Pre-adsorbed Protein Research Articles

Surface modification by pre-adsorption of proteins and polypeptides on Ti substrate with controlled hydrophilicity to improve biocompatibility

The water contact angle (WCA) value of the Ti substrate surfaces was successfully controlled to high hydrophilicity (≤ 20 deg.) by the treatment of UV light irradiation, atmospheric plasma or hydrothermal treatment, and stored in different environments to control the variation in WCA over time. Proteins with various molecular mass were used to investigate the protein adsorption on the Ti substrate controlled WCA. ATR-FTIR was employed to quantify the protein adsorption amount. It was found that all proteins had relatively higher adsorption amount on superhydrophilic surface. The adsorption amount of proteins was similar under the same WCA value. PEGs, as organic molecules without peptide bonds, was unable to be adsorbed on Ti with WCA < 60 deg., while can be adsorbed only on the hydrophobic surfaces with WCA > 90 deg. In particular, we have carried out in vivo tests upon the protein pre-adsorbed Ti specimens. The in vivo tests indicated that the Ti substrate which pre-adsorbed proteins with high molecular mass had much higher osteoconductivity than that without protein adsorption. On the other hand, the PEGs adsorbed substrates showed no effect on osteoconductivity enhancement. This was due to its lack of peptide bonds. It was considered that implants adsorbed polypeptides can greatly enhance the osseointegration, regardless the mass and type of molecules. In general, the adsorption of protein and polypeptide containing amino acid is an effective way to improve osseointegration, before implantation into body environment. It provides crucial guidance to the biocompatibility improvement of the metallic bone implant materials.

Dynamic photoelectrical regulation of ECM protein and cellular behaviors.

Dynamic regulation of cell-extracellular matrix (ECM)-material interactions is crucial for various biomedical applications. In this study, a light-activated molecular switch for the modulation of cell attachment/detachment behaviors was established on monolayer graphene (Gr)/n-type Silicon substrates (Gr/Si). Initiated by light illumination at the Gr/Si interface, pre-adsorbed proteins (bovine serum albumin, ECM proteins collagen-1, and fibronectin) underwent protonation to achieve negative charge transfer to Gr films (n-doping) through π-π interactions. This n-doping process stimulated the conformational switches of ECM proteins. The structural alterations in these ECM interactors significantly reduced the specificity of the cell surface receptor-ligand interaction (e.g., integrin recognition), leading to dynamic regulation of cell adhesion and eventual cell detachment. RNA-sequencing results revealed that the detached bone marrow mesenchymal stromal cell sheets from the Gr/Si system manifested regulated immunoregulatory properties and enhanced osteogenic differentiation, implying their potential application in bone tissue regeneration. This work not only provides a fast and feasible method for controllable cells/cell sheets harvesting but also gives new insights into the understanding of cell-ECM-material communications.

The effects of serum albumin pre-adsorption of nanoparticles on protein corona and membrane interaction: A molecular simulation study

As a platform to deliver imaging and therapeutic agents to targeted sites in vivo, nanoparticles (NPs) have widespread applications in diagnosis and treatment of cancer. However, the poor in vivo delivery efficiency of nanoparticles limits its potential for further application. Once enter the physiological environment, nanoparticles immediately interact with proteins and form protein corona, which changes the physicochemical properties of nanoparticle surface and further affects their transport. In this study, we performed molecular dynamics simulations to study the adsorption mechanism of nanoparticles with various surface modifications and different proteins (e.g., human serum albumin, complement protein C3b), and their interactions with cell membrane. The results show that protein human serum albumin prefers to interact with hydrophobic and positively charged nanoparticles, while the protein C3b prefers the hydrophobic and charged nanoparticles. The pre-adsorption of human serum albumin on the nanoparticle surface obviously decreases the interaction of nanoparticle with C3b. Furthermore, the high amount of protein pre-adsorption could decrease the probability of nanoparticle-membrane interaction. These results indicate that appropriate modification of nanoparticles with protein provides nanoparticles with better capability of targeting, which could be used to guide nanoparticle design and improve transport efficiency.

