Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

QCM-D sensitivity to protein adsorption reversibility

Using a quartz crystal microbalance with dissipative monitoring (QCM-D) we have determined the adsorption reversibility and viscoelastic properties of ribonuclease A adsorbed to hydrophobic self-assembled monolayers. Consistent with previous work with proteins unfolding on hydrophobic surfaces, high protein solution concentrations, reduced adsorption times, and low ammonium sulfate concentrations lead to increased adsorption reversibility. Measured rigidity of the protein layers normalized for adsorbed protein amounts, a quantity we term specific dissipation, correlated with adsorption reversibility of ribonuclease A. These results suggest that specific dissipation may be correlated with changes in structure of adsorbed proteins.     

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.     

Plasmid DNA adsorption on silica: kinetics and conformational changes in monovalent and divalent salts

A quartz crystal microbalance with dissipation (QCM-D) is used to determine the adsorption rate of a supercoiled plasmid DNA onto a quartz surface and the structure of the resulting adsorbed DNA layer. To better understand the DNA adsorption mechanisms and the adsorbed layer physicochemical properties, the QCM-D data are complemented by dynamic light scattering measurements of diffusion coefficients of the DNA molecules as a function of solution ionic composition. The data from simultaneous monitoring of variations in frequency and dissipation energy with the QCM-D suggest that the adsorbed DNA layer is more rigid in the presence of divalent (calcium) cations compared to monovalent (sodium) cations. Adsorption rates are significantly higher in the presence of calcium, attaining a transport-limited rate at about 1 mM Ca2+. Results further suggest that in low ionic strength solutions containing 1 mM Ca2+ and in moderately high ionic strength solutions containing 300 mM NaCl, plasmid DNA adsorption to negatively charged mineral surfaces is irreversible.     

Adsorption of zein on surfaces with controlled wettability and thermal stability of adsorbed zein films

Adsorption characteristics of zein protein on hydrophobic and hydrophilic surfaces have been investigated to understand the orientation changes associated with the protein structure on a surface. The protein is adsorbed by a self-assembly procedure on a monolayer-modified gold surface. It is observed that zein shows higher affinity toward hydrophilic than hydrophobic surfaces on the basis of the initial adsorption rate followed by quartz crystal microbalance studies. Reflection absorption infrared (RAIR) spectroscopic studies reveal the orientation changes associated with the adsorbed zein films. Upon adsorption, the protein is found to be denatured and the transformation of alpha-helix to beta-sheet form is inferred. This transformation is pronounced when the protein is adsorbed on hydrophobic surfaces as compared to hydrophilic surfaces. Electrochemical techniques (cyclic voltammetry and impedance techniques) are very useful in assessing the permeability of zein film. It is observed that the zein moieties adsorbed on hydrophilic surfaces are highly impermeable in nature and act as a barrier for small molecules. The topographical features of the deposits before and after adsorption are analyzed by atomic force microscopy. The protein adsorbed on hydrophilic surface shows rod- and disclike features that are likely to be the base units for the growth of cylindrical structures of zein. The thermal stability of the adsorbed zein film has been followed by variable-temperature RAIR measurements.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.     

Cholesterol biosensors prepared by layer-by-layer technique

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet–visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.     

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose,Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.     

Formation of polysaccharide gel layers in the presence of Ca2+ and K+ ions: measurements and mechanisms

Understanding the adsorption properties of polysaccharides in terms of substrate affinity, kinetics, and layer structure is of paramount importance in numerous industrial and natural systems. The structural growth of the layers of two model polysaccharides--sodium alginate and polygalacturonic acid (PGA)--was characterized by quartz crystal microbalance with dissipation and atomic force microscopy. Monitoring the variations in frequency and dissipation energy provides insights into both the average adsorbed mass and the viscoelastic properties of the adsorbed layer of polyelectrolytes along with the associated ions and water molecules. Both polysaccharides had similar adsorption patterns with increasing ionic strengths and showed significant complexation of calcium ions. In the presence of calcium, the alginate gel layer exhibited significant swelling with an increasing concentration of monovalent salt that the PGA gel layer did not manifest. Basing our discussion on the "egg-box model", we interpreted these different swelling behaviors as resulting from differences in the complexation modes of the two polysaccharides. The dimerization of the polymers by cross-linking and the weaker dimer-dimer associations play major roles in the sensitivity of the polysaccharide gel matrix to high salt concentration environments.     

