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Dynamic Adsorption of Albumin on Nanostructured TiO(2)Thin Films
Abstract
Spectroscopic ellipsometry was used to characterize the optical properties of thin (< 5 nm) films of nanostructured titanium dioxide (TiO2). 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/m2 of albumin adsorbed to nanostructured TiO2. 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.
... The adsorption experiments were performed using bovine serum albumin (BSA), a ''soft" globular protein with an IP of 4.6 [26], that is widely used as model system to study adsorption onto solid surfaces [16,[26][27][28]. The day of each experiment protein solutions were freshly prepared by dissolving a known amount of the protein in PBS (pH = 7.4), gently stirring for 60 min. ...
... The adsorption experiments were performed using bovine serum albumin (BSA), a ''soft" globular protein with an IP of 4.6 [26], that is widely used as model system to study adsorption onto solid surfaces [16,[26][27][28]. The day of each experiment protein solutions were freshly prepared by dissolving a known amount of the protein in PBS (pH = 7.4), gently stirring for 60 min. ...
... Moreover, the HA substrate despite having the highest surface area, S BET = 80.0 m 2 g À1 , presented an insignificant protein adsorbed density at the equilibrium in comparison with its TiO 2 counterpart. Detailed analysis of dilute BSA solutions yields that the molecule acquires a prolate ellipsoidal shape (a = b < c) with a = b = 4 nm and c = 14 nm [26]. Assuming that the prolate ellipsoid molecule is confined within a parallelepiped of a = b = 4 nm side and c = 14 nm high, and that it can be situated parallel or perpendicular to the adsorbent surface without denature, the theoretical equilibrium adsorbed density necessary to complete a monomolecular layer of protein on the adsorbent would be about 1973 and 6900 l BSA m À2 respectively. ...
The bioactivity of an implant is displayed on its ability to induce heterogeneous nucleation of biogenic apatite onto its surface upon immersion in body fluids; forming, through this layer, a stable bond with the host tissue. The present article evaluates the bioactivity of different nanostructured substrates based on synthetic hydroxyapatite (HA) and titania (TiO2) nanoparticles, where we extend the debate regarding the selective roles played by the presence of albumin on the biogenic apatite coating evolution. The substrates bone-bonding potential was evaluated by keeping the materials in contact with Simulated Body Fluid, while the influence of the presence of Bovine Serum Albumin in bioactivity was analyzed by a spectrophotometric technique. Our results show that materials’ surface reactivity and their interfacial hydration are responsible for the bonding-site alteration and surface charge density distribution, which in turn, regulate the protein adsorption process. As a matter of fact, variations on the protein adsorbed density have a directly proportional impact on calcium binding sites, which should be responsible for the initiation of the mineralization process, disturbing the deposition of the interfacial calcium phosphate (Ca-P) mineralized coating.
... At pH close to the IEP of a protein, repulsive interactions between BSA molecules are at the lowest, therefore the loosely bound portion of proteins adsorbed is increased [207]. The highest adsorption was observed near the IEP of the BSA, as it is possible to see in Figure 9 [208]. The mechanisms that can explain the pH effect on protein adsorption on Ti was proposed by Imamura et al. [209]. ...
... Raman spectroscopy is a technique mostly applied for studying adsorption on nanoparticles [36,237], which was also reported to be used, with 2D-correlation analysis, for adsorption on bulk materials [71]. The evolution of the adsorbing layer can be monitored in situ by spectroscopic ellipsometry by measuring the thickness of the layer, according to the variation of the ellipsometric angles Δ and Ψ [86,208,209]. Some amino acids are intrinsically fluorescent, tryptophan and tyrosine in particular, and their emission is sensitive to the chemical environment around them. ...
Titanium and its alloys, specially Ti6Al4V, are among the most employed materials in orthopedic and dental implants. Cells response and osseointegration of implant devices are strongly dependent on the body–biomaterial interface zone. This interface is mainly defined by proteins: They adsorb immediately after implantation from blood and biological fluids, forming a layer on implant surfaces. Therefore, it is of utmost importance to understand which features of biomaterials surfaces influence formation of the protein layer and how to guide it. In this paper, relevant literature of the last 15 years about protein adsorption on titanium-based materials is reviewed. How the surface characteristics affect protein adsorption is investigated, aiming to provide an as comprehensive a picture as possible of adsorption mechanisms and type of chemical bonding with the surface, as well as of the characterization techniques effectively applied to model and real implant surfaces. Surface free energy, charge, microroughness, and hydroxylation degree have been found to be the main surface parameters to affect the amount of adsorbed proteins. On the other hand, the conformation of adsorbed proteins is mainly dictated by the protein structure, surface topography at the nano-scale, and exposed functional groups. Protein adsorption on titanium surfaces still needs further clarification, in particular concerning adsorption from complex protein solutions. In addition, characterization techniques to investigate and compare the different aspects of protein adsorption on different surfaces (in terms of roughness and chemistry) shall be developed.
... Although the surficial BSA greatly reduced after sonicated cleaning, the surface of MWCP11 still adsorbed more BSA than other groups. However due to small size and the flexible feature of BSA [53,54], the sub-nano scale protein may be trapped more in the nano-scale architecture of MW groups by van der Waals force (Fig. 6). On the other hand, the positively charged histone (similar size to BSA) is expected to have a stronger adhesion force (both by Coulomb force and van der Waals force). ...
... Although our research has shown that the protein adsorption is related to the surface potential and nanoscale patterns, it is noticeable that proteins adsorbed on the surface of titanium implants in-vivo will subject to complex protein adsorption kinetics, as proteins will fold/unfold or rearrange, and change their surface charges in different pH [55]. In addition, a large number of albumin in the plasma causing surface "passivation" and protein exchange with time as larger stable-binding protein will replace the smaller one via the "Vroman effect" [53,56]. Therefore, the adsorption of proteins would be dynamically affected by surface potential and nanoscale patterns and their effects on regulating cell osteogenesis need to be further studied in the future. ...
TUsing a novel microwave-assisted hydrothermal (MW) process we created nano-scale anatase on micro-arc-oxidized (MAO) titanium surface. The morphology/crystallinity and surface potential of the anatase which altered by Ca/P ions concentrations and pH in the MW medium were characterized by atomic force microscopy, Kelvin probe force microscopy, Raman spectrometer, and x-ray photoelectron spectroscopy. The surface of MWDD (processed in DD water) and MWCP (in neutral pH medium with Ca/P ions) are covered with anatase spikes, which enhance their nano-roughness, hydrophilicity, and possess lower surface potential than other groups. The nano-precipitates on surface of MWCP9 and MWCP11 (processed in medium containing Ca/P ions at pH 9 or pH 11) were mainly amorphous anatase with less P ions. The protein adsorption of negatively charged bovine serum albumin was higher in MWDD and MWCP groups which possess lower surface potential. The adsorption of positively charged histones on the surface of MWCP11 was higher compared to the other groups. The osteogenic characterizations of D1 mice bone marrow mesenchymal stem cells co-cultured for 1, 7, and 14 days were measured by ALP and osteopontin assays. Although MW groups revealed comparable viability of D1 mice bone marrow mesenchymal stem cells to MAO group, their superior hydrophilicity and higher protein adsorption, thus regulating the differentiation of osteoprogenitor stem cells demonstrating higher ALP and osteopontin secretion after 7 days and 14 days. The nanoscale topography, crystallinity and surface potential change the hydrophilicity and protein adsorption on the MW treated titanium surface, thus regulating the differentiation of osteoprogenitor stem cells in vitro.
... The measurements are complementary to the information obtained by QCM and can be used to determine the optical properties of a substrate and the thickness of multiple layers on the surface. The technique is simple, nondestructive, provides angstrom resolution, and has the capability of allowing the observation of the adsorption process in real time [94,134,135]. The use of imaging ellipsometry can also provide spatial resolution of protein binding [136]. ...
... Because BSA typically exhibits significant surface-induced spreading upon adsorption, a stable layer of the protein can be formed under a wide number of conditions in less than 1 h [135,205,303]. For these reasons (and its low cost), it has been extensively used as a model protein to study adsorption [68,80,110,134,137,139,304,305], to aid in the suspension of CNT [306], and to block the remaining sites of surfaces after the immobilization of a biorecognition element [250,260,294,307]. BSA has also been adsorbed to the surface of PMMA nanoparticles to enhance the subsequent attachment of GOx by electrostatic interactions, retaining most of the activity of the free enzyme [308,309]. ...
... For each sample, the reported contact angle is the average of three measurements performed in different spots on the surface, accompanied by an uncertainty of ± 2°. The thickness of the low surface energy fluorinated layers deposited on untreated Si wafers was evaluated with a Spectroscopic Ellipsometer (J.A. Woollam Co., Lincoln, NE, USA) [36]. ...
Conventional biomedical research models are confined to static cell culture models and animal testing, both of them representing suboptimal preclinical models. Organs-on-chips (OOCs) are novel 3D microfluidic cell culture devices lined with living cells that allow for faithful mimicry of the physiology and function of a vital human organ unit. Bone marrow-on-a-chip (BMoC) systems are currently limited to in vitro maintenance of the hematopoietic potential, supported with 3D scaffold matrices. Herein, we introduce a purely in vitro and scaffold-free bone marrow-on-a-chip device, intended for both the generation and sustainment of the perivascular hematopoietic niche, to serve as a study platform for the chronic autoimmune disease of systemic lupus erythematosus (SLE). The device consists of three layers of poly(dimethylsiloxane) (PDMS), including two cylindrical microchambers separated by an intervening porous membrane, altogether bonded to a glass slide. In this communication, the fabrication steps for a reliable and reproducible BMoC device are presented, from its design to its final assembly. As a first step towards the full recapitulation of the perivascular niche on-chip, the bone marrow stromal niche was replicated inside sealed type I collagen-coated microchambers hosting the in vitro 3D microfluidic culture of mesenchymal stem cells (MSCs) for 8 days. Culture of the MSCs revealed significant cell proliferation and progressive formation of 3D stromal tissue, a promising outcome for the subsequent 3D stromal matrix growth on the bone marrow-on-a-chip platform.