Dynamics of bubble formation in spontaneous microfluidic devices: Controlling dynamic adsorption via liquid phase properties

HypothesisThe interplay of interface evolution and surfactant adsorption determines the formation and stabilization of bubbles, and can be controlled by the liquid phase properties. ExperimentsWe studied bubble formation in an Edge-based Droplet GEneration (EDGE) microfluidic device at relevant length and time scale, allowing investigation of sub-events in a single bubble formation cycle. We vary the properties of the continuous phase that contains whey proteins and study a range of trans-pore pressures (Pd∗). FindingsThe shallow pores highlight the crucial role of the Laplace pressure and dynamic adsorption of proteins to the meniscus. Bubble formation is divided into two regimes by the Laplace pressure of the bare meniscus inside the pore. At Pd∗<1400 mbar, pre-adsorption of proteins is required to lower the Laplace pressure; the bubble formation frequency f0 increases with increasing protein concentration and is hardly affected by velocity and viscosity. At Pd∗≥1400 mbar, bubble formation immediately occurs upon applying pressure, and f0 mainly decreases with increasing viscosity. In both regimes, the initial bubble size d0 mainly increases with the viscosity (~η1/3). Bubble coalescence is only observed at Pd∗≥1400 mbar and can be effectively suppressed by raising protein concentration and viscosity within certain boundaries, yet ultimately this is at the cost of higher polydispersity of the bubbles. Our insights into the formation dynamics of micrometer-sized bubbles at time scales down to tens of microseconds can be used for effective control of bubble formation and stabilization in practical applications.

Protein-Cloaked Nanoparticles for Enhanced Cellular Association and Controlled Pathophysiology via Immunosurveillance Escape.

Weak interactions play an important role in soft corona (SC) formation and thus help in evaluating the biological fate of the nanoparticles (NPs). Preadsorption of specific proteins on the NP surface, leading to SC formation, has been found to help NPs in evading immunosurveillance. However, the role of different preadsorbed biomolecules in determining the NP pathophysiology and cellular association, upon their re-exposure to in vivo conditions, still remains elusive. Here, differently charged gold NPs were precoated with two different blood components, viz. red blood cells and human serum albumin protein, and these were then re-exposed to human serum. Cloaking NPs with protein improved the NP colloidal stability and other physico-chemical properties along with increased cellular association. Detailed proteomic analysis suggested that protein-camouflaged NPs showed a decrease in immune-responsive proteins compared to their bare counterparts. Further, it was also observed that the secondary protein signature on the NP surface was governed by primary protein coating; however, the event was more or less NP charge-independent. This study will pave the path for future strategies to make NPs invincible to the immunosurveillance system of the body.

Preparation of the protein corona: How washing shapes the proteome and influences cellular uptake of nanocarriers

A protein coat, termed the protein corona, assembles around the nanocarriers´ surface once it gets in contact with a biological environment. We show that the media used for the washing of protein corona can be crucial. This is true for the downstream analysis as well as for the pre-coating used in in vitro or in vivo. This has been widely overlooked so far. In this paper we focus on eight different washing media and analyze how they influence the composition of the hard protein corona of several nanocarriers incubated with human blood plasma and serum. SDS-PAGE and LC-MS analysis showed major differences in protein corona profiles when using diverse washing media. While plasma and serum proteins already have different complexities, each washing media changes the composition of proteins detected by downstream methods with different key proteins bound to the nanocarriers´ surface. Furthermore, the protein structure of the most abundant blood proteins incubated in the different media was analyzed with nanoDSF. This also emphasized the importance of the washing media, which had a significant influence on the protein adsorption stability. Lastly, cell uptake experiments for HeLa and RAW 264.7 macrophages also indicated an influence of the washing media. In conclusion, picking a specific washing media is on the one hand an important factor for downstream detection of protein compositions and may on the other hand be used to deliberately tune the protein corona for pre-adsorbed proteins from complex protein compositions. This might further support a guided delivery of the nanocarrier to a desired location within a physiological environment. Statement of SignificanceThe successfully application of nanocarriers as drug delivery vehicles is currently hampered by a limited understanding of the nanocarriers´ behavior in a complex biological environment. Once the nanocarrier comes into contact with blood plasma or serum, biomolecules rapidly adsorb onto their surface, covering the nanocarriers and forming a protein corona, which then dictates their biological identity. Analyzing the composition of this dynamic network of bound molecules, has already been shown to be influenced by various factors. However, the impact of the washing media used for the protein corona preparation has so far been neglected. In the present study, we demonstrate a quantitative influence of the washing media on the composition of the hard corona of different nanocarrier systems, which additionally affects protein stability and cellular uptake behavior.

