Protein adsorption on supported phospholipid bilayers

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO(2) surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed.     

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.     

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.     

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.     

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.     

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.     

Effects of copolymer concentration and chain length on the pH-responsive behavior of diblock copolymer micellar films

The pH-responsive behavior of adsorbed diblock copolymer films of PDMA-PDEA (poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diethylamino)ethyl methacrylate)) on silica has been characterized using a quartz crystal microbalance with dissipation monitoring (QCM-D), an optical reflectometer (OR) and an atomic force microscope (AFM). The copolymer was adsorbed at pH 9 from various copolymer concentrations; QCM-D measurements indicate that the level of desorption when rinsed at pH 9 depends on the initial copolymer concentration. The adsorbed films produced at pH 9 generally have low charge densities; adjusting the solution pH to 4 results in a significant protonation of the constituent copolymers and a related interfacial structural change for the copolymer film. OR studies show no significant change during pH cycling, while QCM-D measurements indicate that the adsorbed mass and dissipation alter dramatically in response to the solution pH. The difference between the QCM-D adsorbed masses and dissipation values at pH 4 and 9 were found to be dependent on the initial copolymer concentration. This is due to differences in the initial conformations within the adsorbed copolymer layers at pH 9. The effect of the PDMA chain length on the pH-responsive behavior has also been studied; both the QCM-D adsorbed mass and dissipation of PDMA54-PDEA24 (shorter PDMA block) at pH 4 and 9 were observed to be greater than those of PDMA9X-PDEA2Y (longer PDMA block). This suggests that the normal extension of the adsorbed PDMA54-PDEA24 copolymer films is more significant than that of the PDMA9X-PDEA2Y films on silica.     

Fibronectin adsorption on gold, Ti-, and Ta-oxide investigated by QCM-D and RSA modelling

The adsorption of fibronectin on gold, Ti-, and Ta-oxide surfaces is investigated by means of the quartz crystal microbalance with dissipation (QCM-D) technique. The surface chemistry (gold, Ti-, and Ta-oxide) is found to influence the frequency shift observed during adsorption of the fibronectin layer with the magnitude being Delta f Au>Delta f Ti-oxide approximately Delta f Ta-oxide. Corresponding variations in the dissipation change normalised to frequency change (Delta D/Delta f) for the layer are observed. The QCM-D data are further analyzed by the random sequential adsorption (RSA) model, and adsorption rate parameter ka and footprint (a) determined, which supported the trend seen in the Delta f and Delta D/Delta f values. The value of ka found by the RSA modelling of the QCM-D resonance frequency data is found to match the ratio between the mass measured by QCM-D and the mass reported by optical techniques in literature. We conclude that comparison of the adsorption rate parameter (ka) obtained by RSA modelling of the QCM-D data with ka values obtained from RSA modelling of data obtained using optical techniques can be a route to determine the degree of hydration of the adsorbed protein layer.     

Adsorption of plasmid DNA to a natural organic matter-coated silica surface: kinetics, conformation, and reversibility

A quartz crystal microbalance with dissipation (QCM-D) has been used to determine the adsorption rate of ampicillin-resistant linear and supercoiled plasmid DNA onto a silica surface coated with natural organic matter (NOM). The structure of the resulting adsorbed DNA layer was determined by analyzing the viscoelastic properties of the adsorbed DNA layers as they formed and were then exposed to solutions of different ionic composition. The QCM-D data were complemented by dynamic light scattering measurements of diffusion coefficients of the DNA molecules as a function of solution ionic composition. The obtained results suggest that electrostatic interactions control the adsorption and structural changes of the adsorbed plasmid DNA on the NOM-coated silica surface. The adsorption of DNA molecules to the NOM layer took place at moderately high monovalent (sodium) electrolyte concentrations. A sharp decrease in solution ionic strength did not result in the release of the adsorbed DNA, indicating that DNA adsorption on the NOM-coated silica surface is irreversible under the studied solution conditions. However, the decrease in electrolyte concentration influenced the structure of the adsorbed layer, causing the adsorbed DNA to adopt a less compact conformation. The linear and supercoiled DNA had similar adsorption rates, but the linear DNA formed a thicker and less compact adsorbed layer than the supercoiled DNA.     