... At this point it is important to point out that this time was selected as a compromise between the time required to prepare the chip and that required for BSA to adsorb and undergo surface-induced structural rearrangements. 26 This consideration is also in line with results reported by our group, [27][28][29][30] specifically addressing the adsorption kinetics of BSA to various substrates. Then (and unless otherwise stated), 15 µL of buffer were spotted in the buffer zone (d = 5 mm) and allowed to wick through the device (through the proteinmodified zone) towards the waste reservoir (d = 10 mm). ...
Adsorption is the most common approach to immobilize biorecognition elements on the surface of paper-based devices. Adsorption is also the route selected to coat the substrate with albumin, therefore minimizing the interaction of other proteins. While similar in nature, the structure of the selected proteins as well as the conditions selected from the immobilization have a significant effect on the amount and distribution of the resulting composites. To illustrate these differences and provide general guidelines to efficiently prepare these devices, this article explores the interaction (adsorption and desorption) of BSA with 3MM chromatography paper. The experimental conditions investigated were the protein concentration, the interaction time, the number of times the protein was spotted, the pH of buffer solution, and the ionic strength of the buffer solution. The proposed approach mimics the steps involved in the fabrication (adsorption) and use (rinsing induced by the sample) of paper-based microfluidic devices. To identify the protein location following the rinsing step, the protein was fixed by dehydration in a convection oven and then stained using Coomassie Blue. The color intensity, which was found to be proportional to the amount of protein immobilized, was determined using a desktop scanner. To highlight the importance of understanding the adsorption process to the rational development of μPADs, results were complemented by experiments performed with lysozyme and immunoglobulin G.
... Close to its IEP, the protein side chains are electrically balanced and will be adsorbed through reorientation of the molecules when they approach the surface to allow electrostatic attraction with the charged surface[39,17]. Many authors[16,[40][41][42]14]have studied the effect of pH on protein adsorption and found that at the IEP of the protein molecules in solution, the surfaces adsorb the highest amount of proteins. At acidic pH, the proteins are positively charged and will be adsorbed on the negatively charged surface via coulombic attractions. ...
The adsorption of Bovine Serum Albumin (BSA) proteins on amorphous silicon (a-Si) surfaces was studied with respect to solution pH. Thin films of a-Si were deposited using radio-frequency magnetron sputtering at room temperature and then treated in a hydrogen ambient to form a hydrogenated a-Si surface layer (a-Si:H). The interactions of the as-deposited and hydrogenated surfaces with the proteins at neutral, acidic, and basic environments was probed by means of Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy, Spectroscopic Ellipsometry (SE), and Atomic Force Microscopy (AFM), to study the influence of the charge of proteins on their adsorption and conformation on the a-Si:H surface, compared with the a-Si surface. The results show that the charge of the proteins has a significant effect on their interactions with these two substrates but in dissimilar ways. For the as-deposited substrate, these interactions are predictably coulombic since the surface is charged. For the hydrogenated substrate, the adsorption of the proteins depends on their conformation which is heavily affected by pH, and the size of their footprint (adsorption mode) on the surface.
... 27 Many researchers have studied the adsorption properties of BSA. It has been reported that the maximum adsorption of BSA occurred at its isoelectric point on various surfaces, including magnetic particles, 28 nanostructured TiO 2 thin films, 29 mesoporous material SBA-15, 30 ordered mesoporous silica, 27 and ultrafine silica particles. 31 Most of these studies mentioned that small lateral repulsion could be the main reason for the maximum adsorption. ...
... For these reasons, researchers have focused on the elucidation of the mechanisms governing protein interactions with various biomaterials including polymers, metals and ceramics (Wehmeyer et al. 2010). A number of surface-sensitive techniques have been used for the quantification of protein adsorption, viz. ...
Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.
... This particular thin film could be explored as photochromic material , optical filter, and sensor. Apart from these, the TiO 2 based thin films have also been employed for several other applications [209][210][211][212][213][214]. ...
Titanium dioxide (TiO2) is one of the most important materials to be employed as a photocatalyst for environmental protection and other applications. Owing to its certain important combination of properties, TiO2 in the form of thin film has been found to be more attractive for a great variety of applications including photocatalytic degradation of organic pollutants in water as well as in air, dye sensitized solar cells (DSSCs), anti-fogging, superhydrophilic, photochromic, and optical applications. Although, a great number of techniques have been employed so far to fabricate TiO2 thin films, the cost of these thin films has not only been found to be dictated by the sophistication of the preparation method involved but also by the quick recombination of photo-generated electron/hole pairs, backward reactions involved, and the poor response of TiO2 to the visible light. Even though, metal loading, metal ion doping, anion doping, dye sensitization, composite semiconducting phenomenon, metal ion-implantation, addition of sacrificial reagents and carbonate salts to the reaction mixtures, etc., have been employed to improve the efficiency of the photocatalytic applications of TiO2 thin films, a clear-cut relationship between the properties of TiO2 thin films and their performance in a given application is yet to be established. In this article, some of the latest developments accomplished in the fabrication techniques, in the characterization and in the understandings of property and performance relationship of TiO2 thin film have been presented and discussed while citing certain of important references.
... The ellipsometer was also used to measure the ellipsometric angles (Ψ and Δ) as a function of the incident wavelength, according to a procedure described elsewhere. 3,4,37,38 In order to calculate the refractive index (n) and extinction coefficient (k) of the film, the collected data were modeled with WVASE (J. A. Woollam Company; Lincoln, NE), using an optical model composed of a layer of Si (bulk; d = 1 mm), a layer of SiO 2 (d = 2.104 nm), and the OTCE. Differences between the experimental and model-generated data were assessed by the mean square error (MSE), 39 a built-in function in WVASE. ...
... In order to obtain information related not only to the adsorption process but also the structure, thickness [21], optical constants, and microstructure of films [22], our lab is now focused on the use of variable angle spectroscopic ellipsometry (VASE) [23,24]. VASE enabled the possibility of extending the scope of the project to DNA [25] and other proteins such as albumin [26], D-amino acid oxidase [27], catalase [28], and glucose oxidase [29]. In general, our results demonstrated that the activity of enzymes adsorbed to CNT is not only exclusively proportional to the adsorbed amount of protein (which is the only variable optimized in most systems) but also to the initial adsorption rate (Figure 4). ...
Recent developments in materials, surface modifications, separation schemes, detection systems and associated instrumentation have allowed significant advances in the performance of lab-on-a-chip devices. These devices, also referred to as micro total analysis systems (µTAS), offer great versatility, high throughput, short analysis time, low cost and, more importantly, performance that is comparable to standard bench-top instrumentation. To date, µTAS have demonstrated advantages in a significant number of fields including biochemical, pharmaceutical, military and environmental. Perhaps most importantly, µTAS represent excellent platforms to introduce students to microfabrication and nanotechnology, bridging chemistry with other fields, such as engineering and biology, enabling the integration of various skills and curricular concepts. Considering the advantages of the technology and the potential impact to society, our research program aims to address the need for simpler, more affordable, faster and portable devices to measure biologically active compounds. Specifically, the program is focused on the development and characterization of a series of novel strategies towards the realization of integrated microanalytical devices. One key aspect of our research projects is that the developed analytical strategies must be compatible with each other; therefore, enabling their use in integrated devices. The program combines spectroscopy, surface chemistry, capillary electrophoresis, electrochemical detection and nanomaterials. This article discusses some of the most recent results obtained in two main areas of emphasis: capillary electrophoresis, microchip-capillary electrophoresis, electrochemical detection and interaction of proteins with nanomaterials.
... The resulting optical model is schematically shown inFigure 1B. In agreement with previous reports describing the adsorption of transparent polymers555657, the optical properties of the enzyme were described using a Cauchy model (A=1.45, B=0.1, C=0). ...
This study is the first to focus on the potential use of carbon nanotube (CNT) scaffolds as enzyme immobilization substrates for analytical purposes. Besides all the well-known advantages of CNT, three-dimensional scaffolds can significantly increase the amount of enzymes adsorbed per unit area, preserve the catalytic activity of the adsorbed molecules, and allow effective exposure to substrates present in the adjacent medium. Additionally, our results indicate that the sensitivity of analytical probes based on enzyme-loaded CNT scaffolds is proportional to the thickness of the scaffold providing 3-fold sensitivity improvements with respect to the control surfaces.
... Among others, it is relevant to mention that at low coverage a) only small changes in the signal (Ψ and Δ as function of time or λ) are obtained, limiting the sensitivity of the experiment; b) the fitting parameters (A, B and C) could be strongly correlated with the thickness; and c) the system cannot distinguish between similar materials (polymers, proteins, etc) competing for the adsorption sites. Such information is crucial for the analysis of the adsorbed layer in many applications such as layer-by-layer deposition [18,19] or the development of biosensors202122, antifouling coatings [15,23] and biocompatible materials [24,25]. Although in some cases, the optical constants or thickness of the underlying substrate can be adjusted to maximize the sensitivity of the analysis [26], a simple and broadly applicable procedure is currently not available. ...
This communication describes a simple way to improve the sensitivity of spectroscopic ellipsometry, when applied to monitor the adsorption of proteins to solid surfaces. The method described herein is based on the reaction of a commercially available dye (Coomassie brilliant blue G) with the adsorbed proteins and the subsequent analysis by spectroscopic ellipsometry. In order to demonstrate the potential advantages of this method, the adsorption of bovine serum albumin to an antifouling coating was also investigated. According to our results, the modification with the dye significantly affects the optical properties of the adsorbed protein layer, which can be represented using a simple optical model (Lorentz). In general, the proposed modification increases the sensitivity of the detection by 2.5 ± 0.4-fold and enables the analysis of thin layers of adsorbed protein not obtainable by conventional methods. These results particularly reveal the importance of the proposed modification for the evaluation of low adsorbing substrates and antifouling coatings.
