Wettability modification and the subsequent manipulation of protein adsorption on a Ti6Al4V alloy by means of CO<Subscript>2</Subscript> laser surface treatment

Improvements in the wettability of the Ti6Al4V alloy following CO₂ laser treatment were identified as being due mainly to the increase in surface roughness, surface oxygen content and surface energy of the material. Untreated and mechanically roughened samples had higher amounts of adsorbed albumin and lower amounts of adsorbed fibronectin than CO₂ laser treated samples. Moreover, as the wettability of the Ti6Al4V alloy increased the adsorbed amounts of fibronectin increased, while the adsorbed amounts of albumin decreased—indicating the controllability of the CO₂ laser process. From this finding it is possible to assert that the wettability of the Ti6Al4V alloy was the prime influence on the observed changes in in vitro protein adsorption. Further, the noted considerable change in the polar component of surface energy, , on the protein adsorption implied that the protein adsorption on the Ti6Al4V alloy was probably due to the polar and chemical interactions. This work has demonstrated that CO₂ laser radiation could be a suitable means to modify the wettability of the Ti6Al4V alloy and thereby manipulate protein adsorption and consequently render the material more bone cell responsive.     

Electrochemical quartz crystal nanobalance to detect solvent displacement by pH-induced conformational changes of proteins at Pt

The electrochemical quartz crystal nanobalance (EQCN) techniques of simultaneous measurements of frequency and cyclic voltammetry (CV) were used to investigate protein adsorption behavior resulting from pH-induced conformational changes at the Pt electrode at 298 K. The adsorption behavior of holo- and apo-alpha-lactalbumin was studied in electrolyte solutions of pH < 2, 7.4, and 11. The EQCN frequency measurements did not directly monitor the mass of the adsorbed protein at anodic potentials, but instead, at a potential characteristic of the double layer for platinum, gave a measure of the extent of solvent displacement by the adsorbed protein (i.e., a "footprint"), which correlated well with known pH-induced conformational changes of the protein. Simultaneous CV charge transfer measurements provided information on the number of layers of protein adsorbed to the surface. This ability of the EQCN to detect solvent displacement by protein adsorption is potentially useful for biosensors to detect and to monitor protein conformational changes in the bulk and during the adsorption process. The Langmuir adsorption isotherm provided the Gibbs energy of adsorption, DeltaG(ADS), and showed excellent agreement between the CV and EQCN frequency measurements.     

Adsorption of proteins on electro-conductive polymer films

Shapable electro-conductive (SEC) polymer films (polyanion-doped polypyrrole films) show several interesting properties for bioelectrochemical applications. The SEC film can be used as an inert, stable and hydrophobic electrode in aqueous solution over a wide potential range. In this study, the physical and the potential-assisted adsorption of various proteins on the SEC film is described. Because of the hydrophobic surface characteristic proteins easily adsorb and retain on the film surface by strong hydrophobic interactions. The amount of the adsorbed protein varies from 2.2 to 4.8 μg cm⁻² depending on the protein when the film is incubated for 22 h in the protein solution. The adsorption is effectively accelerated and enhanced by applying a positive potential in the range from 0.4 V to 1.0 V (vs. Ag/AgCl). The potential-assisted adsorption process is completed by 10–15 min and the amount of the adsorbed protein is nearly doubled as compared to the adsorption without potential. The adsorbed protein is chemically very stable in comparison with the protein in solution. More than 85% of the initial adsorbed proteins retains on the surface after three weeks of incubation in buffer solution. The initial adsorption rate is studied by quartz crystal micro-balance measurements on a thin polymer film coated quartz crystal. In addition, the SEC film surface is etched with air plasma which leads to a four-fold increase of the adsorption of proteins.     

