Human immunoglobulin adsorption investigated by means of quartz crystal microbalance dissipation, atomic force microscopy, surface acoustic wave, and surface plasmon resonance techniques

Time-resolved adsorption behavior of a human immunoglobin G (hIgG) protein on a hydrophobized gold surface is investigated using multitechniques: quartz crystal microbalance/dissipation (QCM-D) technique; combined surface plasmon resonance (SPR) and Love mode surface acoustic wave (SAW) technique; combined QCM-D and atomic force microscopy (AFM) technique. The adsorbed hIgG forms interfacial structures varying in organization from a submonolayer to a multilayer. An "end-on" IgG orientation in the monolayer film, associated with the surface coverage results, does not corroborate with the effective protein thickness determined from SPR/SAW measurements. This inconsistence is interpreted by a deformation effect induced by conformation change. This conformation change is confirmed by QCM-D measurement. Combined SPR/SAW measurements suggest that the adsorbed protein barely contains water after extended contact with the hydrophobic surface. This limited interfacial hydration also contributed to a continuous conformation change in the adsorbed protein layer. The viscoelastic variation associated with interfacial conformation changes induces about 1.5 times overestimation of the mass uptake in the QCM-D measurements. The merit of combined multitechnique measurements is demonstrated.     

Methods for research on immune complement activation on modified sensor surfaces

We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a ---CH₃ end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.     

QCM-D on mica for parallel QCM-D-AFM studies

Quartz crystal microbalance with dissipation monitoring (QCM-D) has developed into a recognized method to study adsorption processes in liquid, such as the formation of supported lipid bilayers and protein adsorption. However, the large intrinsic roughness of currently used gold-coated or silica-coated QCM-D sensors limits parallel structural characterization by atomic force microscopy (AFM). We present a method for coating QCM-D sensors with thin mica sheets operating in liquid with high stability and sensitivity. We define criteria to objectively assess the reliability of the QCM-D measurements and demonstrate that the mica-coated sensors can be used to follow the formation of supported lipid membranes and subsequent protein adsorption. This method allows combining QCM-D and AFM investigations on identical supports, providing detailed physicochemical and structural characterization of model membranes.     

Minimal F-actin cytoskeletal system for planar supported phospholipid bilayers

Preferential binding of F-actin to lipid bilayers containing ponticulin was investigated on both planar supported bilayers and on a cholesterol-based tethering system. The transmembrane protein ponticulin in Dictyostelium discoideum is known to provide a direct link between the actin cytoskeleton and the cell membrane ( Wuestehube, L. J. ; Luna, E. J. J. Cell Biol. 1987, 105, 1741- 1751 ). Purification of ponticulin has allowed an in vitro model of the F-actin cytoskeletal scaffold system to be formed and investigated by AFM, epi-fluorescence microscopy, surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D). Single filament features of F-actin bound to the ponticulin containing lipid bilayer are shown by AFM to have a pitch of 37.3 +/- 1.1 nm and a filament height of 7.0 +/- 1.6 nm. The complementary techniques of QCM-D and SPR were used to obtain dissociation constants for the interaction of F-actin with ponticulin containing bilayers, giving 10.5 +/- 1.7 microM for a physisorbed bilayer and 10.8 +/- 3.6 microM for a tethered bilayer, respectively.     

Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates

Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins behaved similarly on addition of the alkaline-surfactant cleaning solution, in that platinum and gold exhibited an increase, while tungsten, titanium, and stainless steel showed a decrease in weight. According to dissipation measurements with the QCM-D, the adsorbed layer for platinum and gold was rigid, while that for the tungsten, titanium, and stainless steel was much more flexible. The removal efficiency of adsorbed-protein by alkaline solution of SDS depended on the water content of the adsorbed layers for W, Ti, and SS, while for Pt and Au, it depended on secondary structural content. When protein adsorption was high (Pt, Au), protein-protein interactions and protein-surface interactions were dominant and the removal of protein layers was limited. Water content of the adsorbed protein layer was the determining factor for how efficiently the layer was removed by alkaline SDS when protein adsorption was low. Hence, protein-protein and protein-surface interactions were minimal and protein structure was less perturbed in comparison with those for high protein adsorption. Secondary structural content determined the efficient removal of adsorbed protein for high adsorbed amount.     