The development of membranes for protein purification has stringent requirement of disinfection resistance, low protein adsorption and anti-fouling, without changing protein structure. In this study, hydrophilic titanium dioxide (TiO2)/calcium alginate (TiO2/CaAlg) hydrogel membranes were prepared by a simple ionic cross-linking method. The effects of the porogenic agent polyethylene glycol (PEG) concentration, the molecular weight of PEG, and the concentration of TiO2 on the filtration properties were systematically investigated. The TiO2/CaAlg membrane exhibited excellent bovine serum albumin (BSA) rejection and anti-fouling properties. The mechanical properties and surface energy of the TiO2/CaAlg membrane were significantly improved. The chemical bonding mechanism of TiO2 and NaAlg was investigated by molecular dynamic simulation. The TiO2/CaAlg membrane had good chlorine resistance and could be disinfected or cleaned with sodium hypochlorite. The TiO2/CaAlg hydrogel membrane loaded with polyhydroxybutyrate (PHB) nanofibers maintained high flux (136.7 L/m2h) and high BSA rejection (98.0 %) at 0.1 MPa. The results of circular dichroism and synchronous fluorescence indicated that the secondary structure of BSA was maintained after membrane separation. This study provides one method for the preparation of green and environmentally friendly membrane for protein purification.
Out-of-plane structure and surface morphology of the adsorbed thin films of globular protein bovine serum albumin (BSA) are investigated using X-ray reflectivity (XRR) and atomic force microscopy (AFM). BSA adsorption on hydrophilic silicon surface is done by dip coating method in absence and presence of mono- (Na⁺), di- (Ca²⁺) and tri- (La³⁺ and Y³⁺) valent ions in BSA solution for different salt concentrations. Unlike mono- and di-valent ions, BSA shows re-entrant phase transition for tri-valent ions like La³⁺ and Y³⁺ with their concentration variation. XRR study shows that the thickness of the adsorbed BSA layer slightly increases with the increase of mono- and di-valent ions concentration, while for tri-valent ions, considerable amount of thickness is obtained when the film is deposited from the condensed phase under re-entrant condensation and becomes relatively less with salt concentrations lower or higher than that particular salt concentration, however, thickness is again increased for higher salt concentration. Surface morphologies of the films are also modified with the variation of the salt type and their concentration. Thus, structural and morphological changes of the adsorbed protein layer on hydrophilic surface and the probable reason for such modifications are explored in the presence of different valent ions with their concentration variation.
Increasing use of silver nanoparticles (AgNPs) in different consumer products provokes interest in understanding interactions of AgNPs with biological molecules, e.g., proteins. The adsorption of AgNPs and bovine serum albumin (BSA) onto self‐assembled monolayer of mercaptohexylpyridinium (MHP) on gold surface is described. A quartz crystal microbalance coupled with electrochemical measurements were used to study interactions between AgNPs and BSA. It was found that AgNPs adsorbed on MHP can be considered as highly elastic (stiff) film since the dissipation ΔD ∽ 0. Measurements of the mass loss and the increase in dissipation in parallel with the electrochemical oxidation/reduction of AgNPs shows that albumin adsorption on AgNPs highly diminish electrochemical Ag/AgCl conversion. The formation of a less rigid Ag layer than the original MHP‐AgNPs film is also indicated. The 3D assemblies nanostructure observed with SEM revealed clustering of particles after the redox process. This article is protected by copyright. All rights reserved.
Single-molecule imaging of proteins using atomic force microscopy (AFM) is crucially dependent on protein attachment to ultra-flat substrates. The technique of template stripping (TS), which can be used to create large areas of atomically flat gold, has been used to great effect for this purpose. However, this approach requires an epoxy which can swell in solution, causing surface roughening and substantially increasing the thickness of any sample, preventing its use on acoustic resonators in liquid. Diffusion bonding techniques should circumvent this problem but cannot be used on samples containing patterned features with mismatched heights due to cracking and poor transfer. Here, we describe a new technique called pressure forming template stripping (PTS) which permits an ultra-flat (0.35 ± 0.05 nm root-mean-square roughness) layer of gold to be transferred to the surface of a patterned substrate at low temperature and pressure. We demonstrate this technique by modifying a quartz crystal microbalance (QCM) sensor to contain an ultra-flat gold surface. Standard QCM chips have substantial roughness, preventing AFM imaging of proteins on the surface after measurement. With our approach there is no need to run samples in parallel: the modified QCM chip is flat enough to permit high-contrast AFM imaging after adsorption studies have been conducted. The PTS-QCM chips are then used to demonstrate adsorption of bovine serum albumin in comparison to rough QCM chips. The ability to attach thin layers of ultra-flat metals to surfaces of heterogeneous nature without epoxy will have many applications in diverse fields where there is a requirement to observe nanoscale phenomena with multiple techniques, including surface and interfacial science, optics, and biosensing.
Despite the intensive research on protein adsorption in mesoporous materials, the effect of (de)hydration and confinement on the adsorbed protein’s stability and activity is poorly understood. In this paper, we study the effect of differences in structural features (pore size) and drying time on the adsorption and structural stability of horse heart myoglobin (hhMb) on mesoporous titanium dioxide. Infrared spectroscopy (DRIFT) and thermal analysis (TGA) coupled to a quadrupole mass spectrometer (TGA-MS) were used to evaluate the impact of the confinement in different pores and hydration on the myoglobin secondary structure. Electron paramagnetic spectroscopy (EPR) was applied to identify the changes in the heme and its close surrounding. The peroxidase-like activity of myoglobin toward 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of hydrogen peroxide allowed to detect changes in the protein activity after adsorption in pores with different sizes and drying for different periods of time. The results show a clear effect of the pore size and drying time on the secondary structure of hhMb, which is confirmed by differences induced in the catalytic activity of the adsorbed proteins. Therefore, we recommend to evaluate the effect of both hydration and confinement in future application involving biomolecule adsorption in porous matrices.
The evaluation of thickness, refractive index, and optical properties of biomolecular films and self-assembled monolayers (SAMs) has a prominent relevance in the development of label-free detection techniques (quartz microbalance, surface plasmon resonance, electrochemical devices) for sensing and diagnostics. In this framework Spectroscopic Ellipsometry (SE) is an important player. In our approach to SE measurements on ultrathin soft matter, we exploit the small changes of the ellipsometry response (\(\delta \varDelta \) and \(\delta \varPsi \)) following the addition/removal of a layer in a nanolayered structure. So-called \(\delta \varDelta \) and \(\delta \varPsi \) difference spectra allow to recognize features related to the molecular film (thickness, absorptions) and to the film-substrate interface thus extending SE to a sensitive surface UV-VIS spectroscopy. The potential of ellipsometry as a surface spectroscopy tool can be boosted when flanked by other characterizations methods. The chapter deals with the combined application of broad-band Spectroscopic Ellipsometry and nanolithography methods to study organic SAMs and multilayers. Nanolithography is achieved by the accurate removal of molecules from regularly shaped areas obtained through the action of shear forces exerted by the AFM tip in programmed scans. Differential height measurements between adjacent depleted and covered areas provide a direct measurement of film thickness, which can be compared with SE results or feed the SE analysis. In this chapter we will describe the main concepts behind the SE difference spectra method and AFM nanolithograhy. We will describe how SE and AFM can be combined to strengthen the reliability of the determination of thickness and, as a consequence, of the optical properties of films. Examples will be discussed, taken from recent experiments aimed to integrate SE and AFM nanolithography applied to SAMs and nano layers of biological interest. By analysing in detail the changes of the spectroscopic features of compact versus non-compact layers and correlating such changes with the post-lithography AFM analysis of surface morphology SE unravels the specific versus unspecific adsorption of biomolecules on gold surfaces functionalized with suitable SAMs.
Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.
An Au-Ag alloy film surface plasmon resonance (SPR) sensor was studied experimentally and theoretically. SPR chips were prepared by sputtering a glass surface with a 50-nm-thick Au-Ag alloy film, and experiments were carried out with the wavelength-interrogated SPR sensor using the Kretschmann configuration. Aqueous sodium chloride and bovine serum albumin solutions were used to study the refractive index and adsorption sensitivities of the sensor. The results were compared with those obtained using Au film and Ag film SPR sensors. The refractive index sensitivity of the Au-Ag alloy film SPR sensor is higher than that of the Au film SPR sensor, but lower than that of the Ag film SPR sensor. The adsorption sensitivity of the Au- Ag alloy film SPR sensor is similar to that of the Ag film SPR, and three times of the Au film SPR sensor. Theoretical studies showed that that the sensitivity of the Au-Ag alloy film SPR is close to that of the Ag film SPR sensor, and 2.31 times of the Au film SPR sensor. The full width at half maximum of the Au-Ag film sensor is only 0.36 times of the Au or Ag film SPR sensors. The Au-Ag alloy film and Au film SPR sensors are chemically stable, but the Ag film SPR sensor is easily oxidized, so it is not often used. These results show that an Au-Ag alloy film can improve the sensitivity of the sensor, while retaining the accuracy. Au-Ag films could therefore be used as high-sensitivity, low cost, and stable SPR-sensitive materials.
Nowadays, a broad choice of instruments, including dedicated synchrotron radiation beamlines, allows to exploit Spectroscopic Ellipsometry (SE) to investigate the thickness and the dielectric properties of thin films, from the terahertz down to the VUV wavelength range. Instruments combining fast parallel detection, precision, accuracy, are pushing forward real time and in-situ applications, to monitor the dynamics of processes such as e.g. film growth, oxidation, polymerization, electrochemical processes, with a diverging spectrum of scientific and industrial applications in the fields of nano-electronics, coatings, solar cell materials, polymer technology, bio-sensing, photonics, just to name a few. This chapter, beyond presenting the essentials of principles and instrumentation of SE, is intended to place this thin-film technique in the perspective of the surface scientist, through the selection of applications to ultra-thin films and nanostructures. Emphasis is placed on reflection experiments, in the 190–1700 nm wavelength range covered by high-quality commercial instruments, although some infra-red (IRSE) and far UV experiments are also discussed.
The influence of surface curvature on the adsorption of bovine serum albumin (BSA) was evaluated through the combination of two fairly simple techniques: electrophoretic light scattering and UV/vis spectroscopy. Measurements were carried out for a range of protein concentrations (0–320 μg/ml) at pH 3.5, 4.5 and 7 using hydrophobic polystyrene nanospheres of 38.8, 82 and 220 nm in diameter. The results obtained demonstrate that the charge of the BSA molecules in solution dictates the pH-dependent behavior of the protein-coated nanospheres, indicating in all cases a significant adsorption of BSA molecules. At a fixed pH, however, it is the zeta potential that characterizes the uncoated nanospheres normalized by their surface area that primarily controls protein adsorption. In particular, it is found that the rate at which BSA interact with the different nanospheres increases as their negative zeta potential per unit area (or diameter) increases (decreases) regardless of the pH. Moreover, provided that adsorption occurs away from the isoelectric point of the protein, highly curved surfaces are found to stabilize the native-like conformation of BSA upon adsorption by likely reducing lateral interactions between adsorbed molecules.