Adsorption of serum proteins on titania nanotubes and its role on regulating adhesion and migration of mesenchymal stem cells.

Migration and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is an important biological process in tissue regeneration. Nanostructured titanium materials are believed to play a fundamental role in dental and orthopedic applications. However, the protein adsorption on nanostructured titanium materials and its correlation with the subsequent cell behaviors have not been studied. In this work, the titania nanotube arrays with different tubular diameters ranging from 27.3 to 88.2 nm were fabricated by using an electrochemical etching method. The adsorbed amounts and types of cell adhesion-related proteins (such as fibronectin, vitronectin, and laminin) from serum were investigated, revealing that these proteins were preferred to bind onto the surface with nanotubes of a smaller diameter. Adhesion and migration of BMSCs were studied as a function of different nanotube diameters in the presence or absence of serum proteins. Compared with the nanotube surface with a larger tubular diameter (88.2 nm), the surface with a smaller one could better support BMSCs in terms of adhesion and spreading. The pre-adsorbed serum proteins significantly enhanced adhesion and migration abilities of BMSCs. However, the adequate interactions between cells and serum proteins on the nanotubes surface with smallest nanotubes in diameter weakened cell mobility. Arrangement of cytoskeleton and expressions of key genes and proteins were studied, revealing that the nanostructured surfaces and pre-adsorbed proteins jointly mediated the adhesion and migration of BMSCs.

Surface rheology and morphology of beer protein and iso-humulone at air-liquid surface

Foam is one of the unique properties of beer, but how the amphiphilic components stabilize the foam is still not fully understood. This study concerns the main surface active components. For this purpose, protein from barley and iso-humulone from hop, were extracted from raw materials separately to be able to consider their effect separately and in the controlled mixture. The layers were formed in three ways, i.e. single component, pre-mixed solution and sequential addition of the individual components. Drop tensiometry and Brewster angle microscopy were employed to reveal the surface tension effects, rheological properties and morphology of the adsorbed layers.Both protein and iso-humulone showed surface activity and the combination gave synergistic effects in terms increasing the surface pressure. Surface rheology showed the combination of pre-mixed solutions of protein and iso-humulone formed the stiffest surface film. The pre-mixed solution gave higher dilatational moduli than if the two components were added sequentially. Furthermore, protein had a major influence on resisting deformation of the surface layer. The attractive interaction was confirmed by comparing the surface rheology and morphology of adsorbed layers formed by pre-mixed solutions and sequential addition. Iso-humulone also had a significant influence on interfacial properties. But this depends on whether it was pre-mixed with protein before added in to a buffer sub-phase or added to a pre-adsorbed protein layer. BAM reveals different surface morphology between the sequential addition and the pre-mixed sample. Thus, the property of the adsorbed film depends not only on the chemical composition, but also on the addition order of components.

Preparation of the Protein Corona: How Washing Shapes the Proteome and Influences Cellular Uptake

A protein coat, termed the protein corona, assembles around the nanocarriers´ surface once it gets in contact with a biological environment. We show that the media used for washing of the protein corona can be crucial. This is true for downstream analysis as well as for precoating for in vitro or in vivo use. This has been widely overlooked so far. We focus on the choice of eight different washing media and how they influence the composition of the hard protein corona of several nanocarriers incubated with human blood plasma and serum. SDS-PAGE and LC-MS analysis showed major differences in protein corona profiles when using diverse washing media. While plasma and serum proteins already have different complexities, each washing media changes the composition of proteins detected by downstream methods with different key proteins bound to the nanocarriers´ surface. Furthermore, the protein structure of the most abundant blood proteins incubated in the different media was analyzed with nanoDSF. This additionally emphasized the importance of washing media, which had a significant influence on protein adsorption stability. Lastly, cell uptake experiments for HeLa and RAW 264.7 macrophages also indicated an influence of the washing media. In conclusion, picking a specific washing media is on the one hand an important factor for downstream detection of protein compositions and may on the other hand be used to deliberately tune the protein corona for pre-adsorbed proteins from complex protein compositions. This might further support a guided delivery of the nanocarrier to a desired location within a physiological environment.

Preadsorption of Serum Proteins Regulates Bacterial Infections and Subsequent Macrophage Phagocytosis on Biomaterial Surfaces.

