycol) (PEG)-biotin was grafted on the OH-functionalized GaN NW surfaces through active Si-Cl functional groups. Streptavidin protein molecules were then attached to the biotin ends via specific binding. The immobilized streptavidin molecules were examined with scanning electron microscopy, HRTEM, and fluorescent imaging. Results from HRTEM and energy-dispersive X-ray revealed that the nitrogen concentrations on the three ALD coatings were significantly higher than that on the piranha-treated surface. Results from fluorescent imaging further showed that the protein attachments on the Al(2)O(3), TiO(2), and SiO(2) ALD coatings were, respectively, 6.4, 7.8, and 9.8 times that of piranha-treated surface. This study demonstrates that ALD coating can be used as a functionalization strategy for nanobiosensors because it is capable of creating functional groups with much higher density compared to widely used acid modifications, and among the three ALD coatings, ALD-SiO(2) yielded the most promising results in OH content and protein attachment.     

Liposome layers characterized by quartz crystal microbalance measurements and multirelease delivery

Intact liposomes have been immobilized onto solid surfaces by a NeutrAvidin-biotin link. The construction of these layers has been followed up by X-ray photoelectron spectroscopy (XPS) and quartz crystal microbalance (QCM) measurements with energy dissipation monitoring. Also, the simultaneous release of two fluorescent probes from these liposome layers has been investigated with the aim to validate this method in multirelease delivery systems. XPS showed the successful immobilization of the different layers. XPS results also point out the importance of the deactivation method used to reveal the presence of the specific NeutrAvidin-biotin attachment. QCM measurements allowed the buildup of the different layers to be followed in real time and in situ and suggest that biotinylated liposomes stay intact upon surface attachment on NeutrAvidin-covered surfaces and had viscoelastic behavior. QCM experiments also demonstrated that surface-immobilized liposomes were able to resist irreversible adsorption from fetal bovine serum. Release kinetic profiles were studied by monitoring the release of two different fluorescent probes, namely, carboxyfluorescein and levofloxacin, from these liposome layers. These studies showed that it was possible to modulate to some extent the release rates of the two molecules by using different configurations of liposome layers.     

长链DNA在金基底上的固定化和电化学标记

本文提出在金基底上用阳离子聚电解质———聚二烯丙基二甲基胺氯化物 (poly(dial lyldimethylammoniumchloride) ,PDDA)自组装膜固定长链DNA的方法 ,用DiffuseReflectanceIn frared ,XPS和STM技术进行表征 ,并对DNA杂交进行电化学标记     

Covalent attachment of trypsin on plasma polymerized allylamine

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV–vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.     

Collagen-grafted ultra-high molecular weight polyethylene for biomedical applications

A novel material for hard tissue implants has been prepared. The ultra-high molecular weight polyethylene (UHMWPE) was grafted with collagen I, to improve its biocompatibility with soft tissue in case of its usage in bone engineering. Before collagen immobilization, commercial grade UHMWPE was treated with air plasma to introduce hydroperoxides onto the surface and subsequently grafted with carboxylic acid to functionalize the surface. Acrylic acid and itaconic acid were used for surface functionalization. After graft polymerization of carboxylic acids, collagen was immobilized covalently through the amide bonds between residual amino and carboxyl groups in the presence of water-soluble carbodiimide/hydroxysuccinimide cross-linking system. Each step of modification was characterized using spectroscopic (EPR, ATR-FTIR, and XPS), microscopic (SEM and CLSM), and contact angle measurement methods. The experimental results showed that plasma treatment led to a generation of free radicals on the UHMWPE surface resulting in the formation of unstable hydroperoxides. These reactive species were used to graft unsaturated carboxylic acids onto UHMWPE. Consequently, collagen was grafted via the-NH₂ and-COOH reaction. The obtained experimental data along with microscopic observations confirmed the success of graft poly-merization of itaconic as well as of acrylic acid and collagen immobilization onto the UHMWPE surface. Presented at the 1st Bratislava Young Polymer Scientists Workshop, Bratislava, 20–23 August, 2007.     

Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: An efficient platform for 3D IgG immobilization

Immobilized antibodies with oriented and homogeneous patterns are crucial to solid-phase molecular recognition assay. Antibody binding protein-based immobilization can effectively present the desired antibodies. However, steadily installing the stromatoid protein with site-specific attachment manner onto a matrix surface remains to be elucidated. In this study, we present an optimal protocol to tightly attach an immunoglobulin G (IgG)-binding protein (Z-domain) through covalent incorporation of Cys-tag and maleimide group onto polystyrene surface to guarantee site-specific, oriented, and irreversible attachment, resulting in a highly efficient platform for three-dimensional IgG immobilization. The actual IgG-binding characteristic of immobilized Z-Cys was investigated by employing affinity chromatography and size exclusion chromatography. And the efficacy and potential of this platform was demonstrated by applying it to the analysis of interaction between rabbit anti-HRP IgG and its binding partner HRP. The proposed approach may be an attractive strategy to construct high performance antibody arrays and biosensors given that the antibody is compatible with the Z-domain.     