The recent increase in nanomaterial usage has led to concerns surrounding its health risks and environmental impact. The food chain is an important pathway for high-trophic-level organisms absorbing and enriching nanomaterials. Our study therefore simulated nanometer titanium dioxide (nano-TiO2) transfer along a 2-step food chain, from the unicellular alga Scenedesmus obliquus to the water flea Daphnia magna. We also explored the effect of sodium dodecyl benzene sulfonate (SDBS) on nano-TiO2 bioavailability. A suspension of 10 mg/L nano-TiO2 was optimally dispersed in aqueous solutions by 5 mg/L SDBS. After 72 h, S. obliquus growth was not significantly affected by 10 mg/L nano-TiO2, 5 mg/L SDBS and their mixed suspension. SDBS not only improved nano-TiO2 stability in water, but also increased its uptake in S. obliquus and enhanced its accumulation in D. magna. Our study suggests that nano-TiO2 is mildly toxic to S. obliquus, and can be transferred along the aquatic food chain with a biomagnification effect.
An active protein adsorption membrane composed of poly(vinylidenedifluoride) (PVDF) and TiO2 nanoparticles, which surface modified by oleic acid (OA) molecule has been fabricated. The hybrid membranes were obtained using an induced phase inversion method by blending the PVDF casting solution and OA-modified TiO2 nanoparticles. The TiO2 nanoparticles in anatase phase with average size ranged from 10 to 50 nm were entrapped into the pores and deposited on the surface of PVDF films. Furthermore, the presence of TiO2 nanoparticles affected the formation process of PVDF films and resulted in the variation of the pore structure of PVDF films. The adsorption performances of these PVDF hybrid membranes were measured using bovine hemoglobin (BHb) and bovine serum albumin (BSA) as target substances, and the effects of adsorption conditions were systematically studied to determine the optimum conditions. The adsorption results showed that the hybrid membranes had significant enhanced adsorption activity toward BHb and BSA in comparison with pure PVDF, and there was a positive relationship between adsorption capacity of proteins and the loading amounts of TiO2. However, The OA-modification enhanced the BHb adsorption and meanwhile depressed the BSA adsorption. Interestingly, the adsorption capacity of BHb on hybrid membranes was as 3-4 times as that of BSA. Using the mixed protein solution as adsorption object, the SDS-PAGE analysis demonstrated that BHb can be selectively adsorbed on the hybrid membranes. This work showed a prospect application of these hybrid membranes in the selective adsorption and separation of BHb.
Mesoporous materials, especially functionalized ones, have become a promising carrier for enzyme immobilization. We synthesized room temperature ionic liquid-decorated mesoporous SBA-15 (RTIL-SBA-15) for papain immobilization. The results of powder XRD, IR and N2 adsorption–desorption isotherms have confirmed that ionic liquid [Simim+][Cl−] was successfully grafted on the surface of SBA-15. As a consequence of the electrostatic attraction between the cation [Simim+] and the negatively charged papain, RTIL-SBA-15 had an advantage over SBA-15 when papain was immobilized at pH = 9.00. The kinetic study showed that the interaction between papain and the carrier was stronger after ionic liquid modification. In the casein hydrolysis, the papain immobilized on RTIL-SBA-15 showed a higher specific activity than that on SBA-15, implying that the ionic liquid [Simim+][Cl−] was beneficial to improve the activity of the immobilized papain. The optimum pH of the immobilized papain was shifted to higher than that of free enzyme.
This paper describes a simple and inexpensive procedure to produce thin-films of poly(dimethylsiloxane). Such films were characterized by a variety of techniques (ellipsometry, nuclear magnetic resonance, atomic force microscopy, and goniometry) and used to investigate the adsorption kinetics of three model proteins (fibrinogen, collagen type-I, and bovine serum albumin) under different conditions. The information collected from the protein adsorption studies was then used to investigate the adhesion of human dermal microvascular endothelial cells. The results of these studies suggest that these films can be used to model the surface properties of microdevices fabricated with commercial PDMS. Moreover, the paper provides guidelines to efficiently attach cells in BioMEMS devices.
This paper presents an efficient and less expensive method to fabricate high quality titanium dioxide (TiO2) thin films by APCVD technique using TiCl4 as precursor for application as antireflection coatings on monocrystalline silicon solar cells. The structural, electrical and optical properties of the produced coatings were successfully characterized by Atomic Force Microscopy (AFM), Four Point Probe (FPP) and Spectroscopic Ellipsometry (SE) combined with Transmission Spectroscopy, respectively. A perfect agreement between the AFM results and ellipsometric results was confirmed. For ellipsometry modelling process, the TiO2 optical constants were successfully modelled by means of the "Multilayer Structure Model" which offers the best results. The refractive index of our TiO2 thin films was found to be n = 2.25 at the wavelength � = 550 nm, with a thickness of 56.2 nm. The obtained results demonstrate the real opportunity of the APCVD technique to prepare high quality antireflection coatings for high efficiency silicon solar cells.
In this study a label-free proteomic approach was used to investigate the composition of the layer of protein adsorbed to rough titanium (Ti) after exposure to human blood plasma. The influence of the protein layer on the surface free energy (SFE) of the Ti was evaluated by contact angle measurements. Ti discs were incubated with blood plasma for 180 min at 37 °C, and the proteins recovered were subjected to liquid chromatography coupled to tandem mass spectrometry analysis. A total of 129 different peptides were identified and assigned to 25 distinct plasma proteins. The most abundant proteins were fibronectin, serum albumin, apolipoprotein A-I, and fibrinogen, comprising 74.54% of the total spectral counts. Moreover, the protein layer increased the SFE of the Ti (p < 0.05). The layer adsorbed to the rough Ti surface was composed mainly of proteins related to cell adhesion, molecule transportation, and coagulation processes, creating a polar and hydrophilic interface for subsequent interactions with host cells.
The present paper describes the results related to the optical and electrochemical characterization of thin carbon films fabricated by spin coating and pyrolysis of AZ P4330-RS photoresist. The goal of this paper is to provide comprehensive information allowing for the rational the selection of the conditions to fabricate optically-transparent carbon electrodes (OTCE) with specific electro-optical properties. According to our results, these electrodes could be appropriate choices as electrochemical transducers to monitor electrophoretic separations. At the core of this manuscript is the development and critical evaluation of a new optical model to calculate the thickness of the OTCE by variable angle spectroscopic ellipsometry (VASE). Such data was complemented with topography and roughness (obtained by AFM), electrochemical properties (obtained by cyclic voltammetry), electrical properties (obtained by electrochemical impedance spectroscopy), and structural composition (obtained by Raman spectroscopy). Although the described OTCE were used as substrates to investigate the effect of electrode potential on the real-time adsorption of proteins by ellipsometry, these results could enable the development of other biosensors that can be then integrated into various CE platforms. This article is protected by copyright. All rights reserved.
The amperometric glutamate biosensor based on screen-printed electrodes containing carbon nanotubes (CNT), and its integration in a flow injection analysis system, is described herein. The sensor was fabricated by simply adsorbing enzyme glutamate oxidase (GlutOx) on a commercial substrate containing multi-wall CNT. The resulting device displayed excellent electroanalytical properties toward the determination of L-glutamate in a wide linear range (0.01-10 μM) with low detection limit (10 nM, S/N≥3), fast response time (≤5 s), and good operational and long-term stability. The CNT modified screen-printed electrodes have a potential to be of general interest for designing of electrochemical sensors and biosensors.
The adsorption of Yeast Cytochrome c (YCC) on well defined, flat gold substrates has been studied by Spectroscopic Ellipsometry (SE) in the 245-1000 nm wavelength range. The investigation has been performed in aqueous ambient at room temperature, focusing on monolayer-thick films. In situ δΨ and δΔ difference spectra have shown reproducibly well-defined features related to molecular optical absorptions typical of the so-called heme group. The data have been reproduced quantitatively by a simple isotropic optical model, accounting for the molecular absorption spectrum and film-substrate interface effects. The simulations allowed a reliable estimate of the film thickness and the determination of the position and the shape of the so-called Soret absorption peak that, within the experimental uncertainty, is the same found for molecules in liquid. These findings suggest that YCC preserves its native structure upon adsorption. The same optical model was able to reproduce also ex situ results on rinsed and dried samples, dominated by the spectral features associated to the polypeptide chain that tend to overwhelm the heme absorption features.
This paper is the first report on the characterization of the hydrodynamic conditions in a flow cell designed to study adsorption processes by spectroscopic ellipsometry. The resulting cell enables combining the advantages of in situ spectroscopic ellipsometry with stagnation point flow conditions. An additional advantage is that the proposed cell features a fixed position of the "inlet tube" with respect to the substrate, thus facilitating the alignment of multiple substrates. Theoretical calculations were performed by computational fluid dynamics and compared with experimental data (adsorption kinetics) obtained for the adsorption of polyethylene glycol to silica under a variety of experimental conditions. Additionally, a simple methodology to correct experimental data for errors associated with the size of the measured spot and for variations of mass transfer in the vicinity of the stagnation point is herein introduced. The proposed correction method would allow researchers to reasonably estimate the adsorption kinetics at the stagnation point and quantitatively compare their results, even when using different experimental setups. The applicability of the proposed correction function was verified by evaluating the kinetics of protein adsorption under different experimental conditions.
The powdered samples of CuInS2 obtained by a chemical synthesis method and contained either thread-like or belt-like nanowires were studied by a spectroscopic phase-modulated ellipsometer in the photon energy range from 0.8 to 6 e V at room temperature. The samples with thread-like and belt-like nanowires were found to have different pseudodielectric function, which was closer to that of bulky single crystalline CuInS2 in the case of belt-like nanowires. The same value of energy gap revealed by incoherent ellipsometric approach to bulky samples and samples with nanowires was accounted for the presence of some fraction of bulky phase in the nanostructured samples.Reliability of the obtained data and relationship between the ground state shape of the nanowires and the electronic spectrum were discussed. It was substantiated that the obtained data provide a first realistic glimpse on electronic spectrum of nanostructured CuInS2.
