Formation Of Protein Layers Research Articles

Near-Field Terahertz Morphological Reconstruction Nanoscopy for Subsurface Imaging of Protein Layers.

Protein layers formed on solid surfaces have important applications in various fields. High-resolution characterization of the morphological structures of protein forms in the process of developing protein layers has significant implications for the control of the layer's quality as well as for the evaluation of the layer's performance. However, it remains challenging to precisely characterize all possible morphological structures of protein in various forms, including individuals, networks, and layers involved in the formation of protein layers with currently available methods. Here, we report a terahertz (THz) morphological reconstruction nanoscopy (THz-MRN), which can reveal the nanoscale three-dimensional structural information on a protein sample from its THz near-field image by exploiting an extended finite dipole model for a thin sample. THz-MRN allows for both surface imaging and subsurface imaging with a vertical resolution of ∼0.5 nm, enabling the characterization of various forms of proteins at the single-molecule level. We demonstrate the imaging and morphological reconstruction of single immunoglobulin G (IgG) molecules, their networks, a monolayer, and a heterogeneous double layer comprising an IgG monolayer and a horseradish peroxidase-conjugated anti-IgG layer. The established THz-MRN presents a useful approach for the label-free and nondestructive study of the formation of protein layers.

A Facile Method to Synthesize 3D Pomegranate-like Polydopamine Microspheres.

Nanospheres have found versatile applications in the biomedical field; however, their possible harmful effects on immune and inflammatory systems are also a crucial concern. Inspired by a pomegranate structure, we demonstrated a novel structure for the nanostructured microspheres to overcome the drawbacks of nanospheres without compromising their merits. In this study, 3D pomegranate-like polydopamine microspheres (PDAMS) were synthesized by self-oxidative polymerization of dopamine hydrochloride. Herein, controlling the pH during polymerization led to synthesizing homogeneous agglomerated nano-sized spheres (400–2000 nm) and finally forming tunable and monodisperse micron-sized particles (21 µm) with uniform spherical shape porous microstructure. PDAMS interaction with the potential targets, Bone morphogenetic protein-2 (BMP2), Decorin, and Matrilin-1, was investigated via molecular calculations. Theoretical energy analysis revealed that PDAMS interaction with BMP2, Decorin, and Matrilin-1 is spontaneous, so that a protein layer formation on the PDAMS surface suggests application in bone and cartilage repair. It was also observed that PDAMS presented in-vitro degradation within 4 weeks. Here, disappearance of the UV-VIS spectrum peak at 280 nm is accompanied by the degradation of catechol groups. Pomegranate-like PDAMS support the biomimetic formation of hydroxyapatite-like layers, making them appropriate candidates for hard tissue applications. Herein, the appearance of peaks in XRD spectrum at 31.37, 39.57, 45.21, and 50.13° attributed to hydroxyapatite-like layers formation. All these results demonstrated that self-oxidative polymerization under a controllable pH can be a green and straightforward technique for preparing the pomegranate-like PDAMS and providing an innovative basis for further pre-clinical and clinical investigations.

Abstract P-35: Cryo-Electron Tomography of Protein Conjugated Upconverting Nanophosphors