Bacterial infection has been one of the main obstacles for extensive biomedical applications of biomaterial films. Understanding the interactions among macromolecules, cells, and bacteria in the microenvironment located on the film surface at the molecular level is essential for developing antibacterial films. Here we report the distinct influence of several key serum proteins adsorbed on diamond-like carbon (DLC) and traditional Ti films on initial bacterial adhesion, biofilm formation, and corresponding immune responses. Type I collagen, Fn, and IgG were selected as the typical serum proteins. Gram-positive bacterium Staphylococcus epidermidis and Gram-negative bacterium Escherichia coli were used as the model bacteria. Macrophage phagocytosis tests were carried out to examine the impact of adsorbed proteins on the ability of macrophages to clear the adhered pathogens. Results show that it was the specific molecular recognition between adsorbed proteins and bacteria, not the surface physiochemical properties such as surface wettability, surface roughness, and surfaces charge, that decisively affected bacterial adhesion and following biofilm formation. Collagen resisted bacterial adhesion on both DLC and Ti films, even though the molecules exhibited distinct conformations on the two surfaces, whereas for Fn and IgG, the specific molecular recognition was closely related to protein conformations. Fn molecules formed globular aggregates on Ti surfaces that greatly enhanced bacterial adhesion but exhibited a fibril conformation on DLC surfaces that inhibited bacterial adhesion. IgG showed an end-on orientation with free F(ab)2 domains on Ti surfaces, facilitating bacterial adhesion and biofilm formation, while the flattened orientation on DLC films showed little effect on bacterial behaviors. Furthermore, preadsorption of Fn and IgG significantly promoted the phagocytosis ability of macrophages against S. epidermidis and affected the corresponding secretion of inflammatory cytokine. These results would give insights into understanding protein-surface interactions for developing appropriate surface modification techniques for biomaterials with desired anti-inflammatory functions.

Plasma Proteins at the Interface of Dental Implants Modulate Osteoblasts Focal Adhesions Expression and Cytoskeleton Organization.

The host-material interface is a crucial relationship dictating the possibility of successful osseointegration in implant dentistry. The aim of the present study was to characterize the effects of plasma proteins pre-adsorption on the adhesion capacity of osteoblasts, which occurs immediately after implant insertion in vivo. After having pre-adsorbed human plasma proteins on a machined and microrough titanium surface, MC3T3-E1 osteoblasts adhesion was evaluated through crystal violet cell adhesion assay, immunofluorescence staining for cytoskeleton, focal adhesions and cell nuclei, and scanning electron microscopy. The pre-adsorbed protein layer markedly affected the adhesion rate of cells, as well as their morphology and the expression of focal contacts. Moreover, protein adsorption to the underlying titanium surface was found to be correlated to surface pre-wetting. Thus, the early adsorption of serum proteins to the interface of dental implants impacts cell adhesion in terms of strength and of focal adhesions expression.

Inflammation via myeloid differentiation primary response gene 88 signaling mediates the fibrotic response to implantable synthetic poly(ethylene glycol) hydrogels

Synthetic hydrogels, such as poly(ethylene glycol) (PEG), are promising for a range of in vivo applications. However, like all non-biological biomaterials, synthetic hydrogels including PEG elicit a foreign body response (FBR). The FBR is thought to be initiated by adsorbed protein that is recognized by and subsequently activates inflammatory cells, notably macrophages, and culminates with fibrotic encapsulation. However, the molecular mechanisms that drive the FBR are not well understood. Toll-like receptors (TLRs) are key receptors that recognize pathogens, but also recognize altered host proteins that display damage-associated molecular patterns (DAMPs). Thus TLRs may play a role in the FBR. Here, we investigated myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor protein that mediates inflammatory cytokine production induced by most TLRs. An in vitro model was used consisting of macrophages cultured on the surface of synthetic hydrogels, specifically PEG, with pre-adsorbed serum proteins. Our in vitro findings demonstrate that MyD88-dependent signaling is the predominant inflammatory pathway in macrophage activation to synthetic hydrogels. When stimulated with TLR agonists to mimic additional DAMPs present in vivo, MyD88-dependent signaling was also the predominant pathway in macrophage activation. An in vivo model of PEG hydrogels implanted subcutaneously in wild-type and MyD88−/− mice also demonstrated that MyD88 is the key contributor to the recruitment of inflammatory cells and formation of the fibrous capsule surrounding the implanted hydrogel. Taken together, findings from this study identify MyD88-mediated inflammation as being a critical pathway involved not only in the inflammatory response, but in formation of the fibrous capsule to PEG hydrogels. Statement of SignificanceSynthetic hydrogels are promising for in vivo applications but, like all non-biological biomaterials, synthetic hydrogels elicit a foreign body response (FBR). The molecular mechanisms that drive the FBR are not well understood. This work identifies the myeloid differentiation primary response gene 88 (MyD88) as a central mediator to macrophage activation in response to a poly(ethylene glycol) hydrogel with pre-adsorbed proteins in vitro. Moreover, MyD88 was also central to the recruitment of inflammatory cells, which included neutrophils, monocytes, and macrophages, to implanted PEG hydrogels and to fibrous encapsulation. These findings demonstrate that MyD88-mediated inflammation is responsible in part for the formation of the fibrous capsule of the FBR.