Electrical characteristics of sol–gel derived titanium dioxide (TiO2) as insulating layers were investigated by making capacitance and leakage current measurements in metal–insulator–semiconductor configurations. The structure was fabricated by depositing 37 nm thick anatase TiO2 films on p-type silicon (p-Si) substrates. The frequency dispersion of capacitance was attributed to the leaky behaviour of the TiO2 dielectrics. Using an equivalent circuit, values of the frequency-independent dielectric constant, interfacial surface density and threshold voltage were estimated to be 13, 3 × 1014 m−3 and −0.085 V, respectively. The carrier diffusion was found to be primarily responsible for the diode leakage current at room temperature but the increase in the ideality factor with lowering temperature was believed to be due to fluctuations of barrier height at the TiO2/p-Si interface.
This review article summarizes briefly some important achievements of our recent reserach on anatase and/or rutile TiO2 thin films, fabricated by helicon RF magnetron sputtering, with good crystal quality and high density, and gives the-state-of-the-art of the knowledge on systematic interrelationship for fabrication conditions, crystal structure, composition, optical properties, and bactericidal abilities, and on the effective surface treatment to improve the optical reactivity of the obtained films.
Plasma-gas condensation cluster deposition systems have been introduced and applied for preparation of Co/CoO and Co/Si clusters assemblies. In Co/CoO cluster assemblies prepared by the single source PGC system with introduction of O2 gas into the deposition chamber, fcc Co cores are covered with NaCl type CoO shells, showing marked enhancement of unidirectional and uniaxial magnetic anisotropy and a clear cross-over phenomenon in the magnetic relaxation from the high temperature thermal regime to the low temperarure quantum tunneling regime. In Co/Si cluster assemblies prepared by the double source PGC system, fcc Co cores are also covered with amorphous Si rich shells, showing rather small magnetic coercivity. Since Co/CoO and Co/Si core–shell clusters are stable in ambient atmosphere, they will be used as building blocks for novel nano-structure-controlled materials.
This review encompasses the fundamental aspects of electrophoretic deposition technique, factors influencing the deposition process, kinetic aspects, types of EPD, the driving forces, preparation of electrophoretic suspension, stability and control of suspension, mechanisms involved in EPD, multicomponent/composite deposition, drying of deposits obtained by EPD. Numerous applications including coatings, nanoscale assembly, micropatterned thin films, near shape ceramics and glasses, solid oxide fuel cells, laminated or graded materials, hybrid materials, infiltration in porous and woven fibre preforms for preparation of fibre reinforced ceramic matrix composites, etc. have been described. The use of mathematical modeling including kinetic equations for deposit formation and volumetric particle concentration in the suspension, together with brief description of discrete element modeling of EPD process is presented.
Spectroscopic ellipsometry is used to investigate optical properties of cobalt-implanted silica thin films. The films under investigation are 250 nm thick thermal SiO 2 layers on Si substrates implanted with Co + ions at energy of 160 keV and at fluences of 10 17 ions/cm 2 for different temperatures of substrate during implantation (77 and 295 K). Changes due to Co + implantation are clearly observed in the optical response of the films. Optical behaviours are furthermore different for the three implantation temperatures. To understand the optical responses of these layers, the ellipsometric experimental data are compared to different models including interference effects and metal inclusions effects into the dielectric layer. The simulated ellipsometric data are obtained by calculating the interferences of an inhomogeneous layer on a Si substrate. The material within this layer is considered as an effective medium which dielectric function is calculated using the Maxwell-Garnett effective medium approximation. We show that although the structures of these layers are very complicated because of ion-implantation mechanisms, quite simple models can provide relatively good agreement. The possibilities of ellipsometry for the study of the optical properties of such clusters-embedded films are discussed. We especially provide the evidence that ellipsometry can give interesting information about the optical properties of nanostructured layers. This is of special interest in the field of nanostructured layered systems where ellipsometry appears to be a suitable optical characterization technique.
The adsorption of bovine serum albumin (BSA) onto relatively hydrophobic TiO2 surfaces was studied by ellipsometry as a function of pH and BSA concentration. Titanium oxide layers were electrochemically grown on Ti disc electrodes. When fast attachment of BSA onto TiO2 takes place, the adsorption can be considered as occurring in two different steps. The first step is fast and is the result of the direct adsorption of the protein molecules that attach to the surface without changing their conformation. The second process is slow and lasts for several hours. In this process, the adsorbed amount remains constant, whereas the thickness of the layer increases and its refractive index decreases with time. The changes in this second step are due mainly to rearrangements in the adsorbed layer produced by variations in the conformation and structure of the adsorbed molecules. The main conformational changes take place in the direction normal to the surface because lateral molecule–molecule interactions impede significant lateral expansion. Adsorption from BSA solutions of low concentration does not appear to lead to significant reconformation of the protein layer. Comparison with adsorption on powdered TiO2 indicates that the adsorbed amount and the effective area occupied by an adsorbed BSA molecule can remain about constant even when strong surface reconformation takes place.
The thickness resolution and in situ advantage of ellipsometry make this optical technique particularly suitable for studies of thin organic layers of biological interest. Early ellipsometric studies in this area mainly provided thickness quantification, often expressed in terms of surface mass. However, today it is possible to perform monolayer spectroscopy, e.g. of a protein layer at a solid/liquid interface, and also to resolve details in the kinetics of layer formation. Furthermore, complicated microstructures, like porous silicon layers, can be modeled and protein adsorption can be monitored in such layers providing information about pore filling and penetration depths of protein molecules of different size and type. Quantification of adsorption and microstructural parameters of thin organic layers on planar surfaces and in porous layers is of high interest, especially in areas like biomaterials and surface-based biointeraction. Furthermore, by combining ellipsometric readout and biospecificity, possibilities to develop biosensor concepts are emerging. In this report we review the use of ellipsometry in various forms for studies of organic layers with special emphasis on biologically-related issues including in situ monitoring of protein adsorption on planar surfaces and in porous layers, protein monolayer spectroscopy and ellipsometric imaging for determination of thickness distributions. Included is also a discussion about recent developments of biosensor systems and possibilities for in situ monitoring of engineering of multilayer systems based on macromolecules.
An immunosensor for the detection of pathogens was developed using imaging ellipsometry (IE) as a detection method. Yersinia enterocolitica was selected as the target pathogen in this study. A gold surface deposited with a self-assembled layer of 11-mercaptoundecanoic acid (11-MUA) was used as a substrate. For the fabrication of the immunosensor, protein G spots were made on the substrate using an inkjet-type microarrayer, and monoclonal antibody (Mab) was adsorbed onto the protein G spots. Deposition of each layer onto the substrate was confirmed by the measurement of surface plasmon resonance. The ellipsometric image of the protein G spot and the Mab-adsorbed protein G spot were acquired using an off-null ellipsometry type of imaging ellipsometry system. By measuring the ellipsometric angles of the protein layers, the surface concentration of each protein layer was calculated. The change in the mean optical intensity of the protein spot to the various concentrations of Y.enterocolitica was estimated. The immunosensor using imaging ellipsometry could successfully detect Y. enterocolitica in concentrations varying from 10(3) to 10(7) cfu/mL. The proposed immunosensor system has the advantage of allowing label-free detection, high sensitivity, and operational simplicity.
The transient growth and thinning of the passive film on iron was investigated in ethylenediaminetetraacetic acid, disodium salt (EDTA) containing borate buffer solutions of pH 8.4 using real-time spectroscopic ellipsometry under potentiostatic control. EDTA effectively suppressed the formation of the outer layer of the passive film, thereby rendering the barrier layer amenable to direct examination. It was shown that the barrier layer growth was completed in about 10s upon potential stepping in the anodic direction. On the other hand, the thinning of the barrier oxide layer upon potential stepping in the cathodic direction occurred at a rate that was two orders in magnitude lower than the growth rate. The steady-state barrier layer thickness varied linearly with applied potential, whereas the steady-state current density did not depend on the formation potential. These spectroscopic ellipsometric measurements are qualitatively explained by the point defect model (PDM).
Spin-coated poly(o-methoxyaniline) (POMA) thin films on various substrates were investigated using spectroscopic ellipsometry (SE) in the 1.5–4.5 eV photon energy range. Spin-coating process parameters are reported (spin speed and concentration). Substrates with higher surface energy were found to increase polymer film thickness and decrease roughness. An optical model was developed using SE data along with complementary data from atomic force microscopy and UV–vis spectroscopy to obtain optical properties—refractive index n and extinction coefficient k for POMA. The model includes Lorentz oscillators for the POMA film and a Bruggeman effective medium approximation for roughness. In-plane film optical anisotropy was not observed, but a small out of plane anisotropy was detected for the polymer.
An in vitro study focusing on select osteoblast function as well as on resorption by osteoclasts were conducted. The osteoblast functions considered were adhesion, proliferation, and deposition of extracellular calcium. The cytocompatibility of Ca nanophase ceramics was investigated. This showed the potential of nanophase ceramics to serve as the next generation of orthopedic and dental implants.
We report on a complete characterization of the optical dispersion properties of conducting thin films of single-wall carbon nanotubes (SWCNTs). The films studied exhibit sheet resistances between 50 and 1000 Ω∕sq and optical transparencies between 65% and 95% on glass and quartz substrates. These films have the potential to replace transparent conducting oxides in applications such as photovoltaics and flat-panel displays; however, their optical properties are not sufficiently well understood. The SWCNT films are shown to be hole conductors, potentially enabling their use as hole-selective contacts and allowing alternative device designs. The fundamental optical, morphological, and electrical characteristics of the films are presented here, and a phenomenological optical model that accurately describes the optical behavior of the films is introduced. Particular attention is paid to ellipsometry measurements and thorough evaluation of the reflection and absorption spectra of the films.