Analysis of interpenetrating polymer networks via quartz crystal microbalance with dissipation monitoring

A quartz crystal microbalance with dissipation monitoring (QCM-D) was used to assess the physical properties of interpenetrating polymer networks (IPNs) through swelling experiments in ambient humidity and in phosphate-buffered saline (PBS), pH 7.4. The IPNs, based on acrylamide (AAm) and poly(ethylene glycol) (pEG), swell from thin, rigid films when dry (16.7 +/- 5.2 nm on Si/SiO(2)) to expanded, viscoelastic films when hydrated (107 +/- 24.2 nm on Si/SiO2). The dry IPNs could be analyzed using the Sauerbrey relationship, but for the hydrated films it was necessary to interpret QCM-D data with a Kelvin-Voigt viscoelastic model. A complex modulus |G| of 116 +/- 38.1 kPa for the swollen IPN surface on Si/SiO2 was defined by the model. The QCM-D was also employed to quantify the adsorption of human fibrinogen, a protein important in thrombus formation, onto the IPNs. Fibrinogen adsorption studies demonstrated the sensitivity of the QCM-D, as well as confirmed the nonfouling nature of the IPN surface, where less than 5 ng/cm2 of fibrinogen was adsorbed.     

A multitechnique study of liposome adsorption on Au and lipid bilayer formation on SiO2

We have used a new setup for parallel quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) measurements to measure the detailed kinetics of vesicle-to-bilayer transformation on SiO2 and vesicle adsorption on Au, respectively. The combination of SPR and QCM-D, complemented by atomic force microscopy measurements, has enabled a complete, time-resolved separation of vesicle and bilayer coverages, and thus, for the first time, allowed precise quantification of the critical surface coverage of vesicles needed for rupture. We furthermore demonstrate and quantify a previously undetected vesicle-size- and concentration-dependent loss of lipid material during the later stages of the process.     

Viscoelastic properties of fibrinogen adsorbed to the surface of biomaterials used in blood-contacting medical devices

The hemocompatibility of polymeric vascular implants is in part dependent on the propensity of fibrinogen to adsorb to the implant surface. Fibrinogen surface adsorption was measured in real time using a quartz crystal microbalance with dissipation monitoring (QCM-D). Six new, biodegradable tyrosine-derived polycarbonates were used as test surfaces. Stainless steel, poly(L-lactic acid), poly(D,L-lactide-co-glycolide), and poly(ethylene terephthalate) surfaces served as controls and provided a comparison of the test surfaces with those of commonly used biomaterials. Our study addressed the question regarding to which extent systematic variations in polymer structure can be used to optimize X-ray visibility and provide tunable degradation rates while generating protein-repellant surface properties that minimize fibrinogen adsorption. QCM-D revealed surface-dependent changes in fibrinogen layer thickness (2 to 37 nm), adsorbed wet mass (0.2 to 4.3 microg/cm2), and viscosity (0.001 to 0.005 kg/ms). While we did not find an overall correlation between surface air-water contact angle measurements and fibrinogen adsorption (R2 = 0.08), our data demonstrate that gradually increasing the poly(ethylene glycol) content within a subgroup of polymers having the same polymer backbone will lead to decreased fibrinogen adsorption. Within this subgroup of polymers, there was a strong correlation between decreasing air-water contact angles and decreasing fibrinogen adsorption (R2 = 0.95). We conclude that it is possible to minimize fibrinogen adsorption to tyrosine-derived polycarbonates while optimizing X-ray visibility and degradation rates. Some of the tyrosine-derived polycarbonates were identified as useful materials for the design of blood-contacting implants on the basis of their substantially lower levels of fibrinogen adsorption relative to the commonly used controls.     