Background: Over the past decades, significant advances have been made in the field of creating nanobioreagents for solving modern medicine problems (Grebenik et al., JBO, 2013). However, the problem of their low accumulation rate in pathological tissue in vivo experiments still remains. First of all, it is associated with the adsorption of blood proteins on the surface of nanobioreagents and the protein layer formation, which significantly changes the surface properties, which leads to their rapid excretion by the reticuloendothelial system. In particular, it is possible to reduce the blood plasma proteins adsorption and increase the time spent in the circulatory system by forming a coating of proteins. Methods: In situ cryоelectron tomography (Cryo-ET) is the only method that allows the experimental observation of protein structures on the nanoparticle’s surface in their natural functional state. The basic principle of the method is to obtain a series of projections of a vitrified sample thin lamella at different tilt angles related to an incident electron beam. Their further processing leads to obtaining the volumetric information about the structure of the sample. The use of a cryo-focused ion beam (Cryo-FIB) in specimen thinning makes it possible to carry out experiments with thin sections of cellular structures and observe the penetration of nanoparticles into the intracellular environment. Results: Upconverting nanophosphors (AN) were used as a nanoplatform for creating a protein coating. To create a protein coating on the AN surface, they were functionalized using an amphiphilic polymer containing carboxyl groups. Then, conjugation with protein molecules from the class of immunoglobulins was carried out by the method of carbodiimide activation. At each stage of synthesis and modification, AN solutions with different size distribution were vitrified for subsequent tomography. After a series of experiments to study the morphology of nanoparticles, an experiment on their successful absorption by cells of the cancer line A549 was carried out. Conclusion: Within this work, a series of in situ Cryo-ET methods were proposed and applied for structural characterization and visualization of the processes of synthesis, modification, and engulfment of nanoparticles into cellular systems. For the first time in its native form, the engulfment of ANF into the internal environment of the A549 cancer line cells was demonstrated.

Molecular Modeling of Protein Corona Formation and Its Interactions with Nanoparticles and Cell Membranes for Nanomedicine Applications.

The conformations and surface properties of nanoparticles have been modified to improve the efficiency of drug delivery. However, when nanoparticles flow through the bloodstream, they interact with various plasma proteins, leading to the formation of protein layers on the nanoparticle surface, called protein corona. Experiments have shown that protein corona modulates nanoparticle size, shape, and surface properties and, thus, influence the aggregation of nanoparticles and their interactions with cell membranes, which can increases or decreases the delivery efficiency. To complement these experimental findings and understand atomic-level phenomena that cannot be captured by experiments, molecular dynamics (MD) simulations have been performed for the past decade. Here, we aim to review the critical role of MD simulations to understand (1) the conformation, binding site, and strength of plasma proteins that are adsorbed onto nanoparticle surfaces, (2) the competitive adsorption and desorption of plasma proteins on nanoparticle surfaces, and (3) the interactions between protein-coated nanoparticles and cell membranes. MD simulations have successfully predicted the competitive binding and conformation of protein corona and its effect on the nanoparticle–nanoparticle and nanoparticle–membrane interactions. In particular, simulations have uncovered the mechanism regarding the competitive adsorption and desorption of plasma proteins, which helps to explain the Vroman effect. Overall, these findings indicate that simulations can now provide predications in excellent agreement with experimental observations as well as atomic-scale insights into protein corona formation and interactions.

Adsorption of Human Serum Albumin on Graphene Oxide: Implications for Protein Corona Formation and Conformation.

The influence of solution chemistry on the adsorption of human serum albumin (HSA) proteins on graphene oxide (GO) was investigated through batch adsorption experiments and the use of a quartz crystal microbalance with dissipation (QCM-D). The conformation of HSA layers on GO was also examined with the QCM-D. Our results show that an increase in ionic strength under neutral pH conditions resulted in stronger binding between HSA and GO, as well as more compact HSA layers on GO, emphasizing the key role of electrostatic interactions in controlling HSA-GO interactions. Calcium ions also facilitated HSA adsorption likely through charge neutralization and bridging effect. At physiological ionic strength conditions (150 mM), maximum HSA adsorption was observed at the isoelectric point of HSA (4.7). Under acidic conditions, the adsorption of HSA on GO led to the formation of protein layers with a high degree of fluidity due to the extended conformation of HSA. Finally, the attachment of GO to a supported lipid bilayer that was composed of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine, a model for cell membranes, was reduced in the presence of protein coronas. This reduction in GO attachment was influenced by the conformation of the protein coronas on GO.

Adsorption of proteins on gold nanoparticles: One or more layers?