This work compares the spreading and relaxation rates of albumin and fibrinogen, inferred from single-component and competitive adsorption kinetic experiments, on model surfaces of varying hydrophobicity. Kinetics from the single-component studies revealed a constant spreading rate, where the adsorbed protein footprint grew linearly in time for at least 15 min. This spreading rate increased with substrate hydrophobicity (ranging from 0.02 to 0.16 nm2/molecule/s for albumin and from 0.04 to 0.26 nm2/molecule/s for fibrinogen), resulting in a larger extent of footprint growth and a lower ultimate coverage on hydrophobic surfaces when compared with hydrophilic surfaces at the same adsorption conditions. Competitive adsorption studies were in qualitative agreement with the single-component experiments but were able to probe longer spreading time scales. Although spreading appeared to occur initially at a constant rate in the competitive experiments, after 2 h the spreading rate had slowed dramatically and the spreading process had begun to level off.
The generally positive correlation between the extent of protein adsorption and the hydrophobicity of the adsorbent surface suggests that the hydrophobic interaction is a major driving force for adsorption. The customary approach to evaluating the role of this force, comparison of protein adsorption to a spectrum of solid surfaces spanning a range of hydrophobicities, is limited by incomplete control over surface chemical variables other than the overall hydrophobicity. We evaluate the importance of the hydrophobic interaction in protein adsorption, while maintaining constant surface properties, by addition of alcohols known to modulate the strength of the hydrophobic interaction in aqueous solutions. The responses of adsorption isotherms, dynamics, and lateral mobility of adsorbed proteins to changes in solvent composition demonstrate the primary role of the hydrophobic interaction in the adsorption of ribonuclease A to polystyrene surfaces. Manipulating the magnitude of the hydrophobic interaction alters the adsorption mechanism through both fluid-phase transport and protein binding at the interface.
TEM studies have been performed on 50 nm thick monolayer films of sol-gel derived anatase titania (TiO2). It is found that a dense polycrystalline structure, having low porosity and consisting of nanocrystallites ranging from 5-20 nm in diameter, is the result of a sintering process involving temperature ramping up to 550 °C. Ohmic conduction is believed to be due to almost flat band conditions caused by partial depletion of carriers from the crystallites.
The method of spectroscopic ellipsometry has been applied to study in situ the adsorption
of bovine serum albumin (BSA). The porosity and amount of adsorbed BSA were
determined by fitting the ellipsometric data to the Bruggeman effective medium
approximation model. The presence of intermediate adsorbed layers of polyelectrolytes was
found to increase protein adsorption.
Porous silicon layers with a one-dimensional lateral gradient in pore size are prepared by electrochemical etching and characterized by spectroscopic ellipsometry in the visible to near-infrared region. The ellipsometer is equipped with a micro-spot option giving a lateral resolution of approximately 100 μm. By matching multiple-layer-model calculations to the laterally-resolved variable angle of incidence spectroscopic ellipsometry data, the thickness variation along the gradient as well as the in-depth porosity profile is mapped. Upon exposure to a protein solution, protein adsorption occurs on top of the porous silicon layer. At the high-porosity region of the gradient also penetration of protein molecules into the porous layer takes place. Ellipsometry data are recorded after protein exposure and variations of protein adsorption along the porous silicon gradient is modeled as well as the in-depth profile of protein penetration.
Film characterization based on variable-angle spectroscopic ellipsometry (VASE) is desirable in order to understand physical and optical characteristics of thin films. A number of TiO2 film samples were prepared by ion-assisted electron-beam evaporation with 200-nm nominal thickness, 2.0 Å/s deposition rate and 8 sccm oxygen flow rate. The samples were maintained at 250 °C during the deposition, and annealed in air atmosphere afterwards. As-deposited and annealed films were analyzed by VASE, spectrophotoscopy and X-ray diffractometry. From ellipsometry modeling process, the triple-layer physical model and the Cody–Lorentz dispersion model offer the best results. The as-deposited films are inhomogeneous, with luminous transmittance and band gap of 62.37% and 2.95 eV. The 300 °C and 500 °C are transition temperatures toward anatase and rutile phases, respectively. Increasing temperature results in an increase of refractive index, transmittance percentage and band gap energy. At 500 °C, the highest refractive index and band gap energy are obtained at 2.62 and 3.26 eV, respectively. The developed VASE-modeling process should be able to characterize other TiO2 films, using similar physical and optical modeling considerations.
Mixtures of sodium myristate (SM), of 1 or 2 mM, and bovine serum albumin (BSA), from 100 to 10 000 ppm, were studied at the air/water interface using tensiometry, ellipsometry, and infrared reflection-absorption spectroscopy (IRRAS). The equilibrium surface tension of the mixtures ranged from 23 to 40 mN/m, which are values between those of the pure SM (20–25 mN/m) and BSA (50 mN/m), showing that lower tensions are favored and suggesting that SM was primarily on the surface for the concentrations studied. For the mixtures containing 1000 ppm BSA or less, the ellipsometric angles were the same as those of SM alone with surface densities ranging from 3.2 to 4.5 mol/m2×10−6. For mixtures with 10 000 ppm BSA, larger surface densities were determined as a result of the presence of myristic acid from the protonation of SM due to the pH decrease caused by the BSA addition. IRRAS results are consistent with the ellipsometry results, showing that at the lower BSA concentrations only SM was present at the surface, and at the higher BSA concentrations SM was present along with myristic acid. The results demonstrate that at the concentrations studied, SM can exclude BSA completely from the air/water interface.
Under most conditions proteins show a strong tendency to adsorb at interfaces. The general principles underlying the interaction between proteins and solid surfaces in an aqueous environment are discussed. These principles are illustrated by experimental results obtained with well-defined systems. The approach is mainly based on thermodynamic arguments.
The application of ellipsometry of the study of the adsorption behavior of proteins and synthetic macromolecules at the air-water interface has been investigated. It is shown that for macromolecules the amount adsorbed per unit area, Γ, as determined by ellipsometry, only has a well-defined physical meaning if the refractive-index increment remains constant up to high concentrations present in the adsorbed layer. It has been found experimentally that this conditioned is fulfilled for proteins. The ellipsometric Γ values of some protein agree satisfactorily with those obtained by two independent techniques has been used to investigate the adsorption from solution of κ-casein, bovine serum albumin, and polyvinyl alcohol. For bovine serum albumin, Γ reaches a plateau value of 2.9 mg/m2 for concentrations ≥ 0.05 wt%. The thickness of the adsorbed molecules. For κ-casein, Γ steadily increases with increasing centration and multilayers are formed. The technique provides interesting information on conformational changes in adsorbed macromolecules, on the rate of the process, and on the conditions under which these occur.
Osteoblast, fibroblast, and endothelial cell adhesion on nanophase (that is, materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) was investigated using in vitro cellular models. Osteoblast adhesion was significantly (p < 0.01) greater after 4 h on nanophase alumina, titania, and HA than it was on conventional formulations of the same ceramics. In contrast, compared to conventional alumina, titania, and HA, after 4 h fibroblast adhesion was significantly (p < 0.01) less on nanophase ceramics. Examination of the underlying mechanism(s) of cell adhesion on nanophase ceramics revealed that these ceramics adsorbed significantly (p < 0.01) greater quantities of vitronectin, which, subsequently, may have contributed to the observed select enhanced adhesion of osteoblasts. Select enhanced osteoblast adhesion was independent of surface chemistry and material phase but was dependent on the surface topography (specifically on grain and pore size) of nanophase ceramics. The capability of synthesizing and processing nanomaterials with tailored (through, for example, specific grain and pore size) structures and topographies to control select subsequent cell functions provides the possibility of designing the novel proactive biomaterials (that is, materials that elicit specific, timely, and desirable responses from surrounding cells and tissues) necessary for improved implant efficacy. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 51, 475–483, 2000.
A proteolytic enzyme, α-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water-soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water-soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat-induced inactivation process is not identical with the heat-induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.
The electronic properties of titanium dioxide (TiO2) nanocrystalline films, which were prepared by dip coating from Degussa P25 photocatalyst aqueous suspension, have been investigated by surface photovoltage spectroscopy (SPS). As indicated by the positive contact potential difference (CPD) change in the sub-band-gap region, SPS shows that the molecularly adsorbed H2O in the freshly prepared P25 film creates an empty electron state, which is distributed within 0.79 eV below the conduction band edge, and acts as an electron trap and carrier recombination center. With film aging or under a drying atmosphere, the H2O-associated state diminishes, and the occupied electron state due to molecularly adsorbed oxygen, lying within 1.06 eV above the valence band edge, is identified by the reversed polarity of the CPD change in the sub-band-gap region. This information is important in developing a better understanding of real photocatalyst behavior. (c) 2007 American Institute of Physics.
The adsorption of bovine serum albumin (BSA) at the TiO2–aqueous NaCl interface was studied as a function of pH and electrolyte concentration using kinetic and steady-state measurements. The electrokinetic properties of BSA-covered TiO2particles and the acid–base character of the protein were monitored by electrophoretic mobilities and acid–base potentiometric titration, respectively. Some experiments were also outlined to analyze the different affinities of BSA monomers, dimers, and polymers for the surface. The adsorption process is fast; less than 10 min is enough to achieve steady-state conditions. All measured isotherms showed high initial slopes even under adverse electrostatic conditions, resulting in high-affinity isotherms. Adsorption plateau values (Γmax) had a great pH dependence; Γmax–pH curves reached a maximum at around the isoelectric point of BSA and showed a drastic decrease on the acid side. Both structural and electrostatic effects must be invoked to explain the diminution of adsorbed BSA on either side of the isoelectric point. Structural effects were related to the different conformational states that BSA molecules adopt with pH changes, whereas electrostatic effects were analyzed assuming that BSA molecules behave as soft particles. This reasoning also allows us to explain the independence of the Γmax–pH curves from electrolyte concentration.
