用SPR生物传感器研究纤维蛋白原在生物医用材料表面的吸附

材料表面对血浆蛋白的吸附特性 ,是研究和评价生物医用材料血液相容性的重要依据。本文用自制的表面等离激元 (SPR)传感器 ,测量了金膜、磷脂DSPC膜、成都科大Ⅱ型聚氨酯、Pellethane2 36 3 55D聚氨酯及有机玻璃膜表面对纤维蛋白原的动态吸附特性 ,在纤维蛋白原溶液浓度为5mg/ml的相同条件下 ,磷脂DSPC膜表面吸附纤维蛋白原的速度最低 ,饱和吸附浓度也最小 (表面浓度为 1ng/mm2 )。其次是裸金膜 (表面浓度为 3.5ng/mm2 ) ,再其次是成都科大Ⅱ型聚氨酯膜 (表面浓度为 3.8ng/mm2 )和Pellethane 2 36 3 55D聚氨酯 (表面浓度为 4 .3ng/mm2 ) ,吸附速度和吸附量最高的是有机玻璃膜 (表面浓度为 4 .5ng/mm2 )。结果表明 ,材料表面对纤维蛋白原的吸附动力学特性 ,与材料的血液相容性密切相关。表面等离激元技术与本文采用的在金膜上铺展高分子材料的离心铺膜法和LB技术等样品制备技术相结合 ,为生物材料表面对蛋白质吸附特性的实时、动态、原位研究提供了一种新的高灵敏度的方法 ,并可能发展成为一种材料生物相容性的测试和评价的新方法。     

用SPR生物传感器研究纤维蛋白原在生物医用材料表面？…

材料表面对血浆蛋白的吸附特性，是研究和评价生物医用材料血液相容性的重要依据。本文用自制的表面等离激元（ＳＰＲ）传感器，测量了金膜、磷脂ＤＳＰＣ膜、成都科大Ⅱ型聚氨酯、Ｐｅｌｌｅｔｈａｎｅ２３６３－５５Ｄ）聚氨酯及有机玻璃膜表面对纤维蛋白原的动态吸附特性，在纤维蛋白原溶液浓度为５ｍｇ／ｍｌ的相同条件下，磷脂ＤＳＰＣ膜表面吸附纤维蛋白原的速度最低，饱和吸附浓度也最小（表面浓度为１ｎｇ／ｍｍ＾２）。其次     

聚醚型聚氨酯材料表面纤维蛋白原的吸附性研究

本文采用放射性同位素标记的方法研究了嵌段聚醚型聚氨酯在纯纤维蛋白原溶液中和稀释血浆中的表面纤维蛋白原吸附性规律，考察了聚醚型聚氨酯的特性粘数及溶液体系中的ＮａＣｌ浓度对材料表面纤维蛋白原吸附性的影响，结果表明，随着聚合物特性粘数的增大，材料表面的纤维蛋白原吸附量呈降低的趋势；溶液体系中盐浓度的降低导致纤维蛋白原凝固性增强，在纯纤维蛋白原溶液中，材料表面纤维蛋白原的吸附量相应增多，而在稀释血浆中，纤维蛋白原的吸附量相应减少，在达到最低值后又有上升的趋向，表明纤维蛋白原在材料表面的吸附还受血浆中其它大分子的影响。     

血浆蛋白质在聚苯乙烯-g-(十八烷聚氧乙烯)表面吸附的研究

利用放射碘同位素标记技术研究了三种主要血浆蛋白质（白蛋白、免疫球蛋白或纤维蛋白原）在聚苯乙烯－ｇ－（十八烷聚氧乙烯）接枝共聚物表面的吸附动力学、等温吸附及竞争吸附。表面蛋白质吸附量和表面ＳＰＥＯ含量非单调关系，与ＳＰＥＯ侧链的疏水末端基效应密切相关。二元蛋白质竞争吸附结果表明这三种蛋白质的相对竞争吸附能力为纤维蛋白原最大，免疫球蛋白次之，白蛋白最小。     