耗散型石英晶体微天平在生物医用高分子材料中的应用

石英晶体微天平(QCM)是一种基于石英晶体压电效应的分析检测技术,可实时在线提供石英晶体表面吸附层质量、厚度、粘弹性等信息,由此获得表面分子相互作用关系。 耗散型石英晶体微天平(QCM-D)因其独特的对粘弹性的解析,使其在高分子材料中的应用迅速发展,尤其是生物医用高分子材料领域,已用来评价生物医用高分子材料的表界面相互作用,力学和生物相容性等。 本文简单介绍了耗散型石英晶体微天平的基本原理及理论模型,重点综述了近几年QCM-D在高分子链构象、蛋白质吸附、生物大分子相互作用、药物释放以及水凝胶中的应用,并且展望了QCM-D的未来发展趋势。     

A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30–1.35 mg/m² at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.     

The characterization of biomolecular secondary structures by surface plasmon resonance

Recently there has been considerable interest in using surface plasmon resonance (SPR) for the measurement of conformational changes of immobilized biomolecules that are induced by an exogenous analyte. While a number of studies have shown the analytical utility of such measurements, there has been no report which characterizes the specific secondary structure that actuates the change in SPR signal. The use of SPR to indicate the type of secondary structure present in two immobilized polypeptides, poly-L-lysine (PL) and poly-L-glutamic acid (PGA), and a globular protein, concanavalin A (Con A) is described in this report. The PL, PGA and Con A were modified with N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP) to introduce disulfide groups to facilitate the attachment onto gold-coated surfaces via self-assembly. Ethanol and 2,2,2-trifluoroethanol (TFE) were used to induce changes in the secondary structure of the immobilized polypeptides and the protein respectively. Using both circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, it has been demonstrated that it is possible to correlate the signal changes observed in SPR to the secondary conformation of the biomolecule. Both CD and FTIR showed that a decrease in SPR signal corresponded to a high content of beta, turn or unordered structures while an increase corresponded to a high alpha-helical content. The sensitivity of the SPR technique is comparable to that obtained in solution with CD and FTIR spectroscopies. These results are the first demonstration that SPR can be used to characterize secondary structures. There is potential, therefore, for SPR to be used as a technique to study secondary conformational changes of immobilized polypeptides and proteins.     

Quantitative analysis of protein adsorption via atomic force microscopy and surface plasmon resonance

Surface properties have a significant influence on the performance of biomedical devices. The influence of surface chemistry on the amount and distribution of adsorbed proteins has been evaluated by a combination of atomic force microscopy (AFM) and surface plasmon resonance (SPR). Adsorption of albumin, fibrinogen, and fibronectin was analyzed under static and dynamic conditions, employing self-assembled monolayers (SAMs) as model surfaces. AFM was performed in tapping mode with antibody-modified tips. Phase-contrast images showed protein distribution on SAMs and phase-shift entity provided information on protein conformation. SPR analysis revealed substrate-specific dynamics in each system investigated. When multi-protein solutions and diluted human plasma interacted with SAMs, SPR data suggested that surface chemistry governs the equilibrium composition of the protein layer.     

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.     

Complexation chemistry for tuning release from polymer coatings

The strategy of metal ion complexation is employed to design a delivery system for an antifouling agent (AFA) in marine paints. A poly(1-vinylimidazole-co-methyl methacrylate) copolymer (PVM), together with Cu2+ or Zn2+ formed a PVM-M2+ complex. The AFA, Medetomidine, was then coordinated into the complex. The coordination strength was investigated in solution by 1H NMR and on solid surfaces by using the Quartz Crystal Microbalance with Dissipation monitoring technique (QCM-D) and Surface Plasmon Resonance (SPR). From the 1H NMR experiments strong interactions were observed between Cu2+ and the PVM-polymer and between Medetomidine and the PVM-Cu2+ complex. From the QCM-D and SPR measurements it was shown that Cu2+, compared to Zn2+, exhibited a larger affinity for the PVM-copolymer surface that resulted in higher degree of swelling of the polymer film. Large amounts of Medetomidine were adsorbed to the PVM-Cu2+ complex resulting in low desorption rates. However, the adsorbed amount of Medetomidine was lower to the Zn2+ doped polymer and a higher desorption rate was observed. These results indicate the possibility of tuning the release of Medetomidine by altering the coordinating metal ion, which may prove to be favorable in a paint formulation.     