Adsorption of proteins on nanoparticles is a complex and poorly studied process. The mechanisms of protein layer formation can fundamentally differ depending on the composition of the medium, the nanoparticles' structure, the protein's nature, and other factors. In particular, monolayer or multilayer immobilization may occur. In the present work, the composition of conjugates of bovine serum albumin and immunoglobulin G with gold nanoparticles obtained by the Turkevich-Frens method are analyzed. The composition was studied by protein fluorescence measurement for particles ranging in size from 20 to 48 nm, depending on the pH of the immobilization medium (from 4 to 5 to 8-10). It was found that a pH shift of the immobilization medium from acidic to alkaline values is accompanied by a change in the mechanism of protein adsorption on the gold surface. In acidic pH conditions (4-5), effective binding of bovine serum albumin and gold nanoparticles occurs throughout the entire range of studied protein concentrations. In alkaline pH conditions (8-10), however, effective binding occurs only at concentrations of >10 μg/mL. This effect is not observed for immunoglobulin G, which is efficiently adsorbed onto nanoparticles throughout the entire range of studied concentrations and pH values. For acidic pH values, the surface of the particles is saturated with the amount of bound proteins, which approximately corresponds to the amount the monolayer is filled. For neutral and alkaline pH values, saturation is not observed and the amount of adsorbed protein certainly exceeds the monolayer filling, leading to multilayer immobilization.

Molecular trajectories provide signatures of protein clustering and crowding at the oil/water interface.

Using high throughput single-molecule total internal reflection fluorescence microscopy (TIRFM), we have acquired molecular trajectories of bovine serum albumin (BSA) and hen egg white lysozyme during protein layer formation at the silicone oil-water interface. These trajectories were analyzed to determine the distribution of molecular diffusion coefficients, and for signatures of molecular crowding/caging, including subdiffusive motion and temporal anticorrelation of the instantaneous velocity vector. The evolution of these properties with aging time of the interface was compared with dynamic interfacial tension measurements. For both lysozyme and BSA, we observed an overall slowing of protein objects, the onset of both subdiffusive and anticorrelated motion (associated with crowding), and a decrease in the interfacial tension with aging time. For lysozyme, all of these phenomena occurred virtually simultaneously, consistent with a homogeneous model of layer formation that involves gradual crowding of weakly interacting proteins. For BSA, however, the slowing occurred first, followed by the signatures of crowding/caging, followed by a decrease in interfacial tension, consistent with a heterogeneous model of layer formation involving the formation of protein clusters. The application of microrheological methods to single molecule trajectories described here provides an unprecedented level of mechanistic interpretation of interfacial events that occurred over a wide range of interfacial protein coverage.

Synovial fluid protein adsorption on polymer-based artificial hip joint material investigated by MALDI-TOF mass spectrometry imaging

UHMW-PE (ultra-high molecular weight polyethylene), most frequently used material in acetabular cup replacement, is affected by the interaction with its surrounding synovial fluid. It is assumed that protein layer formation is of high importance for lubrication, however alters polymer characteristics. This study investigates in vitro protein adsorption on gamma-irradiated and Vitamin E doped UHMW-PE using synovia as modeling system. SDS-PAGE and MALDI-TOF mass spectrometry imaging showed adsorption of high abundance proteins in a mass range between 2 and 200kDa. Protein layer formation was observed on planar UHMW-PE material, whereas morphologically modified UHMW-PE regions were highly affected by protein aggregation.

Advances on Biomedical Titanium Surface Interactions

When used as an implanted material, titanium (Ti) surface controls the subsequent biological reactions and leads to tissue integration. Cells interactions with the surface, through a protein layer that is being formed from the moment Ti surface comes in contact with blood and its components, and indeed this protein layer formation, are regulated by surface properties such as topography, chemistry, charge and surface energy. Currently, the implementation of nanotechnology, in an attempt to support mimicking the natural features of extracellular matrix, has provided novel approaches for understanding and translating surface mechanisms whose modification and tailoring are expected to lead to enhanced cell activity and improved integration. Despite the fact that there has been extensive research on this subject, the sequence of interactions that take place instantly after the exposure of the implanted material into the biologic microenvironment are not well documented and need further investigation as well as the optimization of characteristics of Ti surface. This review, including theoretical and experimental studies, summarizes some of the latest advances on the Ti surface concerning modifications on surface properties and how these modifications affect biomolecular reactions and also attempts to present the initial adsorption mechanism of water and protein molecules to the surface.