血浆蛋白在聚苯乙烯—g—（二八烷聚氧乙烯）表面吸…

利用放射碘同位素标记技术研究了三种主要血浆蛋白质（白蛋白、免疫球蛋白或纤维蛋白原）在聚苯乙烯－ｇ－（十八烷聚氧乙烯）接枝共聚物表面的吸附动力学、等温吸附及竞争吸附。表面蛋白质吸附量和表面的ＳＰＥＯ含量非单调关系，与ＳＰＥＯ侧链的疏水末端基效应密切相关。二元蛋白质竞争吸附结果表明这三种蛋白质的相对竞争吸附能力为纤维蛋白原最大，免疫球蛋白次之，白蛋白最小。     

聚环氧乙烷硫代物、聚环氧乙烷或氟甲基接枝聚氨酯材料表面上的纤维蛋白原吸附

在探讨血液与医用材料之间的反应和研制与血液相容的医用材料中,研究蛋白质的吸附作用是非常重要的。众所周知,纤维蛋白不但是一种具有高度表面活性的蛋白质,而且也能产生瞬间粘附作用,即“vroman效应”。作者研究已报道具有较好血液相容性的氟化聚氨酯(Pu-PFDA)、聚环氧乙烷(PEO)接枝的聚氨酯(Pu-PEO)以及进一步硫化的Pu(Pu-PEO-SO_3)等材料表面的纤维蛋白原吸附现象。将Pu材料浸入内含14C-标记纤维蛋白原的牛血浆中,拿起后先用PBS缓冲液冲洗,然后再用2%SDS溶液冲洗。采用放射性     

纳米碳改性聚氨酯复合材料的表面抗凝血性能

研究纳米碳改性聚氨酯聚合材料表面的血液相容性。将经过表面处理的纳米碳分散到聚氨酯体系中，制成聚氨酯／纳米碳复合薄膜。通过血小板荧光标记人全血灌注实验和羊全血体外循环等实验，观察和测定血小板在材料表面的粘附作用以及血液中血红蛋白浓度、纤维蛋白原浓度的变化，探讨纳米碳对聚氨酯抗凝血性能的影响。实验结果显示聚氨酯／纳米碳表面血小板的粘附明显低于单纯聚氨酯对照组：体外循环4h后，血液中血红蛋白浓度、纤维蛋白原浓度的变化程度减小。表明纳米碳与聚氨酯的复合可以提高材料的血液相容性。     

单壁碳纳米管无纺膜表面的PEG修饰及蛋白质吸附研究

碳纳米管是一种纳米尺度的新型碳材料，具有独特的物理、化学性质。近年来，碳纳米管在生物医学领域的潜在应用前景已经引起科学界和产业界的极大兴趣与关注。与蛋白质分子的非特异性结合是碳纳米管应用于生物系统中必须考虑的基本问题之一。本研究应用扫描电镜和酶联免疫法作为评价方法，定性和定量地分析了血浆中重要凝血因子纤维蛋白原在单壁碳纳米管薄膜表面的非特异性吸附行为；同时，采用聚乙二醇（PEG）分子对单壁碳纳米管薄膜（SWNT膜）进行了表面修饰，通过x．光电子能谱（XPS）对材料表面的化学组成进行了表征，并初步探讨了PEG修饰对纤维蛋白原分子在SWNT膜表面非特异性吸附的阻止作用。实验结果表明，纤维蛋白原分子在SWNT膜表面有强烈的非特异性结合，吸附于薄膜表面的纤维蛋白原分子仍然保有自身的免疫原性。SWNT膜表面可以被PEG分子修饰，连接在薄膜表面的PEG分子可以在一定程度上抑制一定浓度范围内的纤维蛋白原分子的非特异性结合。     

硅橡胶材料对血浆蛋白及血小板粘附作用的半体内试验

本文是研究三种血浆蛋白(γ—球蛋白、纤维蛋白原、白蛋白)在医用硅橡胶材料表面吸附的半体内同位素标记评价方法,同时由扫描电镜观察材料表面血小板粘附的数量和形态变化,从而评价国内外三种医用热硫化甲基乙烯基硅橡胶材料的血液相容性。研究结果表明:CHGB和DCGBE二种材料血液相容性相近,而STGB材料血液相容性较差。     

