类金刚石薄膜成分变化对蛋白吸附的影响

采用全方位离子注入离子束增强沉积工艺制备类金刚石薄膜 (DL C) ,再将 DL C进行有氧和低压氩气保护条件下的热处理 ,分别得到富石墨相 DL C和富金刚石相 DL C。通过 X光电子能谱分析了三种 DL C的碳相组成 ,用放射性同位素 1 2 5I标记法 ,测定了恒温条件下人血白蛋白 (HSA)、纤维蛋白原 (HFG)和免疫球蛋白 (Ig G)在 3种 DL C材料表面的吸附量。结果 ,经两种不同的热处理工艺 ,DL C中石墨相和金刚石相分别增加了一倍左右 ;随着石墨、金刚石等杂质相的增加 ,DL C对 HSA的吸附能力下降 ,对 HFG和 Ig G的吸附能力显著提高 ,同时蛋白吸附特性也由原来对 3种蛋白的非特异性吸附 ,转变为对 HFG和 Ig G的优先吸附。这一结果表明 ,石墨和金刚石等杂质相将严重影响 DL C的蛋白吸附性能 ,并进而对 DL C的血液相容性产生负面影响。文中还对石墨和金刚石相的转化生成机理进行了探讨     

类金刚石薄膜蛋白吸附与碳相成分关系的定量分析研究

运用灰色系统理论中的 T型关联度分析方法 ,对类金刚石 (DL C)薄膜、富石墨相 DL C薄膜和富金刚石相 DL C薄膜三种 DL C薄膜进行了碳相成分对其白蛋白 (HSA)、纤维蛋白原 (HFG)、免疫球蛋白 (Ig G)三种血浆蛋白吸附量影响的定量分析研究。合理地解释了三种材料蛋白吸附量随碳相成分变化的实验结果 ,并得出如下重要的分析结论 :(1)石墨和 C- H相对 HSA的吸附影响较大 ,随着二者的增加 ,HSA的吸附量下降 ;(2 )与 HFG吸附有较强关联的碳相成分是 DL C相和 C- O相 ,前者呈负相关 ,后者为正相关 ;(3)各碳相成分对 Ig G的吸附均有性质不尽相同的影响 ,但程度有限 ,且彼此间相差不大 ;(4 ) DL C碳相具有增强 HSA吸附、抑制 HFG、Ig G吸附的双重功效 ,其对 DL C薄膜血液相容性的影响远较其它碳相成分更为重要。     

碳素材料表面吸附蛋白的红外光谱分析

为探讨类金刚石薄膜（DLC）、金刚石薄膜（DF）和石墨不同血浆蛋白吸附特性的内在原因，对人血白蛋白（HSA）和纤维蛋白原（HFG）在三种碳素材料表面吸附前后进行了红外光谱分析。结果显示：通过氨基，在HSA与DLC、HFG与DF、HFG与石墨之间形成了氢键，从而有力地支持并合理地解释了DLC具有较高的HSA吸附活性、DF和石墨则优先吸附HFG的前期研究结论。     

生物碳素材料表面血小板黏附的实验研究

为了解生物碳素材料的凝血机制 ,将新鲜抗凝人血离心分离为富血小板血浆 ,在 37℃恒温条件下 ,对类金刚石薄膜 (DL C)、金刚石薄膜和石墨三种碳素材料进行了血小板黏附实验 ,通过扫描电镜对黏附于材料表面的血小板进行形态观察和计数分析 ,用形态指数描述血小板的变形程度。结果显示 ,DL C表面无血小板黏附 ,而金刚石薄膜和石墨表面均黏附有为数不少、呈 ～ 型重度变形的血小板。血小板的黏附量石墨最多 ,而形态指数则金刚石薄膜更大。经与前期研究和文献报道的对比分析 ,得出三个主要结论 :(1)蛋白吸附介导的血小板黏附、变形和聚集是生物碳素材料的主要凝血机制 ;(2 )评价生物碳素材料的血液相容性 ,血小板变形度比血小板消耗率更有价值 ;(3) DL C的纯度越高 ,血液相容性越好。这些结论对改进和设计新型碳素人工心瓣材料具有重要指导意义     