Interfacial Effect-Based Quantification of Droplet Isothermal Nucleic Acid Amplification for Bacterial Infection

Bacterial infection is a widespread problem in humans that can potentially lead to hospitalization and morbidity. The largest obstacle for physicians/clinicians is the time delay in accurately identifying infectious bacteria, especially their sub-species, in order to adequately treat and diagnose such infected patients. Loop-mediated amplification (LAMP) is a nucleic acid amplification method that has been widely used in diagnostic applications due to its simplicity of constant temperature, use of up to 4 to 6 primers (rendering it highly specific), and capability of amplifying low copies of target sequences. Use of interfacial effect-based monitoring is expected to dramatically shorten the time-to-results of nucleic acid amplification techniques. In this work, we developed a LAMP-based point-of-care platform for detection of bacterial infection, utilizing smartphone measurement of contact angle from oil-immersed droplet LAMP reactions. Whole bacteria (Escherichia coli O157:H7) were assayed in buffer as well as 5% diluted human whole blood. Monitoring of droplet LAMP reactions was demonstrated in a three-compartment, isothermal proportional-integrated-derived (PID)-controlled chip. Smartphone-captured images of droplet LAMP reactions, and their contact angles, were evaluated. Contact angle decreased substantially upon target amplification in both buffer and whole blood samples. In comparison, no-target control (NTC) droplets remained stable throughout the 30 min isothermal reactions. These results were explained by the pre-adsorption of plasma proteins to an oil-water interface (lowering contact angle), followed by time-dependent amplicon formation and their preferential adsorption to the plasma protein-occupied oil-water interface. Time-to-results was as fast as 5 min, allowing physicians to quickly make their decision for infected patients. The developed assay demonstrated quantification of bacteria concentration, with a limit-of-detection at 102 CFU/μL for buffer samples, and binary target or no-target identification with a limit-of-detection at 10 CFU/μL for 5% diluted whole blood samples.

3-D printed Ti-6Al-4V scaffolds for supporting osteoblast and restricting bacterial functions without using drugs: Predictive equations and experiments

Conditions resulting from musculoskeletal deficiencies (MSDs) are wide-ranging and retain the likelihood for restricting motion or producing pain, especially in the lower back, neck, and upper limbs. Engineered scaffold devices are being produced to replace antiquated modalities that suffer from structural and mechanical deficiencies in the treatment of MSDs. Here, as-fabricated Ti-6Al-4V-based Hive™ interbody fusion scaffolds, commercialized by HD Lifesciences LLC, were assayed for their osteogenicity and antibacterial potential using a series of characterization and in vitro tests, as well as by quantitative analyses. A topographical assessment of the Hive™ meshes indicated that the elementally pure substrates are microscopically porous and rough, in addition to displaying structural heterogeneity. Roughness estimations and static contact angle measurements recommended the use of the as-fabricated Ti-6Al-4V substrates for supporting osteoblast attachment, especially, due to the improved surface roughness and wettability values of these scaffolds relative to the unembellished Ti-6Al-4V surfaces. Quantitative correlations relating the surface properties of roughness and energy were applied to predict cellular behaviors. Cell growth suppositions were experimentally corroborated. Critical in vitro data indicated the competencies of the Hive™ scaffolds for promoting the adhesion and proliferation of human fetal osteoblasts (hFOBs), accumulating substantial calcium deposition from metabolizing hFOBs, and restricting the attachment of bacteria. The model system that investigated the pre-adsorption of casein proteins along the Hive™ test substrates additionally furthered the notion that bacterial attachment may be restricted, with short-scale adhesion dynamics serving as the theoretical basis for this hypothesis. In this manner, this study showed that through predictive models and experiments, these novel 3D printed Ti-based scaffolds can increase bone cell while decreasing bacteria functions without using drugs. Statement of SignificanceSintered Ti-6Al-4V spinal fusion devices (Hive™) manufactured and marketed by HD Lifesciences LLC were assessed for their biocompatibility and antibacterial performance. A mixed methods approach was employed, whereby quantitative measures were used to predict the ability for Hive™ substrates to adsorb specialized proteins and to restrict bacterial surface colonization. In vitro tests that evaluated bone cell and bacterial adhesion, calcium deposition, and protein adsorption supported quantitative predictions. The data herein presented demonstrate the following: (1) surface energy is an important predictor of implant-cell interactions, (2) strong correlations exist between surface energy and surface roughness, (3) mathematical models can be used to improve and predict implant device perofrmance, and (4) porous, rough, 3D-printed materials perform well in terms of biocompatibility and antimicrobial efficacy.