等离子体表面改性涤纶材料的血浆蛋白吸附与血小板黏附行为研究

采用等离子体表面接枝改性技术在涤纶 (聚对苯二甲酸乙二醇酯 ,PET)材料表面接枝不同分子量的聚乙二醇 (PEG) ,从表面能与界面自由能的角度分析了血浆蛋白 (纤维蛋白原和白蛋白 )在材料表面的竞争吸附关系 ,结果表明接枝了 PEG长链分子的 PET材料具有优先吸附白蛋白的性质 ,其中接枝 PEG6 0 0 0的 PET优先吸附倾向最明显。预接触白蛋白和纤维蛋白原的 PET材料表面的血小板黏附实验表明 :吸附白蛋白的表面能够显著抑制血小板的黏附和聚集 ,表现出好的血液相容性 ,而吸附了纤维蛋白原的材料表面具有降低血液相容性的性质。     

Surface-dependent fibrinopeptide A accessibility to thrombin

Fibrinogen adsorption and more recently fibrin formation at interfaces has been reported to depend on surface properties of the underlying substrate. To provide insight into the surface-dependent mechanism of fibrinopeptide A (FpA) release and fibrin formation, the accessibility and susceptibility of FpA to thrombin-catalyzed fibrinopeptide cleavage were examined using polyclonal anti-FpA IgG binding and surface plasmon resonance (SPR). The amount of accessible FpA on adsorbed fibrinogen was significantly influenced by surface properties of the underlying substrate (methyl- and carboxyl-terminated self-assembled monolayers). Roughly 2.7 times more FpA was available on fibrinogen adsorbed at the hydrophobic vs. negatively charged surface. Upon exposure of adsorbed fibrinogen to thrombin, 100% of the available FpA was enzymatically cleaved at both surfaces, indicating that the extent of FpA release and fibrin formation is a function of the surface-dependent FpA availability. The results presented herein suggest negatively charged surfaces impair FpA accessibility, and therefore lead to reduced FpA release and subsequent fibrin formation. As such, negatively charged surfaces may be useful in minimizing surface-induced thrombosis initiated via fibrin formation thereby aiding in the development of more biocompatible blood-contacting devices.     

Mechanisms of fibrinogen domains : biomaterial interactions

Spontaneous adsorption of fibrinogen is critical to the pathogenesis of biomaterial-mediated inflammatory responses. However, the mechanism by which adsorbed fibrinogen affects phagocyte responses is still not clear. To investigate the molecular interaction between fibrinogen and biomaterials, fibrinogen fragments (D 100 and E50) were generated and used in the present study. The results indicate that biomaterial : D100 interaction is essential to fibrinogen-mediated inflammatory responses, because biomaterials precoated with D100, but not E50, prompt strong inflammatory responses. Furthermore, the results from in vitro studies show that whole molecule fibrinogen and D100 exhibit very similar protein:surface interactions. Specifically: (1) both D100 and fibrinogen have high affinity for biomaterial surfaces; and (2) the retention rates of adsorbed D 100 in both in vivo and in vitro environments are as high as that for adsorbed fibrinogen. On the other hand, E50 does bind to biomaterials but with low affinity because, once bound, it is not tightly adherent to the biomaterial surfaces. Taken together, the results suggest that the mechanism of fibrinogen-mediated inflammatory responses may involve the following three consecutive events: (1) after contact with blood or tissue fluid, the D domain tends to interact with biomaterial surfaces and is important in the tight binding of fibrinogen to implant surfaces; (2) the biomaterial surface then promotes conformational changes within the D domain, exposing P1 epitope (y 190-202, which interacts with phagocyte Mac-1 integrin); and (3) the engagement of Mac-1 integrin with P1 epitope then triggers subsequent phagocyte adherence and reactions.     

Protein-Resistant Materials via Surface-Initiated Atom Transfer Radical Polymerization of 2-Methacryloyloxyethyl Phosphorylcholine

Poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) was grafted from various polymeric substrates to prepare protein-resistant materials. The poly(MPC) chain length was adjusted via the ratio of monomer to sacrificial initiator in solution. The surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). The protein-resistant properties of the poly(MPC)-grafted surfaces were evaluated by single adsorption experiments with fibrinogen and lysozyme. It was shown that the simple three-step grafting method could be applied to modify various biomaterial surfaces including polyurethane and silicones. The adsorption of fibrinogen and lysozyme to the modified surfaces was greatly reduced compared to the unmodified surfaces, and adsorption decreased with increasing poly(MPC) chain length. On polyurethane film grafted with poly(MPC) of chain length 100, the reduction in adsorption was approx. 96% for lysozyme and approx. 99% for fibrinogen.     