碳相成分对DLC薄膜血液相容性影响的能量机制

以6个不同工艺条件制备的DL C薄膜为样本,在前期碳相成分与表面能量参数测定、碳相成分和表面能量参数与血小板黏附特性关联分析的基础上,用T型关联度法,进一步对碳相成分和表面能量参数进行了灰色关联分析。结果显示:(1)在6个表面能量参数中,临界表面张力与碳相成分的整体关联最大,其次为表面张力的色散分量,其它则关联较小;(2 ) DL C碳相与临界表面张力和表面张力的色散分量有较大的负关联度(- 0 .5 7,- 0 .33) ,而与亲水性的其它表面能量参数的关联度均为小于0 .2 0的正值;(3) C- H碳相和C- O碳相与临界表面张力呈较大的正相关(0 .4 8,0 .2 5 )。据此得出分析结论:(1) DL C碳相主导DL C薄膜的血液相容性,其作用机制是对表面润湿性的强抑制和亲水性的有限增强;(2 ) C- H碳相和C- O碳相促使血小板变形的能量机制是增大DL C薄膜的临界表面张力;(3)评价DL C薄膜的血液相容性,既可用临界表面张力作指标设立标准,也可以对DL C碳相的含量要求、辅以对C- H碳相和C- O碳相的含量限定设立标准。本研究为用表面性质表征DL C薄膜的血液相容性提供了理论依据。     

DLC血小板黏附特性与其表面能量的灰色关联分析

以不同工艺条件制备的 DL C材料为样本 ,进行了血小板黏附实验 ,测定了乙醇、水和不同组分比乙醇 /水溶液在各样本表面的平衡接触角 ,由此计算出表面张力、临界表面张力、界面张力、粘黏功 4个表面能量参数及表面张力的色散和极性分量 ,通过 T型关联度计算 ,分析了各参数对血小板黏附量和形态指数的影响。结果显示 :(1) 4个表面能量参数均与血小板的黏附量呈正相关 ,而形态指数则与表面张力、、界面张力和黏附功呈负相关 ;(2 )血小板的黏附量和形态指数与表面张力色散、极性分量的关联度异号 ,极性分量对血小板的黏附有利 ,而色散分量则于血小板的变形多功 ;(3)临界表面张力与血小板的黏附量和形态指数均有较大的正关联度 ;(4)表面张力极性分量对血小板黏附量和形态指数的影响与表面张力、界面张力和黏附功步调一致。由此得出 2个重要结论 :(1)血小板在 DL C表面的黏附特性与其表面界面性能密切相关 ,DL C的血液相容性取决于其亲水性和有限润湿的平衡 ,存在一个以临界表面张力为指标的血液相容性区域 ;(2 )血小板在 DL C表面的黏附和变形具有不同的能量机制 :亲水性的表面有利于血小板黏附 ,而黏附血小板的变形则要借助于表面的疏水作用。     

类金刚石/酪蛋白磷酸肽复合薄膜修饰钛合金表面成骨细胞的增殖

背景：钛合金表面沉积类金刚石薄膜可提高其摩擦和抗腐蚀性能,但缺乏生物活性,在其表面接枝生物蛋白分子是一种新的生物化学改性途径。 目的：观察成骨细胞在类金刚石/酪蛋白磷酸肽复合薄膜修饰钛合金表面的增殖与黏附。 方法：采用非平衡磁控溅射和多步组装方法在钛合金表面制备类金刚石/酪蛋白磷酸肽复合薄膜。将对数生长期的成骨细胞悬液接种于类金刚石/酪蛋白磷酸肽复合薄膜修饰的钛合金与纯钛合金试件上。 结果与结论：类金刚石/酪蛋白磷酸肽复合薄膜修饰钛合金组细胞增殖率和黏附数量高于纯钛合金组(P < 0.05)。扫描电镜显示,类金刚石/酪蛋白磷酸肽复合薄膜修饰钛合金组成骨细胞胞体显著增大,表面粗糙,边界模糊不清,细胞呈充分的铺展状态;纯钛合金组成骨细胞胞体光滑,边界线条锐利清晰,伸展不良。表明类金刚石/酪蛋白磷酸肽复合薄膜可促进钛合金表面成骨细胞的增殖与黏附。     