In-depth characterization and computational 3D reconstruction of flagellar filament protein layer structure based on in situ spectroscopic ellipsometry measurements

In this study, we have reconstructed the statistical 3D structure of hundreds of nanometers thick surface immobilized flagellar filament protein layers in their native environment, in buffer solution. The protein deposition onto the surface activated Ta 2O 5 film was performed in a flow cell, and the immobilization process was followed by in situ spectroscopic ellipsometry. A multilayer optical model was developed, in that the protein layer was described by five effective medium sublayers. Applying this method, an in-depth analysis of the protein layer formation was performed. Based on the kinetics in the distribution of the surface mass density, the statistical properties of the filamentous film could be determined computationally as a function of the measurement time. It was also demonstrated that the 3D structure of the protein layer can be reconstructed based on the calculated in-depth mass density profile. The computational investigation revealed that the filaments can be classified into two individual groups in approximately equal ratio according to their orientation. In the first group the filaments are close to laying position, whereas in the second group they are in a standing position, resulting in a significantly denser sublayer close to the substrate than at a larger distance.

Dynamics of protein aggregation at the oil–water interface characterized by single molecule TIRF microscopy

Single molecule total internal reflectance fluorescence microscopy was used to observe the dynamic mechanisms of bovine serum albumin layer formation at the silicone oil–water interface. After an initial induction period, the mean diffusion coefficient of protein objects was observed to decrease as a function of exposure time. Simultaneously, the total adsorption rate was observed to decrease; both observations are consistent with protein layer formation. Importantly, the distribution of diffusion coefficients broadened systematically with exposure time, suggesting the presence of surface objects with a wide range of hydrodynamic radii ranging from a few nm (consistent with monomers) to ∼180 nm. The spatial distribution of the adsorption rate, which was initially uniform, gradually became inhomogeneous, also consistent with the existence of large aggregates. The kinetics of protein layer formation was dependent on the bulk protein concentration in the aqueous phase, and a kinetic population balance model was used to explain the nonlinear concentration dependence.

Brownian dynamics of colloidal probes during protein-layer formation at an oil–water interface

We employ the Brownian motion of spherical colloids to investigate the microrheology of layers of the protein β-lactoglobulin adsorbing at a decane–water interface. Using an experimental design in which the protein concentration in the aqueous subphase increases in a diffusion-limited manner, we obtain a time-resolved characterization of the interfacial rheology. After undergoing a period of increasing interfacial viscosity, the layers become viscoelastic with a rheology that indicates an approach to a sol–gel transition. This evolution, which depends on subphase pH, contrasts in several significant ways with layer formation by β-lactoglobulin at an air–water interface. Comparison of the diffusivity of colloids of two sizes as the interfacial viscosity increases illustrates the transformation in particle mobility from a three-dimensional character in which the surrounding bulk fluids dominate to a two-dimensional form dictated by the drag from the interfacial layer. This transformation is similarly revealed in the two-point correlations of the particle motion, which show a change in spatial dependence indicating a crossover from three-dimensional to two-dimensional hydrodynamics.

Whey protein fouling of large pore-size ceramic microfiltration membranes at small cross-flow velocity

Microfiltration of whey protein solutions by tubular ceramic membranes, under constant cross-flow and trans-membrane pressure, with periodic backwashing, is investigated using a fully instrumented pilot unit. Relatively large nominal membrane pore size (0.8 μm) insures very high protein transmission, which is desirable in applications such as microbial load reduction. In the first of a sequence of three filtration-backwashing cycles, irreversible and reversible fouling are identified, over the tested pressure range of 5–17.5 psi. Early in the first cycle, especially at the higher pressures, a pore constriction/blocking mechanism appears to be responsible for the irreversible fouling. In the other two cycles only the reversible fouling is significant, possibly due to some kind of protein layer formation on the membrane surface. The permeate flux level tends to increase by increasing trans-membrane pressure up to a near-optimum value of 10 psi, beyond which pressure has a negative effect. This interesting trend is attributed to the interplay of cross-flow velocity, which tends to reduce fouling by promoting re-suspension and breakage of colloidal protein agglomerates, with the trans-membrane pressure (and related flux) which leads to protein layer formation on the membrane and may impose compressive stresses, thereby increasing its resistance to permeation.