Chemisorption of proteins and their thiol derivatives onto gold surfaces: characterization based on electrochemical nonlinearity

This study was conducted to monitor the electrochemical responses of two proteins (bovine serum albumin (BSA) and gelatin) and their thiol derivatives adsorbed onto gold (Au) electrodes, which were analyzed by a "nonlinear" impedance method. A sinusoidal voltage is applied to a protein-containing aqueous solution and the waveform of the output current is analyzed by fast Fourier transformation (FFT). The intensities of the higher harmonics in the FFT varied with the species of protein and their thiol derivatives, and with time. From the higher harmonics, voltage-dependent capacitance and conductance were quantitatively evaluated to differentiate the state of adsorbed protein. Adsorption and desorption characteristics of BSA and its thiol derivative on the Au surface were continuously measured by a quartz crystal microbalance (QCM) in situ. The microscopic state of thiol-derivatized BSA adsorbed onto the Au surface was imaged by atomic force microscopy (AFM). In general, thiol-derivatized proteins were tightly adsorbed on the Au surface and showed no desorption. The present electrochemical measurements clearly differentiated adsorption characteristics of physically adsorbed (physisorbed) and chemically adsorbed (chemisorbed) proteins on Au surfaces.     

Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei

Hydrophobins are surface-active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and as fusion proteins. Both hydrophobins were produced as C-terminal fusions to the core of the hydrolytic enzyme endoglucanase I from the same organism. It was shown that as a fusion partner, HFBI causes the fusion protein to efficiently immobilize to hydrophobic surfaces, such as silanized glass and Teflon. The properties of the surface-bound protein were analyzed by the enzymatic activity of the endoglucanase domain, by surface plasmon resonance (Biacore), and by a quartz crystal microbalance. We found that the HFBI fusion forms a tightly bound, rigid surface layer on a hydrophobic support. The HFBI domain also causes the fusion protein to polymerize in solution, possibly to a decamer. Although isolated HFBII binds efficiently to surfaces, it does not cause immobilization as a fusion partner, nor does it cause polymerization of the fusion protein in solution. The findings give new information on how hydrophobins function and how they can be used to immobilize fusion proteins.     

A stereocomplex platform efficiently detecting antigen-antibody interactions

Ultrathin poly(methyl methacrylate) (PMMA) stereocomplex films with macromolecularly double-stranded regular nanostructures were prepared by layer-by-layer assembly of isotactic and syndiotactic PMMAs on solid surfaces. Antibodies were immobilized through the Fc region-capturing protein A, which had been physically adsorbed on the complex film, and the binding of antigens to immobilized antibodies was quantitatively investigated by the quartz crystal microbalance technique. Greater amounts of protein A with native forms were adsorbed on the complex film than those on conventional single-component PMMA films. Antibodies with high target-binding activities were also immobilized on the complex film. A greater amount of antigens could be detected on the complex film. The activity of protein A was maintained on the complex for a long time even within a dried state. The mechanism for the preservation of protein native forms on the complex surface was speculated by analyzing the physical adsorption of proteins with various secondary structures. Stereocomplex films can be utilized as novel coating nanomaterials for efficiently detecting protein-protein interactions.     

Characterization of QCM sensor surfaces coated with molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolol in aqueous buffer.     