类金刚石薄膜表面界面特性表征方法以及检测技术

类金刚石薄膜作为一种新型生物材料得到越来越广泛的应用,其表面界面特性决定了生物相容性的好坏.本文阐述了生物材料表面界面特性与生物相容性的关系以及类金刚石薄膜表面界面参数的表征方法和检测技术.     

氮掺杂类金刚石薄膜的制备及生物相容性研究

目的利用等离子体增强化学气相沉积(Plasma Enhanced Chemical Vapor Deposition,PECVD)技术,在钛合金表面制备含氮类金刚石薄膜(DLC∶N),并对其生物相容性进行研究。方法采用扫描电子显微镜、X-射线光电子能谱仪、接触角测量仪、拉曼光谱仪对样品的表面形貌特征、组成元素和表面润湿性进行表征;利用MTT比色法、荧光染色法进行生物学行为的评价。结果成骨细胞在掺氮类金刚石薄膜(DLC∶N)表面不论是增殖黏附状态还是细胞数量都优于其他实验组(0.05)。结论在DLC中加入氮元素,能够提升其生物相容性,促进成骨细胞黏附和增殖。     

喷砂预处理钛合金表面类金刚石薄膜的制备及性能研究

目的利用等离子体增强化学气相沉积(plasma enhanced chemical vapor deposition,PECVD)技术,在不同粗糙度医用钛合金表面制备类金刚石薄膜(diamond-like carbon film,DLC),并对类金刚石薄膜的膜-基结合力、电化学腐蚀行为及生物学行为进行研究,探索基底材料喷砂预处理对于表面制备的类金刚石薄膜是否是一种有效改性方法。方法通过不同粒度的Al2O3在一定条件下喷砂钛合金表面,制备出不同粗糙度表面的钛合金片,采用PECVD技术在其表面制备类金刚石薄膜。通过扫描电子显微镜、X-射线光电子能谱仪、拉曼光谱仪、接触角测量仪、表面性能测试仪和电化学方法检测各组样品的物理化学性能表征;利用MTT比色法、荧光染色法进行薄膜表面生物学行为的评价。结果将DLC沉积在喷砂预处理的钛合金表面,可以明显增强类金刚石的膜-基结合力,最高达到45N;耐腐蚀性研究中,表面沉积DLC的钛合金实验组腐蚀电位均正向大于未沉积DLC的钛合金组(P0.05);生物学行为研究中,在60#Al2O3喷砂处理钛合金表面沉积DLC的S60实验组细胞增殖状况最佳,明显大于未喷砂S0组(P0.05)。结论将钛合金基底喷砂预处理后沉积DLC,可以提高类金刚石薄膜的膜-基结合力、耐腐蚀性及生物相容性,是一种有效的医用钛合金表面改性方法,具有潜在的临床应用价值。     

Competitive adsorption of serum proteins at microparticles affects phagocytosis by dendritic cells