Adsorption layers of two human plasma proteins (albumin-HSA, fibrinogen-FGN) on hydrophobic fluorohydrocarbon polymer (CHF) films were characterized in situ and ex situ by spectroscopic ellipsometry. The adsorbed layers were formed in phosphate buffered saline solutions of varied protein concentrations. Different optical five layer models were compared with respect to the evaluation of protein layers based on ellipsometric data. The Maxwell–Garnett effective medium approximation was concluded to be advantageous in providing a more realistic description of the layer structure as compared to the assumption of optical homogeneous layers. The applied models coincided with respect to the determination of the adsorbed amount of protein. Both the equilibrium surface concentration and the adsorption dynamics of HSA and FGN were found to depend on the solution protein concentration. The maximum adsorbed protein concentrations of the two proteins differed by ratios (HSA/FGN) of 1/4.5 in mass units and 1/0.83 in molar units (HSA: 1.0 mg m−2=15 nmol m−2; FGN: 4.5 mg m−2=12.5 nmol m−2). No reversibility of the adsorption of the two globulins on the polymer surface was observed upon dilution of the protein solutions with pure buffer. Coverage of the polymer surface with respect to the adsorbed molecules was achieved by different amounts of HSA or FGN depending on the transport conditions in the adsorption process. The observed variations of the surface area occupied by a given protein were apparently related to re-orientations and/or intramolecular changes of the adsorbed molecules. Structural parameters of the protein layers gained by the evaluation of the ellipsometric data support this conclusion.
Select functions of osteoblasts (bone-forming cells) on nanophase (materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) were investigated using in vitro cellular models. Compared to conventional ceramics, surface occupancy of osteoblast colonies was significantly less on all nanophase ceramics tested in the present study after 4 and 6 days of culture. Osteoblast proliferation was significantly greater on nanophase alumina, titania, and HA than on conventional formulations of the same ceramic after 3 and 5 days. More importantly, compared to conventional ceramics, synthesis of alkaline phosphatase and deposition of calcium-containing mineral was significantly greater by osteoblasts cultured on nanophase than on conventional ceramics after 21 and 28 days. The results of the present study provided the first evidence of enhanced long-term (on the order of days to weeks) functions of osteoblasts cultured on nanophase ceramics; in this manner, nanophase ceramics clearly represent a unique and promising class of orthopaedic/dental implant formulations with improved osseointegrative properties.
Total internal reflection ellipsometry (TIRE) in spectroscopic mode in the wavelength range 400–1200 nm is employed in situ at a solid/liquid interface for investigation of protein adsorption on thin semitransparent gold films. In this configuration, the surface plasmon resonance phenomenon gives a large enhancement of the thin film sensitivity. Adsorption of a monolayer of the protein ferritin is monitored kinetically in situ and results in a change in the ellipsometric parameter Δ of more than 90° compared to 3° in similar ellipsometric measurements on gold substrates. This large sensitivity demonstrates a potential for sensor applications. The ferritin layer optical function is modeled with a Cauchy dispersion model resulting in a layer thickness of 9.2 nm, in good agreement with the dimension of the ferritin molecules. A transition layer between the protein film and the gold layer is necessary to introduce in the model to account for interactions between the protein layer and the gold film. The large sensitivity of TIRE for thin layers opens up a pathway to analyze in detail the structure of thin protein layers provided that a further development of the experimental setup and the model for the protein layer is carried out.
Osteoblast adhesion on nanophase alumina (Al2O3) and titania (TiO2) was investigated in vitro. Osteoblast adhesion to nanophase alumina and titania in the absence of serum from Dulbecco’s modified Eagle medium (DMEM) was significantly (P<0.01) less than osteoblast adhesion to alumina and titania in the presence of serum. In the presence of 10% fetal bovine serum in DMEM osteoblast adhesion on nanophase alumina (23 nm grain size) and titania (32 nm grain size) was significantly (P<0.05) greater than on conventional alumina (177 nm grain size) and titania (2.12 μm grain size), respectively, after 1, 2, and 4 h. Further investigation of the dependence of osteoblast adhesion on alumina and titania grain size indicated the presence of a critical grain size for osteoblast adhesion between 49 and 67 nm for alumina and 32 and 56 nm for titania. The present study provides evidence of the ability of nanophase alumina and titania to simulate material characteristics (such as surface grain size) of physiological bone that enhance protein interactions (such as adsorption, configuration, bioactivity, etc.) and subsequent osteoblast adhesion.
The adsorption of proteins and particles onto surfaces carrying firmly adsorbed or covalently bound chains of poly(ethylene oxide) (PEO) is generally very low. This makes it of fundamental and practical interest to learn about the structure of PEO coatings and how PEO-coated surfaces interact with each other and, for example proteins. A prerequisite for such studies is, of course, that stable PEO-coated surfaces can be obtained. For this purpose we employed a two-step method to coat negatively charged surfaces, such as mica or silica, with PEO. In the first reaction step, cationic poly(ethylene imine) is adsorbed onto the negatively charged surface. In the next step, the adsorbed polyelectrolyte is reacted with a functionalized PEO chain. Both reaction steps were followed both by ellipsometry and by direct measurement of surface forces. From these measurements we obtained information concerning the adsorbed amount and the layer thickness, as well as the dependence on range and distance of the interaction between two PEO-coated surfaces.
Oxidative-acid-treated nanodiamonds exhibit high affinity for proteins, a property well suited for immobilization of enzymes for biotechnological application. Using lysozyme as an example, this work demonstrates that the enzyme can retain much of its activity after physical adsorption to the surfaces of 100-nm diamond crystallites. The activity relative to that of free lysozyme in solution is ∼ 60% at the maximum surface coverage of 50% and pH 5. While the enzymatic activity decreases as the surface coverage is lowered, it can be recovered by blocking the empty sites on the surface with supplementary proteins such as cytochrome c to create a more “crowded” environment. A relative activity up to 70% can be attained at a partial coverage of 20%.
The adsorption kinetics of three model proteins—human serum albumin, fibrinogen and hemoglobin—has been measured and compared using three different experimental techniques: optical waveguide lightmode spectroscopy (OWLS), ellipsometry (ELM) and quartz crystal microbalance (QCM-D). The studies were complemented by also monitoring the corresponding antibody interactions with the pre-adsorbed protein layer. All measurements were performed with identically prepared titanium oxide coated substrates. All three techniques are suitable to follow in-situ kinetics of protein–surface and protein–antibody interactions, and provide quantitative values of the adsorbed adlayer mass. The results have, however, different physical contents. The optical techniques OWLS and ELM provide in most cases consistent and comparable results, which can be straightforwardly converted to adsorbed protein molar (‘dry’) mass. QCM-D, on the other hand, produces measured values that are generally higher in terms of mass. This, in turn, provides valuable, complementary information in two respects: (i) the mass calculated from the resonance frequency shift includes both protein mass and water that binds or hydrodynamically couples to the protein adlayer; and (ii) analysis of the energy dissipation in the adlayer and its magnitude in relation to the frequency shift (c.f. adsorbed mass) provides insight about the mechanical/structural properties such as viscoelasticity.
Carbon-based thin films, such as amorphous carbon (a-C) and amorphous hydrogenated diamond-like carbon (a-C:H or DLC) are considered as excellent candidates for use as bicompatible coatings on biomedical implants. This arises not only due to their excellent properties, but also due to their chemical composition containing only carbon and hydrogen, which are biological compatibles. The aim of this work is the comparative study of the haemocompatibility of the carbon-based thin films developed by magnetron sputtering under various deposition conditions. Haemocompatibility is one of the most important properties, together with the tissue compatibility and corrosion and wear resistance, that determine the biocompatibility of the artificial implants.
In surface biology there are numerous studies carried out using single wavelength ellipsometry, especially in the area of macromolecular adsorption on solid surfaces. The results obtained contribute significantly to the understanding of the basic mechanisms of adsorption and surface dynamics of organic molecules, especially of proteins. An example of an area of great importance is biomaterials, where ellipsometry is used as a tool in the process of acquiring knowledge about the biological acceptance of new as well as currently used implant materials. In the area of affinity biosensors, ellipsometry has been suggested as a potential readout principle. Ellipsometry is also a tool in emerging technologies, such as surface molecular engineering with the aim to construct molecular superstructures with predesigned biological functions and to interface biology with electronics. However, in most cases when ellipsometry is applied in biology, it has been used for surface mass determination. The potential in using spectroscopic data for resolving microstructural and dynamic information has not been exploited fully. From the above perspective, this report reviews the use of spectroscopic ellipsometry for studies in surface biology and highlights the advantages it offers. Two main themes are developed. The first is spectroscopy on monolayers of macromolecules with emphasis on determination of their dielectric functions and microstructure. A specific example discussed is ferritin adsorption on gold. The results, including dynamics of both the surface mass and layer microstructure, indicate an adsorption model based on a two-state adsorption mechanism. The second theme is ellipsometrically based biosensor systems. The discussion covers aspects of what imaging ellipsometry can provide in this context and is exemplified by results from affinity biosensor and gas sensor systems.
The albumin adsorption on non-hydrogenated amorphous carbon films with different diamond-like character (i.e. sp3 content) is addressed. The films were produced by ion beam assisted deposition and by filtered cathodic vacuum arc deposition to obtain a wide range of sp3 contents. A combination of the spectroscopic ellipsometry, Raman spectroscopy and elastic recoil detection analysis was used for characterization of the films. It is shown that an increase of the deposition temperature leads to a decrease of the film band gap, density and a shift of the Raman G-band position. The wettability of the film is not influenced by its sp3 content. Albumin adsorption on the surface depends more on its wetting behavior than on the sp3 content. In addition, Ar ion treatment of the layers can be used to reduce the amount of adsorbed proteins.
The effects of pH and temperature on the equilibrium and the kinetics of bovine serum albumin (BSA) adsorption onto titanium dioxide (TiO2) were investigated. Adsorption rate and equilibrium experiments were carried out at pH conditions of 4, 5, 10 and in a temperature range of 20–40 °C. Adsorption rate was increased with decreasing pH and increasing temperature. The Langmuir isotherm constants corresponding to adsorption capacity, Q0, were 35.8, 40.0 and 42.6 mg/g for 20, 30 and 40 °C and pH 4; and 24.5, 29.1 and 33.4 mg/g for 20, 30, 40 °C and pH 5, respectively. The BSA adsorption capacities of TiO2 were higher for pH 4 at 40 °C. Adsorption of BSA on TiO2 at different pH and temperatures was found to be first order kinetics. At pH 4, k1 has values of 0.018 and 0.019 min−1 at 20 and 40 °C, respectively. For pH 5, k1 is 0.013 min−1 at 20 °C and 0.019 min−1 at 40 °C. The first order rate constants are higher at pH 4 than the values at pH 5. Higher values of the adsorption rate constants at pH 4, could be due to the change of pore structure of TiO2 or denaturation by conformational change of BSA with increasing pH.