Hydroxyapatite Nanoparticle Coating on Polymer for Constructing Effective Biointeractive Interfaces

Bone is an organic-inorganic composite with the ability to regenerate itself. Thus, several studies based on artificial organic-inorganic interface sciences have been tried to develop capable materials for effective regenerative bone tissues. Hydroxyapatite nanoparticles (HAp NPs) have extensively been researched in bone tissue engineering due to the compositional and shape similarity to the mineral bone and excellent biocompatibility. However, HAp alone has low mechanical strength, which limits its applications. Therefore, HAp NPs have been deposited on the biocompatible polymer matrix, obtaining composites with the enhanced mechanical, thermal, and rheological properties and with higher biocompatibility and bioactivity. For developing new biomedical applications, polymer-HAp interfacial interactions that provide biofusion should be investigated. This paper reviewed common coating techniques for obtaining HAp NPs/polymer fusion interfaces as well as in vitro studies of interfacial interactions with proteins and cells, demonstrating better biocompatibility. Studies based on interfacial interactions between biomolecules and HAp NPs were highlighted, and how these interactions can be affected by specific protein preadsorption was also summarized.

Tuning protein adsorption on charged polyelectrolyte brushes via salinity adjustment

Adsorption and desorption of biomolecules on/from polyelectrolyte surfaces play a critical role in numerous biomedical and engineering applications. Though some weak polyelectrolytes have been developed for protein adsorption and desorption by regulating the salinity and pH, limited reports are available about the regeneration of strong polyelectrolyte surfaces between protein-attractive and protein-repulsive states, which is highly desirable but challenging to realize as it is more difficult to fully release the pre-adsorbed proteins from strong polyelectrolytes due to their permanent charges comparing to weak polyelectrolytes. Here we report a strategy to facilely tune the resistance to nonspecific protein adsorption onto a charged cationic poly([2-(methacryloyloxy) ethyl] trimethylammonium chloride (PMTAC) or anionic Poly(3-sulfopropyl methacrylate potassium salt) (PSPMA)) strong polyelectrolyte brush coating via salinity adjustment. It has been demonstrated that the charged polyelectrolyte coating displays strong adhesion to proteins at low salinity (e.g., 0.1mM NaCl) but protein-repellent property at high salinity (e.g., 1.0M NaCl). The adsorbed proteins on the strong polyelectrolyte coatings under low salinity condition could be readily removed via rinsing with high-salinity water, demonstrating the excellent surface regeneration capability.

Artificial chaperones based on mixed shell polymeric micelles: insight into the mechanism of the interaction of the chaperone with substrate proteins using Förster resonance energy transfer.

Controlled and reversible interactions between polymeric nanoparticles and proteins have gained more and more attention with the hope to address many biological issues such as prevention of protein denaturation, interference of the fibrillation of disease relative proteins, removing of toxic biomolecules as well as targeting delivery of proteins, etc. In such cases, proper analytic techniques are needed to reveal the underlying mechanism of the particle-protein interactions. In the current work, Förster Resonance Energy Transfer (FRET) was used to investigate the interaction of our tailor designed artificial chaperone based on mixed shell polymeric micelles (MSPMs) with their substrate proteins. We designed a new kind of MSPMs with fluorescent acceptors precisely placed at the desired locations as well as hydrophobic domains which can adsorb unfolded proteins with a propensity to aggregate. Interactions of such model micelles with a donor-labeled protein-FITC-lysozyme, was monitored by FRET. The fabrication strategy of MSPMs makes it possible to control the accurate location of the acceptor, which is critical to reveal some unexpected insights of the micelle-protein interactions upon heating and cooling. Preadsorption of native proteins onto the hydrophobic domains of the MSPMs is a key step to prevent thermo-denaturation by diminishing interprotein aggregations. Reversible protein adsorption during heating and releasing during cooling have been confirmed. Conclusions from the FRET effect are in line with the measurement of residual enzymatic activity.