Mapping interfacial chemistry induced variations in protein adsorption with scanning force microscopy

In this work, we demonstrate the sensitivity of scanning force microscopy (SFM), operated in friction force mode, to adsorbed protein conformation or orientation. We employ patterned films of methyl- and carboxylate-terminated alkanethiolate monolayers on gold as substrates for protein adsorption to observe the effect of each functional group in the same image. Infrared spectroscopic and SFM studies of bovine fibrinogen (BFG) adsorption to single-component monolayers indicate that complete films of BFG that are stable to imaging are formed at each functional group. After adsorption of BFG to a patterned monolayer, we observe a contrast in friction images due to differences in adsorbed BFG conformation or orientation induced by each functional group. We also observe frictional contrast in films of other proteins adsorbed on patterned monolayers. These observations lead to the conclusion that SFM-measured friction is sensitive to adsorbed protein state.     

复用血液透析器（人工肾）膜表面粘附蛋白的实验研究

以Fresenius F6聚砜膜复用透析器为研究对象,对其膜表面粘附的蛋白进行定量分析研究.用4种不同的化学清洗剂及超声波的方法,对膜表面粘附的蛋白进行清洗,以获得最佳清洗方法;对透析器不同部位膜上的蛋白进行测定,获得不同部位膜上蛋白含量的差异性;用双向电泳的方法检测膜上粘附的蛋白含量及其性质,获得不同种类蛋白与聚砜膜结合的特异性关系.结果显示:使用5次后每克纤维膜表面粘附的蛋白量至少为0.1 mg,且在透析器各部位分布不同;超声波是一种用来清洗膜上残留物质的有效方法;膜表面粘附的蛋白主要是中性或偏酸性的蛋白,中、大分子量的蛋白容易造成膜孔堵塞且不易去除,而小分子量的蛋白较易去除.     

In vitro and in vivo evaluation of silicated hydroxyapatite and impact of insulin adsorption

This study evaluates the biological behaviour, in vitro and in vivo, of silicated hydroxyapatite with and without insulin adsorbed on the material surface. Insulin was successfully adsorbed on hydroxyapatite and silicated hydroxyapatite bioceramics. The modification of the protein secondary structure after the adsorption was investigated by means of infrared and circular dichroism spectroscopic methods. Both results were in agreement and indicated that the adsorption process was likely to change the secondary structure of the insulin from a majority of α-helix to a β-sheet form. The biocompatibility of both materials, with and without adsorbed insulin on their surface, was demonstrated in vitro by indirect and direct assays. A good viability of the cells was found and no proliferation effect was observed regardless of the material composition and of the presence or absence of insulin. Dense granules of each material were implanted subcutaneously in mice for 1, 3 and 9 weeks. At 9 weeks of implantation, a higher inflammatory response was observed for silicated hydroxyapatite than for pure hydroxyapatite but no significant effect of adsorbed insulin was detected. Though the presence of silicon in hydroxyapatite did not improve the biological behaviour, the silicon substituted hydroxyapatite remained highly viable.     

Cellular Interactions and Modulated Osteoblasts Functions Mediated by Protein Adsorption

The rapid adsorption of proteins is one of the first few events that occur when a biomedical device is implanted and strongly affects attachment and proliferation of cells on the material surface. We describe here for the first time the favorable modulation of osteoblasts functions and cell–substrate interactions induced by protein adsorption (bovine serum albumin) on silicone elastomers. It was intriguing that the cell–substrate interactions on protein‐adsorbed silicone elastomer were significantly different from those observed in stand alone silicone. The origin and differences in cell–substrate interactions in terms of cell attachment, viability, and proliferation and assessment of proteins actin, vinculin, and fibronectin are addressed. Cell attachment and proliferation of cells were significantly enhanced on protein‐adsorbed surface in comparison to the bare surface. Similarly, the expression level of fibronectin, actin, and vinculin was appreciably greater on the protein‐adsorbed surface. The study underscores that protein adsorption on artificial biomedical devices can be envisaged as one of the methods to enhance the bioactivity on the surface and regulate biological functions.     

The Adsorption of Hydroxyl ions to Silver Bromide and its Photographic Implications*

It is analytically shown that hydroxyl ions are adsorbed to a silver-bromide precipitate (silver body), the degree of the adsorption increasing with increasing pH. At the highest ph accessible to the analytical procedure (pH 9.5) roughly one OH-ion is adsorbed per 5 surface ion-sites. Although OH-ion adsorption to the bromide body could not be readily shown by the analytical procedure its existence is by no means excluded. The possible correlation between the postulated silver-hydroxyl surface complex and photographic sensitivity is briefly discussed and the hypothesis of a dual mechanism of latent image formation is reiterated.     