Hydrogels based on poly(ethylene oxide) and poly(tetramethylene oxide) or poly(dimethyl siloxane): synthesis, characterization, in vitro protein adsorption and platelet adhesion

In vitro protein adsorption, platelet adhesion and activation on new hydrogel surfaces, composed of poly(ethylene oxide) (PEO) and poly(tetramethylene oxide) (PTMO) or poly(dimethyl siloxane) (PDMS), were investigated. By varying PEO length (MW = 2000 or 3400), hydrophobic components (PTMO or PDMS) or polymer topology (block or graft copolymers), various physical hydrogels were produced. Their structures were verified by 1H NMR and ATR-IR and the molecular weights were determined by gel permeation chromatography. The hydrogels were soluble in a variety of organic solvents, while absorbed a significant amount of water with preserved three-dimensional structure by physical crosslinking. The dynamic contact angle measurement revealed that the surface hydrophilicity increased by incorporating longer PEO, PEO grafting, and adopting PDMS as a hydrophobic segment instead of PTMO. It was observed from in vitro protein adsorption study that the hydrogels exhibited significantly lower adsorption of human serum albumin (HSA), human fibrinogen (HFg), and IgG, when compared with Pellethane, a commercial polyurethane taken as a control. The hydrogels were attractive for HSA but not sensitive to HFg and IgG. And more than 65% of the proteins detected on the surfaces of the hydrogels were reversibly detached by being treated with an SDS solution. It was evident that the hydrogels synthesized in this study were much more resistant to platelet adhesion than the control, which might depend on the composition of proteins adsorbed on the surfaces and their degree of denaturation. Among the hydrogels tested, PEO3,4kPDMS exhibited albumin-rich and platelet-resistant surfaces, implying a potential candidate for biomaterial.     

Transient adsorption of fibrinogen on foreign surfaces: similar behavior in plasma and whole blood

The adsorption of fibrinogen from both human whole blood and plasma to a number of "foreign" surfaces is reported. Adsorption was measured as a function of plasma or blood dilution using radioiodine labeling. We showed previously that adsorption of fibrinogen from plasma exhibits a maximum at a plasma dilution of about 100:1, and have attributed this behavior to competition from other plasma proteins. (The same phenomenon is manifest as a time transient in fibrinogen adsorption.) In the present work we show that exactly the same trends are observed in whole blood. For each of the four surfaces, glass, siliconized glass, collagen-coated glass and polyethylene, the adsorption of fibrinogen as a function of dilution is the same in whole blood as in plasma. Each of these surfaces shows a unique dependence of fibrinogen adsorption on plasma or blood dilution. On cuprophane and a hydrophilic polyether urethane there is essentially no adsorption of fibrinogen from blood or plasma. For the hydrophilic polyurethane this result may be artifactual, but the absence of fibrinogen binding to cuprophane in blood or plasma is real since fibrinogen is found to be adsorbed in monolayer amounts from buffer.     

Human erythrocyte adhesion and spreading on protein-coated polymer surfaces

Protein adsorption is the first event which occurs when polymer surfaces are exposed to blood. The adsorption of proteins modifies the surface properties of the substrates and therefore influences subsequent cell-surface interactions. In an attempt to elucidate the fundamental mechanisms governing cell-proteinated-surface interactions, the extent of fresh human erythrocyte adhesion and spreading on protein-coated surfaces was examined. Five human serum proteins (albumin, fibrinogen, immunoglobulin G, fibronectin, and transferrin) were used at bulk concentrations ranging from 0.01 mg/mL to 50 mg/mL. Polymer substrates covering a wide range of wettability were employed. Protein adsorption significantly reduces erythrocyte adhesion and spreading on all test surfaces with minimum adhesion observed on fibrinogen: IgG greater than albumin greater than fibronectin greater than transferrin greater than fibrinogen. The extent of these effects is dependent on the nature of the adsorbed protein, the protein bulk concentration, and the surface properties of the underlying polymer substrates.     