Functionalization of solid surfaces with thermoresponsive protein-resistant films

Pulsed plasma polymerization of N-isopropylacrylamide leads to the deposition of thermoresponsive films. The reversible (switching) behavior of these poly(N-isopropylacrylamide) surfaces has been exemplified by screening the adsorption of fibrinogen and fluorescein isothiocyanate labeled bovine serum albumin proteins by surface plasmon resonance (SPR) and fluorescence microscopy at low and elevated temperatures.     

Bovine serum albumin adsorption on nano-rough platinum surfaces studied by QCM-D

The adsorption of bovine serum albumin (BSA) on platinum surfaces with a root-mean-square roughness ranging from 1.49nm to 4.62nm was investigated using quartz crystal microbalance with dissipation (QCM-D). Two different BSA concentrations, 50microg/ml and 1mg/ml, were used, and the adsorption studies were complemented by monitoring the antibody interaction with the adsorbed BSA layer. The adsorption process was significantly influenced by the surface nano-roughness, and it was observed that the surface mass density of the adsorbed BSA layer is enhanced in a non-trivial way with the surface roughness. From a close examination of the energy dissipation vs. frequency shift plot obtained by the QCM-D technique, it was additionally observed that the BSA adsorption on the roughest surface is subject to several distinct adsorption phases revealing the presence of structural changes facilitated by the nano-rough surface morphology during the adsorption process. These changes were in particular noticeable for the adsorption at the low (50microg/ml) BSA concentration. The results confirm that the nano-rough surface morphology has a significant influence on both the BSA mass uptake and the functionality of the resulting protein layer.     

A Au nanoparticle and polydopamine co-modified biosensor: A strategy for in situ and label-free surface plasmon resonance immunoassays

This article reports a surface plasmon resonance (SPR) strategy capable of label-free yet amplified in situ immunoassays for sensitive and specific detection of human IgG (hIgG), a serum marker that is important for the diagnosis of certain diseases. Primarily, a wavelength-modulated Kretschman configuration SPR analyzer was constructed, and Au film SPR biosensor chips were fabricated. Specifically, based on Au nanoparticles (AuNPs) adsorbed on the surface of the Au film, the AuNP/Au film was coated with polydopamine (PDA) to fix streptavidin (SA), and then the biotinylated antibodies were connected to the surface of the biosensor chip. The SPR analyzer was utilized for in situ real-time monitoring of hIgG. Due to the immunological recognition between the receptor and target, the surface plasmon waves produced by the attenuated total reflection were affected by the changes in the surface of the biosensor chip. The resonance wavelength (λ_R) of the output spectra gradually redshifted, and the redshift degrees were directly related to the target concentration. The biosensor can realize the in situ detection of hIgG, displaying satisfactory sensitivity, excellent specificity and stability. Briefly, by monitoring the shift in λ_R after specific binding, a new SPR immunoassay can be customized for label-free, in situ and amplified hIgG detection. The operating principle of this research could be extended as a common protocol for many other targets of interest.     

Quartz crystal microbalance with dissipation monitoring and surface plasmon resonance studies of carboxymethyl cellulose adsorption onto regenerated cellulose surfaces

Adsorption of anionic polyelectrolytes, sodium salts of carboxymethyl celluloses (CMCs) with different degrees of substitution (DS = 0.9 and 1.2), from aqueous electrolyte solutions onto regenerated cellulose surfaces was studied using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) experiments. The influence of both calcium chloride (CaCl(2)) and sodium chloride (NaCl) on CMC adsorption was examined. The QCM-D results demonstrated that CaCl(2) (divalent cation) caused significantly greater CMC adsorption onto regenerated cellulose surfaces than NaCl (monovalent cation) at the same ionic strength. The CMC layers adsorbed onto regenerated cellulose surfaces from CaCl(2) solutions exhibited greater stability upon exposure to flowing water than layers adsorbed from NaCl solutions. Both QCM-D and SPR results showed that CMC adsorption onto regenerated cellulose surfaces from CaCl(2) solutions increased with increasing CaCl(2) concentration up to the solubility limit (10 mM). Voigt-based viscoelastic modeling of the QCM-D data indicated that the CMC layers adsorbed onto regenerated cellulose surfaces had shear viscosities of η(f) ≈ 10(-3) N·s·m(-2) and elastic shear moduli of μ(f) ≈ 10(5) N·m(-2). Furthermore, the combination of SPR spectroscopy and QCM-D showed that the CMC layers contained 90-95% water. Adsorption isotherms for CMCs in CaCl(2) solutions were also obtained from QCM-D and were fit by Freundlich isotherms. This study demonstrated that CMC adsorption from CaCl(2) solutions is useful for the modification of cellulose surfaces.     