基于QCM-D技术的合成高分子对蛋白的吸附行为

采用AFM(原子力显微镜)和QCM-D(带损耗测量的石英晶体微天平)等技术研究了聚乳酸、聚碳酸酯和聚甲基丙烯酸甲酯三种合成高分子材料经旋转成膜后的表面形貌、亲疏水性、水合过程及蛋白吸附行为。结果发现,薄膜的疏水性越强,蛋白吸附量越多,蛋白层的粘弹性越小;且吸附在材料表面的蛋白分为可逆吸附和不可逆吸附两部分,可逆部分可被PBS(磷酸盐缓冲溶液)洗脱,而不可逆吸附蛋白由于在疏水力作用下构象发生变化,变得致密无法脱附,粘弹性比发生可逆吸附的蛋白更小。用Langmuir吸附速率方程可较好地拟合薄膜的蛋白吸附动力学曲线,发现薄膜疏水性越强,白蛋白吸附速率越快。     

Myoglobin adsorption onto poly(glycidyl methacrylate) microbeads with surface functionalized iminodiacetic acid

In this study, poly(glycidyl methacrylate) [PGMA] microbeads with surface modified iminodiacetic acid (IDA) were used for myoglobin (Mb) adsorption from buffer solutions at different pHs and ionic strengths in a packed-bed column. Attenuated Total Reflectance Fourier Transformed Infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM) measurements before and after the adsorption process confirmed the structural stability of adsorbed Mb. The effects of initial concentration, flow-rate, pH and ionic strength on the adsorption were investigated. The results showed that the maximum amount of protein was adsorbed at a pH 7.0, which is the protein isoelectric point. The adsorption is rationalized in terms of local electrostatic forces acting between the protein and the IDA modified PGMA surface as well as hydrophobic interactions close to the protein isoelectric point, whereas at low pH the global changes give rise to protein–protein repulsion and at high pH protein-support material repulsion.     

A pairwise interaction approach to the adsorption of oriented overgrowths

The adsorption energies and stable configurations of adlayers on different substrates of various orientations are calculated by means of a pairwise interaction model based on Lennard-Jones potentials. The adlayer is adsorbed onto a substrate consisting of a variable number of relaxed surface layers surmounting a much larger number of “rigid” layers fixed at bulk spacing. Special consideration is given to the adsorption of Cu, Ag and Pb on W and the adsorption of Ag on Cu.     

Dynamic Adsorption of Albumin on Nanostructured TiO(2)Thin Films

Spectroscopic ellipsometry was used to characterize the optical properties of thin (<5 nm) films of nanostructured titanium dioxide (TiO(2)). These films were then used to investigate the dynamic adsorption of bovine serum albumin (BSA, a model protein), as a function of protein concentration, pH, and ionic strength. Experimental results were analyzed by an optical model and revealed that hydrophobic interactions were the main driving force behind the adsorption process, resulting in up to 3.5 mg/m(2) of albumin adsorbed to nanostructured TiO(2). The measured thickness of the adsorbed BSA layer (less than 4 nm) supports the possibility that spreading of the protein molecules on the material surface occurred. Conformational changes of adsorbed proteins are important because they may subsequently lead to either accessibility or inaccessibility of bioactive sites which are ligands for cell interaction and function relevant to physiology and pathology.     