Fabrication of hydroxyapatite ultra-thin layer on gold surface and its application for quartz crystal microbalance technique

We present a method for coating gold quartz crystal microbalance with dissipation (QCM-D) sensor with ultra-thin layer of hydroxyapatite nanocrystals evenly covering and tightly bound to the surface. The hydroxyapatite layer shows a plate-like morphology and less than 20 nm in thickness. The hydroxyapatite sensor operated in liquid with high stability and sensitivity. The in-situ adsorption mechanism and conformational change of fibrinogen on gold, titanium and hydroxyapatite surfaces were investigated by QCM-D technique and Fourier-transform infrared spectroscopy. The change of secondary structures of fibrinogen adsorbed on the surfaces depended on the adsorbed amounts of protein. The secondary structure of fibrinogen adsorbed on the surfaces changes with increasing coverage. This is explained by repulsion among fibrinogens, affecting water structure and thus the strength of fibrinogen interactions on the surface. The study indicates that the hydroxyapatite sensor is applicable for qualitative and conformational analysis of protein adsorption.     

Antithrombogenicity of hydrophilic polyurethane-hydrophobic polystyrene IPNs. II. In vitro and ex vivo studies

To investigate the effect of hydrophilic and hydrophobic surfaces with phase separated structure on their blood responses, interpenetrating polymer networks (IPNs) composed of hydrophilic polyurethane (PU) and hydrophobic polystyrene (PS) were prepared by simultaneous polymerization. In vitro protein adsorption, in vitro platelet adhesion, and ex vivo A-A shunt test were carried out to evaluate the blood compatibility of the PU/PS IPNs. The results of protein adsorption on the PU/PS IPN surfaces indicated that albumin preferentially adsorbed on the hydrophilic surface (PU), while fibrinogen preferentially adsorbed on the hydrophobic surface (PS). The PU/PS IPNs exhibited suppressive properties for both platelet adhesion and activation. The occlusion time of U50S50 IPN containing 50 wt% of PS was twice as long as that of the PU control (50 min), indicating enhanced blood compatibility, presumably due to the selective adsorption of plasma proteins and the suppression of the adhesion and activation of platelets.     

类金刚石薄膜血液相容性与蛋白吸附的关系研究

以金刚石薄膜 ( DF)和石墨为参比材料 ,采用放射性同位素 1 2 5I标记技术 ,研究了人血白蛋白 ( HSA)、纤维蛋白原 ( HFG)和免疫球蛋白 ( Ig G)在类金刚石薄膜 ( DL C)表面单一蛋白的等温吸附和二元蛋白体系的竞争吸附。结果显示 :( 1)随着蛋白浓度的增加 ,三种蛋白在三种材料表面的吸附量增加 ,并趋于吸附平衡 ;( 2 )石墨对三种蛋白的吸附量远高于 DL C和 DF;( 3 ) DL C对 HSA的吸附活性高于 DF,而 DF、石墨对 HFG、Ig G的吸附活性则明显高于 DL C;( 4) DL C对三种蛋白的吸附能力相差不大 ,而 DF和石墨对 HFG、Ig G的吸附量则显著高于 HSA;( 5 )三种蛋白在 DF和石墨表面的相对竞争吸附能力为 HFG>Ig G>HSA,而对于 DL C,这一顺序则为 HFG≈ HSA>Ig G,HFG对 HSA没有表现出明显的竞争吸附优势。这些结果表明 :DL C对三种血浆蛋白的吸附是非特异性的 ,而DF和石墨则不同程度地优先吸附 HFG和 Ig G,从而在分子水平上阐释了 DL C血液相容性好于 DF和石墨的内在原因     

Rapid postadsorptive changes in fibrinogen adsorbed from plasma to segmented polyurethanes

Fibrinogen adsorbed to biomaterials plays a key role in mediating platelet interactions that can lead to blood clotting so its behavior on surfaces is of fundamental interest. In previous work showing that fibrinogen adsorbed to surfaces quickly becomes non-displaceable upon exposure to blood plasma, the fibrinogen was adsorbed from buffer, so we performed new studies in which the displaceability of fibrinogen adsorbed from plasma was characterized. Fibrinogen was adsorbed from 1% plasma to seven different surfaces for 1-64 min and then transferred to 100% plasma lacking radiolabeled fibrinogen and the amount adsorbed before and after transfer measured. The surfaces were glass, Silicone rubber, and five different polyurethanes. As adsorption time increased, the fibrinogen became increasingly resistant to displacement during the 100% plasma step, but the rate of increase in resistance varied greatly with surface type. Fibrinogen adsorbed from 1% plasma evidently undergoes rapid, surface dependent transitions. This work shows that the transitions that occur when the fibrinogen is adsorbed from blood plasma are similar to what we have previously observed for fibrinogen adsorbed from buffer.