The effect of pre-adsorption of OVA or WPC on subsequent OVA or WPC fouling on heated stainless steel surface

Fouling on the heat exchanger surface during food processing has been researched extensively due to its great importance in energy efficiency, product quality and food safety. The nature of heat exchanger surface has an effect on the initial deposition behavior and deposit removal behavior to some degree. Protein adsorption on surface is considered to be the initial stage in fouling. In the current study, protein ‘pre-adsorption’ at room temperature on stainless steel has been investigated as a means to influence the behavior of protein fouling at pasteurization temperatures. Pre-adsorption was carried out with whey protein concentrate (WPC) and ovalbumin (OVA), respectively, which reduced the fouling of OVA (∼20–30% energy saving in the processing time examined). However, the pre-adsorption had little effect on fouling of whey protein concentrate. Contact angles were measured to show the surface change due to protein pre-adsorption. Protein pre-adsorption made the surfaces more hydrophilic.

Tensiometry and dilational rheology of mixed β-lactoglobulin/ionic surfactant adsorption layers at water/air and water/hexane interfaces

Oscillating drop tensiometry was applied to study adsorbed interfacial layers at water/air and water/hexane interfaces formed from mixed solutions of β-lactoglobulin (BLG, 1μM in 10mM buffer, pH 7 – negative net charge) and the anionic surfactant SDS or the cationic DoTAB. The interfacial pressure Π and the dilational viscoelasticity modulus |E| of the mixed layers were measured for mixtures of varying surfactant concentrations. The double capillary technique was employed which enables exchange of the protein solution in the drop bulk by surfactant solution (sequential adsorption) or by pure buffer (washing out). The first protocol allows probing the influence of the surfactant on a pre-adsorbed protein layer thus studying the protein/surfactant interactions at the interface. The second protocol gives access to the residual values of Π and |E| measured after the washing out procedure thus bringing information about the process of protein desorption.The DoTAB/BLG complexes exhibit higher surface activity and higher resistance to desorption in comparison with those for the SDS/BLG complexes due to hydrophobization via electrostatic binding of surfactant molecules. The neutral DoTAB/BLG complexes achieve maximum elastic response of the mixed layer. Mixed BLG/surfactant layers at the water/oil interface are found to reach higher surface pressure and lower maximum dilational elasticity than those at the water/air surface. The sequential adsorption mode experiments and the desorption study reveal that binding of DoTAB to pre-adsorbed BLG globules is somehow restricted at the water/air surface in comparison with the case of complex formation in the solution bulk and subsequently adsorbed at the water/air surface. Maximum elasticity is achieved with washed out layers obtained after simultaneous adsorption, i.e. isolation of the most surface active DoTAB/BLG complex. These specific effects are much less pronounced at the W/H interface.

Pre-adsorption of protein on electrochemically grooved nanostructured stainless steel implant and relationship to cellular activity.

The successful integration of a biomedical device is governed by the surface properties of the material and also depends on the interaction with the physiological fluid involving adsorption of proteins on the surface. Pre-adsorbed proteins act as pilots for cell adhesion and subsequently govern cellular activity. In this regard, nanograined materials are excellent vehicles to obtain an unambiguous understanding of protein adsorption, which regulate cell adhesion and cellular activity. Toward this end, we have used the concept of phase reversion-induced nanograined structure to understand grain structure-induced self-assembly of a model protein, bovine serum albumin. Furthermore, in the context of bio-mechanical interlocking between implant and bone, and osseointegration of the implant, grain boundaries were electrochemically grooved and studied for osteoblast functions. Experiments indicated that the significant differences in cell attachment, proliferation, and expression level of prominent proteins (actin, vinculin, and fibronectin) is related to synergistic effects of grain structure, pre-adsorbed protein, and grooving of grain boundaries such that the osteoblasts functions and cellular activity is promoted on the nanostructured surface in relation to the coarse-grained counterpart.