A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly(tert-butyl methacrylate)

The adsorption and covalent immobilization of human immunoglobulin (HIgG) and lysozyme (LYZ) on surface-modified poly(tert-butyl methacrylate) PtBMA films have been evaluated using x-ray photoelectron spectroscopy (XPS), ellipsometry and atomic force microscopy (AFM). Surface modification of PtBMA (UV irradiation) afforded surfaces suitable for both the physical and covalent attachment of proteins. The XPS and ellipsometry results showed good correlation in terms of variable-dense/thickness protein layer formation between physisorbed and covalently bound proteins. The amount of physisorbed HIgG ranged from 23.0 ± 1.6 ng mm2 on PtBMA, with corresponding film thicknesses 17.0 ± 1.2 nm. Covalent immobilization mediated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) coupling chemistry, afforded 5.6–8 ng mm2 of HIgG with a corresponding thickness of 5.9 ± 0.6 nm on PtBMA. The attachment of LYZ to modified PtBMA surface was similarly translated, where adsorption yielded up to 15 ng mm2, while covalent immobilization afforded typically 7–8 ng mm2. The thickness of the adsorbed LYZ protein layer was 11.0 ± 3.2 nm (PtBMA), suggesting the greater portion of protein adsorbs on surface-modified PtBMA.

Structure of Protein Layers during Competitive Adsorption

The formation of protein layers during competitive adsorption was studied with ellipsometry. Single, binary, and ternary protein solutions of human serum albumin (HSA), IgG, and fibrinogen (Fgn) were investigated at concentrations corresponding to blood plasma diluted 1/100. As a model surface, hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer modified silica was used. By using multiambient media measurements of the bare substrate prior to protein adsorption the adsorbed amount as well as the thickness and refractive index of the adsorbed protein layer could be followedin situand in real time. Under conditions used in these experiments neither IgG nor fibrinogen could fully displace serum albumin from the interface. The buildup of the protein layer occurred via different mechanisms for the different protein systems. Fgn adsorbed in a rather flat orientation at low adsorbed amounts, while at higher surface coverage the protein reoriented to a more upright orientation in order to accommodate more molecules in the adsorbed layer. IgG adsorption proceeded mainly end-on with little reorientation or conformational change on adsorption. Finally, for HSA an adsorbed layer thickness greater than the molecular dimensions was observed at high concentrations (although not at low), indicating that aggregates or multilayers formed on HMDSO plasma polymer surfaces. For all protein mixtures the adsorbed layer structure and buildup indicated that Fgn was the protein dominating the adsorbed layer, although HSA partially blocked the adsorption of this protein. At high surface concentration, HSA/Fgn mixtures show an abrupt change in both adsorbed layer thickness and refractive index suggesting, e.g., an interfacial phase transition of the mixed protein layer. A similar but less pronounced behavior was observed for HSA/IgG. For IgG/Fgn and HSA/IgG/Fgn a buildup of the adsorbed layer similar to that displayed by Fgn alone was observed.

Protein layer formation on artificial membranes.

Protein layer formation on artificial membranes.

Fourier-transform infrared versus conventional grating-infrared-attenuated total reflectance spectroscopic investigation of the adsorption of blood proteins on polymer surfaces

The advantage of Fourier-Transform-Infrared (FTIR) over conventional grating infrared spectrometry in the field of analysis of organic blood-contact surfaces has been demonstrated. It has been shown that the increase in signal-to-noise ratio by a factor of up to 100 (because of the multiplexing and throughput advantage of FTIR) and the perfect wavenumber calibration of the FTIR-system (Connes advantage) allows the study of adsorbed layers on polymer surfaces and also monitoring of the formation of protein layers on these surfaces directly from aqueous solutions. The results of clinical experiments with polyurethane and polyurethane-siloxane shunt samples are discussed.