Serum protein adsorption to the surface of particulate synthetic drug carrier systems has a major influence on their uptake by phagocytes. The influence of alpha2-human serum glycoprotein (alpha2GP) on the phagocytosis of various surface modified microparticles was studied in dendritic cells (DC) and was compared with a potent opsonin, IgG, and a dysopsonin, human serum albumin (HSA). The microparticles were administered to DC before and after the incubation with alpha2GP, IgG and HSA in single, binary or ternary protein systems and in whole blood serum. Phagocytosis of microparticles was vastly affected by the surface character of the microparticles themselves and by the adsorption of the proteins. Poly-L-lysine (PLL)-modified microparticles were under all conditions internalized with highest efficiency which is suggested to be mediated by their positive surface charge. The adsorption of commonly phagocytosis promoting proteins reduced the uptake of PLL-modified particles and is explained by compensation of the positive surface charge by the adsorbed negatively charged proteins. In all other particle types tested, freshly adsorbed alpha2GP was found to exhibit a strong phagocytosis promoting activity which was comparable to that of adsorbed IgG. Interestingly, this opsonic activity was lost already 2 h after adsorption to the particle surface. Protein adsorption from binary and ternary protein systems and from whole blood serum occurred in a competitive manner. Significant inhibition of phagocytosis was observed, even when HSA was combined with strong opsonins such as alpha2GP or IgG or in mixtures of all three proteins, indicating the importance of studying the influence of protein adsorption in protein mixtures.     

氧化钛薄膜的血液相容性机理探讨

考究了影响氧化钛薄膜血液相容性的因素,提出了血液相容性机理的看法,认为血液相容性是表面能和功函数共同作用的结果。表面能决定蛋白质吸附,而功能函决定蛋白质的分解,并给出了一个表面能区域,指出一个良好血液相容性材料,不仅要有合 表面能,还要有较小的功函数。     

Stickiness to hydrophobic surfaces varies widely among peptides and proteins

To determine whether there are differences in stickiness to hydrophobic surfaces among peptides and proteins under immunoassay conditions, peptides and proteins were radio-labeled with (125)I and competitive adsorption with human serum albumin (HSA) in polystyrene or polypropylene tubes was used to determine the IC (50), the concentration of HSA required to reduce the adsorption of the labeled polypeptides to 50% of maximal. Stickiness was defined as log(10)(10(9) IC (50)). Stickiness varied significantly between the labeled polypeptides (p < 0.00001) and ranged (±sem) from 0.99 ± 0.07 for angiotensin II to 5.30 ± 0.07 for tyr(0)-urocortin II. The stickiness of HSA and γ globulin was 1.62 ± 0.09 and 1.92 ± 0.05, respectively. No significant difference in stickiness between polystyrene and polypropylene was found. We conclude that some peptides are sufficiently sticky to risk adsorptive loss during sampling and analysis, and there may exist peptides so sticky that they remain uncharacterized.     

Selective protein adsorption modulates platelet adhesion and activation to oligo(ethylene glycol)-terminated self-assembled monolayers with C18 ligands

This study focuses on the selective binding of albumin to a nanostructured surfaces to inhibit other blood proteins from adsorbing thereby reducing platelet adhesion and activation. Tetra (ethylene-glycol)-terminated self-assembled monolayers (EG4 SAMs) with different percentages of C18 ligands on the surface were characterized by contact angle measurements, X-ray photoelectron microscopy, infrared reflection-absorption spectroscopy, and ellipsometry. A specific surface (2.5% C18 SAM) was found to be selective for human serum albumin (HSA) in the presence of both albumin and fibrinogen (HFG). The importance of this concentration of C18 ligands was stressed in reversibility studies since that surface exchanged almost all the preadsorbed HSA by HSA in solution, but not by HFG. The effect of protein adsorption in the subsequent adhesion and activation of platelets was studied by pre-immersing the surfaces in albumin and plasma before contact with platelets. Scanning electron microscopy and glutaraldehyde induced fluorescence technique images showed that as surfaces got more hydrophobic due to the immobilization of C18 ligands, the number of adherent platelets increased and their morphology changed from round to fully spread. Pre-immersion in HSA led to an 80% decrease in platelet adhesion and reduction of activation. Pre-immersion in 1% plasma was only relevant in 2.5% C18 SAMs since this was the only surface that demonstrated less adhesion of platelets comparing with buffer pre-immersion. However, they still adsorb more platelets then when HSA was preadsorbed. This was confirmed in competition studies between HSA and plasma that suggested that other plasma proteins were also adsorbing to this surface.     