Crystal structures of hydrophobin HFBII in the presence of detergent implicate the formation of fibrils and monolayer films

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous self-assembly of hydrophobins leads to the formation of an amphiphilic layer that remarkably reduces the surface tension of water. We have determined by x-ray diffraction two new crystal structures of Trichoderma reesei hydrophobin HFBII in the presence of a detergent. The monoclinic crystal structure (2.2A resolution, R = 22, R(free) = 28) is composed of layers of hydrophobin molecules where the hydrophobic surface areas of the molecules are aligned within the layer. Viewed perpendicular to the aligned hydrophobic surface areas, the molecules in the layer pack together to form six-membered rings, thus leaving small pores in the layer. Similar packing has been observed in the atomic force microscopy images of the self-assembled layers of class II hydrophobin, indicating that the crystal structure resembles that of natural hydrophobin film. The orthorhombic crystal structure (1.0 A resolution, R = 13, R(free) = 15) is composed of fiber-like arrays of protein molecules. Rodlet structures have been observed on amphiphilic layers formed by class I hydrophobins; fibrils of class II hydrophobins appear by vigorous shaking. We propose that the structure of the fibrils and/or rodlets is similar to that observed in the crystal structure.     

Adsorption of arabinoxylan on cellulosic surfaces: influence of degree of substitution and substitution pattern on adsorption characteristics

This study presents results that show that the fine structure of arabinoxylan affects its interaction with cellulosic surfaces, an important understanding when designing and evaluating properties of xylan-cellulose-based materials. Arabinoxylan samples, with well-defined structures, were prepared from a wheat flour arabinoxylan with targeted enzymatic hydrolysis. Turbidity measurements and analyses using NMR diffusometry showed that the solubility and the hydrodynamic properties of arabinoxylan are determined not only by the degree of substitution but also by the substitution pattern. On the basis of results obtained from adsorption experiments on microcrystalline cellulose particles and on cellulosic model surfaces investigated with quartz crystal microbalance with dissipation monitoring, it was also found that arabinoxylan adsorbs irreversibly on cellulosic surfaces and that the adsorption characteristics, as well as the properties of the adsorbed layer, are controlled by the fine structure of the xylan molecule.     

Protein adsorption at polymer-grafted surfaces: comparison between a mixture of saliva proteins and some well-defined model proteins

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1 h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, beta-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface.     

Surface specific kinetics of lipid vesicle adsorption measured with a quartz crystal microbalance.

We have measured the kinetics of adsorption of small (12.5-nm radius) unilamellar vesicles onto SiO2, oxidized gold, and a self-assembled monolayer of methyl-terminated thiols, using a quartz crystal microbalance (QCM). Simultaneous measurements of the shift in resonant frequency and the change in energy dissipation as a function of time provide a simple way of characterizing the adsorption process. The measured parameters correspond, respectively, to adsorbed mass and to the mechanical properties of the adsorbed layer as it is formed. The adsorption kinetics are surface specific; different surfaces cause monolayer, bilayer, and intact vesicle adsorption. The formation of a lipid bilayer on SiO2 is a two-phase process in which adsorption of a layer of intact vesicles precedes the formation of the bilayer. This is, to our knowledge, the first direct evidence of intact vesicles as a precursor to bilayer formation on a planar substrate. On an oxidized gold surface, the vesicles adsorb intact. The intact adsorption of such small vesicles has not previously been demonstrated. Based on these results, we discuss the capacity of QCM measurements to provide information about the kinetics of formation and the properties of adsorbed layers.     

Role of structure and glycosylation of adsorbed protein films in biolubrication

Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy), we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation of adsorbed protein films, providing an important clue to design effective therapeutics to restore biolubrication in patients with insufficient biolubrication.     

The influence of cross-linking on protein-protein interactions in a marine adhesive: the case of two byssus plaque proteins from the blue mussel

The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was further investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO(4)), Cu(2+), or mushroom tyrosinase. With this coupling strategy it is shown that a requirement for attraction between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp-1 as well as for the subsequent binding of Mefp-2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.     

CMC-modified cellulose biointerface for antibody conjugation

In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulose-based biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir-Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.