氧化钛薄膜的血液相容性研究

本文研究了离子束增强沉积技术制备的氧化钛薄膜的血液相容性与薄膜的结构，成分，表面能，以及蛋白质在薄膜表面的吸附之间的关系。实验表明，血液相容性是表面能和功函数共同作用的结果。表面能决定蛋白质的吸附量，功函数决定蛋白质的变性。     

Surface spectroscopy of adsorbed proteins: input of data treatment by principal component analysis

X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (ToF–SIMS), two surface-sensitive spectroscopic methods, are commonly used to characterize adsorbed protein layers. Principal component analysis (PCA) is a statistical method which aims at reducing the number of variables in complex sets of data while retaining most of the original information. The aim of this paper is to review work carried out in our group regarding the use of PCA with a view to facilitate and deepen the interpretation of ToF–SIMS or XPS spectra acquired on adsorbed protein layers. ToF–SIMS data acquired on polycarbonate membranes after albumin and, or insulin adsorption were treated with PCA. The results reveal the preferential exposure of particular amino acids at the outermost surface depending on the adsorption conditions (nature of the substrate and of the proteins involved, concentration in solution), giving insight into the adsorption mechanisms. PCA was applied on XPS data collected on three different substrates after albumin or fibrinogen adsorption, followed in some cases by a cleaning procedure with oxidizing agents. The results allow samples to be classified according to the nature of the substrate and to the adsorbed amount and, or the level of surface coverage by the protein. Chemical shifts of particular interest are also identified, which may facilitate further peak decomposition. It is useful to recall that the outcome of PCA strongly depends on data selection and normalisation.     

Tritium-Probe Method in a Study of Adsorption Layers of Lysozyme on the Surface of Detonation Nanodiamonds

Radiochemistry - Tritium-labeled lysozyme was used to determine its adsorption on the surface of nanodiamonds produced by detonation synthesis. It was found that the amount of the adsorbed protein...     

Attenuation of protein adsorption on static and oscillating magnetostrictive nanowires

The research described here investigates the hypothesis that nanoarchitecture contained in a nanowire array is capable of attenuating the adverse host response generated when medical devices are implanted in the body. This adverse host response, or biofouling, generates an avascular fibrous mass transfer barrier between the device and the analyte of interest, disabling the implant if it is a sensor. Numerous studies have indicated that surface chemistry and architecture modulate the host response. These findings led us to hypothesize that nanostructured surfaces will inhibit the formation of an avascular fibrous capsule significantly. We are investigating whether arrays of oscillating magnetostrictive nanowires can prevent protein adsorption. Magnetostrictive nanowires were fabricated by electroplating a ferromagnetic metal alloy into the pores of a nanoporous alumina template. The ferromagnetic nanowires are made to oscillate by oscillating the magnetic field surrounding the wires. Radiolabeled bovine serum albumin, enzyme-linked immunosorbent assay (ELISA), and other protein assays were used to study protein adhesion on the nanowire arrays. These results display a reduced protein adsorption per surface area of static nanowires. Comparing the surfaces, 14-30% of the protein that absorbed on the flat surface adsorbed on the nanowires. Our contact angle measurements indicate that the attenuation of protein on the nanowire surface might be due to the increased hydrophilicity of the nanostructured surface compared to a flat surface of the same material. We oscillated the magnetostrictive wires by placing them in a 38 G 10 Hz oscillating magnetic field. The oscillating nanowires show a further reduction in protein adhesion where only 7-67% of the protein on the static wires was measured on the oscillating nanowires. By varying the viscosity of the fluid the nanowires are oscillated in, we determined that protein detachment is shear-stress modulated. We created a high shearing fluid with dextran, which reduced protein adsorption on the oscillating nanowires by 70% over nanowires oscillating in baseline viscosity fluid. Our preliminary studies strongly suggest that the architecture in the static nanowire arrays and the shear created by oscillating the nanowire arrays would attenuate the biofouling response in vivo.     

Statistical Mechanical Modeling of Protein Adsorption

We present rationale for and a derivation of a statistical mechanical model of protein adsorption. Proteins are modeled as rigid geometric objects adsorbing initially in a reversible manner and subsequently undergoing an irreversible change in shape to a permanently adsorbed state. Both adsorption and shape change occur subject to energetic interactions with previously adsorbed proteins. We evaluate the model quantitatively for proteins with disk‐shaped projections within the scaled particle theory and compare the predictions to experimental measurements taken via optical waveguide lightmode spectroscopy.     