Modulation of proteins adsorption onto the surface of chitosan complexed with anionic copolymers. Real time analysis by surface plasmon resonance

The interpolyelectrolyte complex formation between chitosan and anionic polyacrylic derivatives, bearing sulfonic moieties, as well as the protein adsorption onto the chitosan/polyacrylic complexes were studied by surface plasmon resonance (SPR) optical biosensor. This unique technique allows a real time monitoring of different surface molecular interactions with very high sensitivity. The acrylic macromolecules are two families of copolymers of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and, respectively, 2-hydroxyethylmethacrylate (HEMA) and N,N'-dimethylacrylamide (DMAA). The complexation process was evaluated through the SPR measurements resulting from the flowing of polyacrylic aqueous solution over the sensor previously coated with chitosan. The SPR was able to differentiate strong ionic bonds from other weak and reversible interactions. By means of the coated sensors (uncomplexed and the whole series of complexed chitosan), SPR cold be used for a simple "in vitro" protein adsorption analysis, by flowing aqueous solutions of albumin and fibrinogen. While both proteins were adsorbed on the uncomplexed chitosan, the complexed coatings exhibited different and very promising behaviors. In particular, they showed no adsorption or only selective adsorption of albumin.     

QCM-D研究蛋白质与多糖的相互作用

应用具有耗散因子功能的石英晶体微天平（QCM-D）检测了多糖多层膜的构筑过程以及蛋白质在多层膜上的吸附行为。 研究结果表明,通过层层组装的方法,可以实现多糖卡拉胶多层膜的构筑,并且随着卡拉胶电荷密度的增加,其组装量减少。 研究了牛体血清蛋白(BSA)在卡拉胶上的吸附行为。 结果发现,BSA的吸附行为强烈地依赖于卡拉胶的电荷密度和基体的粗糙度。 通过QCM-D实时监测了BSA在卡拉胶上的动力学吸附曲线,同时根据ΔD值推测了BSA在不同的基底表面上所形成的结构及吸附行为。     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Adsorption-induced conformational changes of proteins onto ceramic particles: differential scanning calorimetry and FTIR analysis

Three model proteins, bovine serum albumin, hen's egg lysozyme and bovine serum fibrinogen, were adsorbed from aqueous solution onto finely dispersed ceramic particles, namely different kinds of alumina and hydroxyapatite particles. The influence of adsorption on protein secondary structure was investigated. The FTIR spectroscopic findings were compared with the results of DSC measurements. In almost all cases it was found that adsorption results in destabilisation and structural loss of the bound protein. A decrease in transition enthalpy is correlated with a loss in alpha-helical structure, which seems to be the most sensitive structure on adsorption-induced rearrangements. A total collapse of structure in the adsorbed proteins was not determined on any ceramic surface. Some residual structure is always retained. Structural changes in the D- or E-domains of fibrinogen could be independently observed by two different calorimetric signals. The two techniques applied in the present study -- micro-DSC and FTIR spectroscopy -- can be concluded to provide complementary information on adsorption-induced structural changes on both the molecular (thermal stability, overall structure) and the sub-molecular level (secondary structure).     