Haemocompatibility is one of the most important properties, together with the tissue compatibility, corrosion and wear resistance that determine the biocompatibility of the artificial implants. Carbon-based thin films, such as amorphous carbon and amorphous hydrogenated diamond-like carbon (a-C:H or DLC) are considered as excellent candidates in order to be used as biocompatible coatings on biomedical implants. The aim of this work is to develop a methodology in order to study the protein adsorption phenomenon on thin films and to explore the optical properties of two basic blood plasma proteins, human serum albumin (HSA) and fibrinogen (Fib) and their adsorption mechanisms on amorphous hydrogenated carbon (a-C:H) thin films. Two techniques advantageous for the study of biological samples are used: Vis–UV spectroscopic ellipsometry (SE) and atomic force microscopy (AFM).a-C:H Films are grown with rf reactive magnetron sputtering. Static and real-time SE measurements are made. In the energy range of Vis–UV, proteins are almost transparent, while they present an absorption peak at higher energies. Different protein adsorption behaviour is observed on amorphous hydrogenated carbon films deposited under different conditions. This is probably due to their different surface structure, composition and topography of the surface and its interaction with the protein molecule. Adsorption phenomenon is studied through AFM technique as well. AFM results are in accordance with those derived by SE. The combination of the two techniques provides us a more accurate description of protein adsorption mechanisms.
Optical anisotropy in single-walled carbon nanotube thin film networks is reported. We obtain the real and imaginary parts of the in-(parallel) and out-of-plane (perpendicular) complex dielectric functions of the single-walled carbon nanotube (SWNT) thin films by combining transmission measurements at several incidence angles with spectroscopic ellipsometry data on different substrates. In sparse networks, the two components of the real part of the complex dielectric constant (epsilon1 parallel and epsilon1 perpendicular) were found to differ by 1.5 at 2.25 eV photon energy. The resulting angular dependence (from 0 to 70 degrees incidence angles) of transmittance is reflected in the conversion efficiency of organic solar cells utilizing SWNT thin films as the hole conducting electrodes. Our results indicate that, in addition to the transparency and sheet resistance, factors such as the optical anisotropy must be considered for optical devices incorporating SWNT networks.
A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, endosseous implants should result in formation of a characteristic interfacial layer and bone matrix with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the interface and of the effects biomaterials have on bone and bone cells is needed. Such knowledge is essential for developing strategies to optimally control osseointegration. This paper reviews current knowledge of the bone-biomaterial interface and methods being investigated for controlling it. Morphological studies have revealed the heterogeneity of the bone-implant interface. One feature often reported, regardless of implant material, is an afibrillar interfacial zone, comparable to cement lines and laminae limitantes at natural bone interfaces. These electron-dense interfacial layers are rich in noncollagenous proteins, such as osteopontin and bone sialoprotein. Several approaches, involving alteration of surface physicochemical, morphological, and/or biochemical properties, are being investigated in an effort to obtain a desirable bone-implant interface. Of particular interest are biochemical methods of surface modification, which immobilize molecules on biomaterials for the purpose of inducing specific cell and tissue responses or, in other words, to control the tissue-implant interface with biomolecules delivered directly to the interface. Although still in its infancy, early studies indicate the value of this methodology for controlling cell and matrix events at the bone-implant interface.
Chemical vapor deposition (CVD) is an important technique for surface modification of powders through either grafting or deposition of films and coatings. The efficiency of this complex process primarily depends on appropriate contact between the reactive gas phase and the solid particles to be treated. Based on this requirement, the first part of this review focuses on the ways to ensure such contact and particularly on the formation of fluidized beds. Combination of constraints due to both fluidization and chemical vapor deposition leads to the definition of different types of reactors as an alternative to classical fluidized beds, such as spouted beds, circulating beds operating in turbulent and fast-transport regimes or vibro-fluidized beds. They operate under thermal but also plasma activation of the reactive gas and their design mainly depends on the type of powders to be treated. Modeling of both reactors and operating conditions is a valuable tool for understanding and optimizing these complex processes and materials. In the second part of the review, the state of the art on materials produced by fluidized bed chemical vapor deposition is presented. Beyond pioneering applications in the nuclear power industry, application domains, such as heterogeneous catalysis, microelectronics, photovoltaics and protection against wear, oxidation and heat are potentially concerned by processes involving chemical vapor deposition on powders. Moreover, simple and reduced cost FBCVD processes where the material to coat is immersed in the FB, allow the production of coatings for metals with different wear, oxidation and corrosion resistance. Finally, large-scale production of advanced nanomaterials is a promising area for the future extension and development of this technique.
Protein adsorption at solid-liquid interfaces is critical to many applications, including biomaterials, protein microarrays and lab-on-a-chip devices. Despite this general interest, and a large amount of research in the last half a century, protein adsorption cannot be predicted with an engineering level, design-orientated accuracy. Here we describe a Biomolecular Adsorption Database (BAD), freely available online, which archives the published protein adsorption data. Piecewise linear regression with breakpoint applied to the data in the BAD suggests that the input variables to protein adsorption, i.e., protein concentration in solution; protein descriptors derived from primary structure (number of residues, global protein hydrophobicity and range of amino acid hydrophobicity, isoelectric point); surface descriptors (contact angle); and fluid environment descriptors (pH, ionic strength), correlate well with the output variable-the protein concentration on the surface. Furthermore, neural network analysis revealed that the size of the BAD makes it sufficiently representative, with a neural network-based predictive error of 5% or less. Interestingly, a consistently better fit is obtained if the BAD is divided in two separate sub-sets representing protein adsorption on hydrophilic and hydrophobic surfaces, respectively. Based on these findings, selected entries from the BAD have been used to construct neural network-based estimation routines, which predict the amount of adsorbed protein, the thickness of the adsorbed layer and the surface tension of the protein-covered surface. While the BAD is of general interest, the prediction of the thickness and the surface tension of the protein-covered layers are of particular relevance to the design of microfluidics devices.
We have investigated the interaction of d-amino acid oxidase (DAAO) with single-walled carbon nanotubes (CNT) by spectroscopic ellipsometry. Dynamic adsorption experiments were performed at different experimental conditions. In addition, the activity of the enzyme adsorbed at different conditions was studied. Our results indicate that DAAO can be adsorbed to CNT at different pH values and concentrations by a combination of hydrophobic and electrostatic interactions. Considering that the highest enzymatic activity was obtained by adsorbing the protein at pH 5.7 and 0.1 mg x mL(-1), our results indicate that DAAO can adopt multiple orientations on the surface, which are ultimately responsible for significant differences in catalytic activity.
Adsorption isotherms of four plasma proteins (fibrinogen, IgG, human serum albumin, and bovine serum albumin) using four different types of small particles as substrates (siliconized glass, Teflon, polyvinylchloride, and Nylon-6,6) are reported. The suspending liquid medium was phosphate-buffered saline, with a surface tension higher than that of any of the proteins. In keeping with the thermodynamic expectations for these systems, protein adsorption decreases for all solids in sequence from fibrinogen (the most hydrophobic) to IgG, human serum albumin, and bovine serum albumin (the most hydrophilic). Furthermore, the extent of protein adsorption also decreases from the low surface tension (hydrophobic) to the higher surface tension solids, again as expected on thermodynamic grounds. There is one minor yet interesting exception to the thermodynamic pattern: In spite of the slightly lower surface tension of siliconized glass, the extent of protein adsorption is slightly higher to Teflon than to siliconized glass. This result is attributed to the theoretically well known phenomenon of "screening."
The effect of ionic strength and ethylene glycol on the adsorption of bovine serum albumin (BSA) or lysozyme by a commercial aluminium hydroxide or aluminium phosphate adjuvant was studied at pH 7.4 and 25 degrees C. The adsorption of BSA by aluminium hydroxide adjuvant and lysozyme by aluminium phosphate adjuvant was found to be inversely related to ionic strength. This indicates that electrostatic attractive forces contribute to adsorption. The adsorption of lysozyme by aluminium phosphate adjuvant was reduced by the addition of ethylene glycol. However, no change in the adsorption of BSA by aluminium hydroxide adjuvant was noted when up to 40% ethylene glycol was present. This behaviour indicates that hydrophobic forces contribute to the adsorption of lysozyme but not of BSA. However, virtually no adsorption was observed when the protein and the adjuvant had the same surface charge. Thus, attractive forces may not be sufficient to produce adsorption of an antigen by an aluminium-containing adjuvant if electrostatic repulsive forces are present.
Appropriate surface modification has significantly improved the blood compatibility of polymeric biomaterials. This article reviews methods of surface modification with water-soluble polymers, such as polyethylene oxide (PEO), albumin, and heparin. PEO is a synthetic, neutral, water-soluble polymer, while albumin and heparin are a natural globular protein and an anionic polysaccharide, respectively. When grafted onto the surface, all three macromolecules share a common feature to reduce thrombogenicity of biomaterials. The reduced thrombogenicity is due to the unique hydrodynamic properties of the grafted macromolecules. In aqueous medium, surface-bound water-soluble polymers are expected to be highly flexible and extend into the bulk solution. Biomaterials grafted with either PEO, albumin, or heparin are able to resist plasma protein adsorption and platelet adhesion predominantly by a steric repulsion mechanism.
The interaction of proteins with surfaces is important in separation and purification procedures as well as in metabolism and its regulation. The degree of binding to a given surface in principle depends on the precise amino acid composition of the protein, although very little is presently known about the relationship between amino acid sequence and binding. Here we report accurate measurements of the kinetics of adsorption of two closely homologous serum albumins (human and bovine) to a hydrated metal oxide surface, using an accurate integrated optics technique. Marked differences between the two proteins are observed. The results are analyzed using a model involving two bound forms, reversible and irreversible. The two forms are identified as two orientations of the protein with respect to the surface which make differing numbers of hydrogen bonds to the surface. These numbers were estimated on the basis of the measured desorption rate constants. The interfacial binding energy was calculated from the quotient of the adsorption and desorption rate constants and compared with the value calculated from surface energy available data. Remarkably, substitution of phosphate buffer for HEPES buffer causes dramatic changes in the adsorption, abolishing the irreversible mode completely for human serum albumin.
A proteolytic enzyme, alpha-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water-soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water-soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat-induced inactivation process is not identical with the heat-induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.