Volumetric interpretation of protein adsorption: ion-exchange adsorbent capacity, protein pI, and interaction energetics

Adsorption of lysozyme (Lys), human serum albumin (HSA), and immunoglobulin G (IgG) to anion- and cation-exchange resins is dominated by electrostatic interactions between protein and adsorbent. The solution-depletion method of measuring adsorption shows, however, that these proteins do not irreversibly adsorb to ion-exchange surfaces, even when the charge disparity between adsorbent and protein inferred from protein pI is large. Net-positively-charged Lys (pI=11) and net-negatively-charged HSA (pI=5.5) adsorb so strongly to sulfopropyl sepharose (SP; a negatively-charged, strong cation-exchange resin, -0.22 mmol/mL exchange capacity) that both resist displacement by net-neutral IgG (pI=7.0) in simultaneous adsorption competition experiments. By contrast, IgG readily displaces both Lys and HSA adsorbed either to quaternary ammonium sepharose (Q; a positively-charged, strong anion exchanger, +0.22 mmol/mL exchange capacity) or to octadecyl sepharose (ODS; a neutral hydrophobic resin, 0 mmol/mL exchange capacity). Thus it is concluded that adsorption results do not sensibly correlate with protein pI and that pI is actually a rather poor predictor of affinity for ion-exchange surfaces. Adsorption of Lys, HSA, and IgG to ion-exchange resins from stagnant solution leads to adsorbed multi-layers, into or onto which IgG adsorbs in adsorption competition experiments. Comparison of adsorption to ion-exchange resins and neutral ODS leads to the conclusion that the apparent standard free-energy of adsorption Delta Gads( degrees ) of Lys, HSA, and IgG is not large in comparison to thermal energy due to energy-compensating interactions between water, protein, and ion-exchange surfaces that leaves a small net Delta Gads( degrees ). Thus water is found to control protein adsorption to a full range of substratum types spanning hydrophobic (poorly water wettable) surfaces, hydrophilic surfaces bearing relatively-weak Lewis acid/base functionalities that wet with (hydrogen bond to) water but do not exhibit ion-exchange properties, and surfaces with strong Lewis acid/base functional groups that exhibit ion-exchange properties in the conventional chemistry sense of ion-exchange.     

Volumetric interpretation of protein adsorption: competition from mixtures and the Vroman effect

A Vroman-like exchange of different proteins adsorbing from a concentrated mixture to the same hydrophobic adsorbent surface is shown to arise naturally from the selective pressure imposed by a fixed interfacial-concentration capacity (w/v, mg/mL) for which protein molecules compete. A size (molecular weight, MW) discrimination results because fewer large proteins are required to accumulate an interfacial w/v concentration equal to smaller proteins. Hence, the surface region becomes dominated by smaller proteins on a number-or-mole basis through a purely physical process that is essentially unrelated to protein biochemistry. Under certain conditions, this size discrimination can be amplified by the natural variation in protein-adsorption avidity (quantified by partition coefficients P) because smaller proteins (MW<50 kDa) have been found to exhibit characteristically higher P than larger proteins (MW<50 kDa). The standard depletion method is implemented to measure protein-adsorption competition between two different test proteins (i and j) for the same hydrophobic octyl sepharose adsorbent particles. SDS-gel electrophoresis is used as a multiplexing, separation-and-quantification tool for this purpose. Identical results obtained using sequential and simultaneous competition of human immunoglobulin G (IgG, protein j) with human serum albumin (HSA, protein i) demonstrates that HSA was not irreversibly adsorbed to octyl sepharose over a broad range of competing solution concentrations. A clearly observed exchange of HSA for IgG or fibrinogen (Fib) shows that adsorption of different proteins (i competing with j) to the same hydrophobic surface is coupled whereas adsorption among identical proteins (i or j adsorbing from purified solution) is not coupled. Interpretive theory shows that this adsorption coupling is due to competition for the fixed surface capacity. Theory is extended to hypothetical ternary mixtures using a computational experiment that illustrates the profound impact size-discrimination has on adsorption from complex mixtures such as blood.