Viscoelastic modeling of highly hydrated laminin layers at homogeneous and nanostructured surfaces: quantification of protein layer properties using QCM-D and SPR

The adsorption of proteins at material surfaces is important in applications such as biomaterials, drug delivery, and diagnostics. The interaction of cells with artificial surfaces is mediated through adsorbed proteins, where the type of protein, amount, orientation, and conformation are of consequence for the cell response. Laminin, an important cell adhesive protein that is central in developmental biology, is studied by a combination of quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) to characterize the adsorption of laminin on surfaces of different surface chemistries. The combination of these two techniques allows for the determination of the thickness and effective density of the protein layer as well as the adsorbed mass and viscoelastic properties. We also evaluate the capacity of QCM-D to be used as a quantitative technique on a nanostructured surface, where protein is adsorbed specifically in a nanopattern exploiting PLL-g-PEG as a protein-resistant background. We show that laminin forms a highly hydrated protein layer with different characteristics depending on the underlying substrate. Using a combination of QCM-D and atomic force microscopy (AFM) data from nanostructured surfaces, we model laminin and antibody binding to nanometer-scale patches. A higher amount of laminin was found to adsorb in a thicker layer of a lower effective density in nanopatches compared to equivalent homogeneous surfaces. These results suggest that modeling of QCM-D data of soft viscoelastic layers arranged in nanopatterns may be applied where an independent measure of the "dry" mass is known.     

Vesicle adsorption on mesoporous silica and titania

Lipid bilayer formation via vesicle fusion on mesoporous silica and mesoporous titania was investigated using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescent recovery after photobleaching (FRAP). Results showed that lipid bilayers were formed on mesoporous silica and that intact vesicle adsorption was obtained on mesoporous titania. From the FRAP results, it could be concluded that the lipid bilayer was fluid; however, it had a smaller diffusivity constant compared to bilayers supported on a nonporous silica.     

Composition and asymmetry in supported membranes formed by vesicle fusion

The structure and formation of supported membranes at silica surfaces by vesicle fusion was investigated by neutron reflectivity and quartz crystal microbalance (QCM-D) measurements. The structure of equimolar phospholipid mixtures of DLPC-DPPC, DMPC-DPPC, and DOPC-DPPC depends intricately on the vesicle deposition conditions. The supported bilayer membranes exhibit varying degrees of compositional asymmetry between the monolayer leaflets, which can be modified by the deposition temperature as well as the salt concentration of the vesicle solution. The total lipid composition of the supported bilayers differs from the composition of the vesicles in solution, and the monolayer proximal to the silica surface is always enriched in DPPC compared to the distal monolayer. The results, which show unambiguougsly that some exchange and rearrangement of lipids occur during vesicle deposition, can be rationalized by considering the effects of salt screening and temperature on the rates of lipid exchange, rearrangement, and vesicle adsorption, but there is also an intricate dependence on the lipid-lipid interactions. Thus, although both symmetric and asymmetric supported bilayers can be prepared from vesicles, the optimal conditions are sensitive to the lipid composition of the system.     

Protein interactions with model chromatographic stationary phases constructed using self-assembled monolayers

Model surfaces representative of chromatographic stationary phases were developed by immobilising an homologous series (C2-C18) of n-alkylthiols, mixed monolayers of C4/C18 and thioalkanes with alcohol, carboxylic acid, amino and sulphonic acid terminal groups onto a flat, silver-coated glass surface using self-assembled monolayer (SAM) chemistry. The processes of adsorption and desorption of serum albumins onto the monolayer surfaces was monitored in real-time using surface plasmon resonance (SPR). Alkyl-terminated SAMs all showed a strong adsorption of bovine serum albumin which was largely independent of alkyl chain length, the ratio of mixed C4/C18 SAMs or the solution pH/ionic strength. The adsorption of human serum albumin to carboxylic and amine terminated SAMs was shown to be predominantly via non-electrostatic interactions (hydrophobic or hydrogen bonding). However, sulphonic acid terminated SAMs showed almost exclusively electrostatic interactions with human serum albumin. This preliminary work using self-assembled monolayer chemistry confirms the usefulness of well characterised SAMs surfaces for investigating protein adsorption and desorption onto/from model chromatography surfaces and gives some guidance for selecting appropriate functionalities to develop better surfaces for chromatography and electrophoresis.