Determination of adsorbed protein concentration in aluminum hydroxide suspensions by near-infrared transmittance spectroscopy

Analysis of aluminum hydroxide based vaccines is difficult after antigen adsorption. Adsorbed protein is often assessed by measuring residual unadsorbed protein for quality control. A new method for the direct determination of adsorbed protein concentration in suspension using near-infrared (NIR) transmittance spectroscopy is proposed here. A simple adsorption system using albumin from bovine serum (BSA) and aluminum hydroxide as a model system is employed. The results show that the NIR absorbance at 700-1300 nm is correlated to the adsorbed BSA concentration, measured by the ultraviolet (UV) method, using the partial least square regression (PLSR) method to construct a calibration model. The linear concentration range of adsorbed BSA is from 0 to 1.75 mg/mL by using 10 mm path length cuvettes. The influence of the sedimentation in suspension, different buffers, and different aluminum hydroxide batches was investigated in this study. It shows that the batch variation is the main influence factor of this method, while the buffer variation has no influence. However, the pretreatment of spectral data by subtracting spectra of BSA blank control (aluminum hydroxide without BSA) can significantly reduce the batch influence, and the NIR predicted results show good agreement with the reference values. The NIR method might be the only direct method for the determination of adsorbed protein concentration in suspension so far. It is a nondestructive method, and it has great advantage for use in vaccine production as a method for quality control and quality assurance.     

Preliminary study on human protein adsorption and leukocyte adhesion to starch-based biomaterials

In this study, the adsorption of human serum albumin (HSA), fibronectin (FN) and vitronectin (VN) onto the surface of novel biodegradable materials was evaluated by immunostaining. Specifically, polymeric blends of corn starch with cellulose acetate (SCA), ethylene vinyl alcohol copolymer (SEVA-C), and polycaprolactone (SPCL) were immersed in unitary and competitive systems; that is, binary and more complex protein solutions. For binary solutions, different HSA and FN protein distribution patterns were observed depending on the starch-based surface. Furthermore, the relative amount of proteins adsorbed onto starch-based surfaces was clearly affected by protein type: a preferential adsorption of VN and FN as compared to HSA was observed. On tests carried out with unitary, binary and more complex solutions, it was found that vitronectin adsorption ability was enhanced in competitive systems, which was associated with a lower amount of adsorbed albumin. In order to assess the effect of these human proteins on cell behavior, a mixed population of human lymphocytes and monocytes/macrophages was cultured over pre-coated SEVA-C surfaces. Through anti-CD3 and CD-14 monoclonal antibody labeling and cell counting, leukocyte adhesion onto pre-coated SEVA-C surfaces was analyzed. Based on the results, it was possible to detect albumin long-term effects and fibronectin short-term effects on cell adhesion proving that previously adsorbed proteins modulate leukocyte behavior.     

Characterization of proteins and fibroblasts on thin inorganic films

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and material microstructure. One of the goals in the development of silicon-based biomedical devices such as biosensors or drug delivery systems is improved biocompatibility which may be achieved through the deposition or adsorption of thin films. In this study, films of single crystal silicon, stoichiometric and low stress silicon nitride, doped and undoped polysilicon, as well as Arg-Gly-Asp (RGD) peptide adsorbed surfaces characterized in terms of protein adsorption or cellular adhesion for a period of four days. Protein adsorption studies using fibrinogen and albumin, two proteins implicated in cellular adhesion and surface activity, reveal that low stress silicon nitride surfaces have a 223%±2.50% greater protein adsorption compared to undoped polysilicon surfaces, followed by silicon nitride, unmodified silicon, and doped polysilicon surfaces, respectively. The thickness of the adsorbed albumin and fibrinogen layer on various thin films was measured by ellipsometry and compared to contact angle measurements. The greatest cellular adhesion was observed on undoped polysilicon, followed by unmodified (control) silicon, low stress silicon nitride, silicon nitride, and doped polysilicon surfaces. Cellular binding supports the differential protein adsorption found on modified and unmodified silicon surfaces. Understanding the biological response to thin films will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.