QCM and EC-SPR Studies of Cytochrome cSelf-assembled on Au Electrode and Enhancement of SPR Signal by Au Nanoparticles

Quartz crystal microbalance(QCM) and cyclic voltammetry(CV) were used to characterize the monolayer of cytochrome c(Cyt c), which was adsorbed on gold film modified with alkanethiol mixed monolayer. A direct comparison of protein surface coverages calculated from QCM and cyclic voltammetric measurements illustrates that the ratio of the electroactive Cyt c to the total surface-confined Cyt cis 34%, which suggests that the orientation is a main factor affecting the electroactivity of Cyt c. Moreover, surface plasmon resonance(SPR) measurement combined with CV “in situ” was used to investigate the conformational change of Cyt c in the redox process. Besides, Au nanoparticles(Au NPs) were adsorbed on the surface of Cyt c. The result indicates that Au NPs promote electron transfer between Cyt c and the gold electrode, and SPR result suggests Au NPs enhance SPR signal.     

Interfacial phase transition of an environmentally responsive elastin biopolymer adsorbed on functionalized gold nanoparticles studied by colloidal surface plasmon resonance

The change in optical properties of colloidal gold upon aggregation has been used to develop an experimentally convenient colorimetric method to study the interfacial phase transition of an elastin-like polypeptide (ELP), a thermally responsive biopolymer. Gold nanoparticles, functionalized with a self-assembled monolayer (SAM) of mercaptoundecanoic acid onto which an ELP was adsorbed, exhibit a characteristic red color due to the surface plasmon resonance (SPR) of individual colloids. Raising the solution temperature from 10 degrees C to 40 degrees C thermally triggered the hydrophilic-to-hydrophobic phase transition of the adsorbed ELP resulting in formation of large aggregates due to interparticle hydrophobic interaction. Formation of large aggregates caused a change in color of the colloidal suspension from red to violet due to coupling of surface plasmons in aggregated colloids. The surface phase transition of the ELP was reversible, as seen from the reversible change in color upon cooling the suspension to 10 degrees C. The formation of colloidal aggregates due to the interfacial phase transition of adsorbed ELP was independently verified by dynamic light scattering of ELP-modified gold colloids as a function of temperature. Colloidal SPR provides a simple and convenient colorimetric method to study the influence of the solution environment, interfacial properties, and grafting method on the transition properties of ELPs and other environmentally responsive polymers at the solid-water interface.     

Surface electric field manipulation of the adsorption kinetics and biocatalytic properties of cytochrome c on a 3D macroporous Au electrode

Upon adsorbing on a solid-state substrate, water-soluble proteins are prone to denaturation and deterioration of their functions due to the conformation change. The surface electric field of a conductive substrate is one of the important factors that influence the character of adsorbed proteins. In this work, a 3D macroporous gold electrode has been prepared and served as the working electrode to study the influence of surface electric field on the adsorption kinetics and conformation of the adsorbed cytochrome c (cyt-c) with the help of electrochemical, in situ electrochemical IR spectroscopic, atomic force microscopic, and contact angle measurements. The external electric field creates excess surface charge which can manipulate the adsorption rate of proteins on the substrate by the enhanced electrostatic interactions between the electrode and protein patches by coupling with complementary charges. The amount of immobilized cyt-c with electrochemical activity on the 3D macroporous gold electrode showed a minimum at potential of zero charge (PZC) and it increased with increasing net excess surface charge. Higher electric field could influence the conformation and the corresponding properties such as direct electrochemistry, bioactivity, and surface character of the adsorbed cyt-c molecules. However, high external electric field leads to damage of the protein secondary structure. This study provides fundamentals for the fabrication of biomolecular devices, biosensors, and biofuel cells through electrostatic interactions. Figure Two cases are illustrated for the protein immobilized on electrode surfaces: a retention of protein structure under moderate excess surface charge, b denaturation and conformation change of proteins adsorbed at high excess surface charge, e.g., due to the higher external electric field.     

The effect of surface coverage on conformation changes of bovine serum albumin molecules at the air-solution interface detected by sum frequency generation vibrational spectroscopy

The air-BSA solution interface has been investigated by various techniques for years. From these studies we know that BSA molecules segregate at the BSA solution-air interface, and the surface coverage increases with the increase of the bulk solution concentration. However, questions still remain as to whether the protein changes conformation, orientation, or a combination of the two upon adsorption. In this paper, by using sum frequency generation (SFG) vibrational spectroscopy we found that the conformation of interfacial BSA molecules changes dramatically at the solution-air interface, compared to that of the native BSA in solution. The hydrophobic methyl groups of BSA molecules at this interface tend to align along the surface normal. The degree of such conformational changes of surface BSA molecules depend on the surface coverage, indicating that the protein-protein interaction plays a very important role in determining the conformation of interfacial protein molecules. At very low surface concentration, the adsorbed BSA molecules unfold substantially. Our results can provide a molecular interpretation of results obtained from other studies such as protein layer thickness and surface tension measurements of protein solution.     

Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.