In vitro haemocompatibility and stability of two types of heparin-immobilized silicon surfaces

Heparin was covalently immobilized onto a silicon surface by two different methods, carbodiimide-based immobilization and photo-immobilization. In the former method, a (3-aminopropyl) trimethoxysilane (APTMS) self-assembled monolayer (SAM) or multilayer was first coated onto the silicon surface as the bridging layer, and heparin was then attached to the surface in the presence of water-soluble carbodiimide. In the latter method, an octadecyltrichlorosilane (OTS) SAM was coated on the silicon surface as the bridging layer, and heparin was modified by attaching photosensitive aryl azide groups. Upon UV illumination, the modified heparin was then covalently immobilized onto the surface. The hydrophilicity of the silicon surface changed after each coating step, and heparin aggregates on APTMS SAM and OTS SAM were observed by atomic force microscopy (AFM). In vitro haemocompatibility assays demonstrated that the deposition of APTMS SAM, APTMS multilayer and OTS SAM enhanced the silicon's haemocompatibility, which was further enhanced by the heparin immobilization. There is no evident distinction regarding the haemocompatibility between the heparin-immobilized surfaces by both methods. However, heparin on silicon with APTMS SAM and multilayer as the bridging layers is very unstable when tested in vitro with a saline solution at 37 °C, due to the instability of APTMS SAM and multilayer on silicon. Meanwhile, photo-immobilized heparin on silicon with OTS SAM as the bridging layer showed superb stability.     

Polymer microspheres for replacement of biological carriers in test systems operating on the principle of latex agglutination reaction

The results of the study of various modification methods of polymer microspheres for their use in immunochemical reactions as bioligand carriers are reported. Ion etching and electron microscopy were used to show that the copolymer microspheres have a porous structure non-uniform in density. The Maillard reaction was used for the first time in the modification of copolymer microspheres by dextrans of different molecular weight as a simplest way of covalent immobilization of saccharides on their surface. The physicochemical properties of polystyrene-divinylbenzene and polyglycidyl methacrylate-ethylene glycol dimethacrylate microspheres modified with diamines and dextrans were determined for the first time. The conditions under which the bioligand (diphtheria toxoid) immobilized on their surface retained the native conformation and the diagnostics obtained on their basis have high sensitivity were revealed.

     

Immobilization of cellulase using acrylamide grafted acrylonitrile copolymer membranes

Immobilization of cellulase onto acrylamide grafted acrylonitrile copolymer (PAN) membranes by means of glutaraldehyde has been studied. The bound cellulase was verified by X-ray photoelectron spectroscopy. The activities of free cellulase and immobilized cellulase are determined by measuring the amount of glucose made from carboxymethyl cellulase in the given conditions. Results show that immobilization conditions had some effects on the activity of immobilized cellulase. The immobilized cellulase had a higher K_m than free cellulase (0.02 mg/ml) did. The immobilized cellulase had better stability with respect to pH or temperature than free cellulase.     

Improved preparation of chiral stationary phases via immobilization of polysaccharide derivative‐based selectors using diisocyanates

The classical method for the preparation of immobilized polysaccharide‐based chiral stationary phases (CSPs) with a diisocyanate was improved. Cellulose or amylose was directly coated onto 3‐aminopropyl silica gel after it was dissolved in a mixture of N,N‐dimethylacetamide, LiCl, and pyridine, then immobilized onto silica gel with a diisocyanate, and finally allowed to react with an excess of corresponding isocyanate. Four polysaccharide derivatives, 3,5‐dimethylphenylcarbamate and 3,5‐dichlorophenylcarbamate of cellulose, and 3,5‐dimethylphenylcarbamate and 5‐chloro‐2‐methylphenylcarbamate of amylose, were immobilized onto silica gel utilizing this method. Compared with the classical diisocyanate method, the improved procedure avoided the derivatization and regeneration of 6‐hydroxyl groups of cellulose and amylose, and thus showed an advantage for simple and economical preparation. The relationships among the amount of diisocyanate used, immobilization efficiency, and enantioseparation on the cellulose‐based CSPs were investigated. Also, the solvent durability of the obtained CSPs was examined with eluents containing chloroform or THF. By utilizing these eluents, the chiral recognition abilities of the obtained CSPs for some of the tested racemates were improved.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.