Protein adsorption and surface patterning

Surface patterning has become an important discipline of biologically oriented surface science over the past decades. Many methods have been developed that allow the formation of patterns on the micro- and nanoscale. This Opinion discusses the role of protein adsorption in patterning technologies, highlighting how it can be used as an integrated part of the patterning process, how it can be controlled by patterns with appropriate properties, and how it may lead to disruption of formed patterns if not properly accounted for. Recent examples from literature are used to emphasize some of the most interesting developments in the field, such as novel surface chemistries only allowing specific protein adsorption, directed self-sorting adsorption of proteins on patterned surfaces, and control of protein adsorption through nanopatterning.     

Effect of surface charge on adsorption of bovine serum albumin as studied by ellipsometry 1. Adsorption on cationic monolayer

The adsorption of bovine serum albumin (BSA) onto a cationic monolayer (N,N-dimethyl-N,N-dialkylammonium chloride) spread at the air/water interface was studied by ellipsometry. Both thicknesses and refractive indices of the BSA layer adsorbed at the monolayer/solution interface are estimated from the observed change in phase difference and the ratio of reflection coefficients. The amount of adsorption of BSA resembles a Langmuir type isotherm. The adsorption changes with pH asymmetrically with respect to the pH of maximum adsorption, which was calculated to be 5.06 ± 0.47 mg/m². The amount of maximum adsorption implies that the BSA molecule adsorbs to the surface in a mode intermediate between side-on and end-on.     

Degradation patterns and surface wettability of electrospun fibrous mats

Degradation profiles and surface wettability are critical for optimal application of electrospun fibrous mats as drug carriers, tissue growth scaffolds and wound dressing materials. The effect of surface morphologies and chemical groups on surface wettability, and the resulting matrix degradation profiles were firstly assessed for electrospun poly(d,l-lactide) (PDLLA) and poly(d,l-lactide)-poly(ethylene glycol) (PELA) fibers. The air entrapment between the fiber interfaces clarified the effects of various surface morphologies on the surface wettability. Chemical groups with lower binding energy were enriched on the fiber surface due to the high voltage of the electrospinning process, and a surface erosion pattern was detected in the degradation of electrospun PDLLA fibers, which was quite different from the bulk degradation pattern for other forms of PDLLA. Contributed by the hydrophilic poly(ethylene glycol) segments, the degradation of electrospun PELA fibers with hydrophobic surface followed a pattern different from surface erosion and typical bulk degradation.     

Polymerizable ionic liquid copolymer P(MMA-co-BVIm-Br) and its effect on the surface wettability of PVDF blend membranes

Polymerizable ionic liquid copolymer P(MMA-co-BVIm-Br) was synthesized by radical polymerization technique, and characterized by Fourier transform infrared spectrometry(FTIR), 1H Nuclear magnetic resonance(1H-NMR) and gel permeation chromatography(GPC). The resulting copolymer was used to prepare poly(vinylidene fluoride)(PVDF) blend membranes via a phase inversion method. The effects of the copolymer on the polymorphism, surface wettability and zeta potential(ζ) of the blend membranes were investigated by ATR-FTIR, contact angle instrument and zeta potential analyzer. Scanning electron microscopy(SEM and SEM-EDS) was also applied to investigate the morphology and the surface element changes of the fabricated membranes. The results indicated that P(MMA-co-BVIm-Br) copolymer existed on the surface of the membrane which made the blend membrane have a positive surface during the experimental p H range. The copolymer was also in favor of the formation of β crystal phase in PVDF membranes. The contact angle experiment indicated that P(MMA-co-BVIm-Br) copolymer could switch the wettability of the blend membranes from hydrophilic to hydrophobic by exchanging Br-anion with PF-6. Compared with pure PVDF membranes, the water flux and water recovery flux of the blend membranes were enhanced obviously. The results from the flux recovery ratio(FR) and total fouling ratio(Rt) all suggested that the blend membranes had good anti-fouling properties.     

Study of the effect of chemical composition on the surface wettability of three-dimensional graphene foams

The chemical composition obviously affects the surface wettability of a three-dimensional(3D) graphene material apart from its surface energy and microstructure.In the hydrothermal preparation,the heteroatom doping changes the chemical composition and wettability of the 3D graphene material.To realize the controllable surface wettability of graphene materials,aminobenzene sulfonic acid(ABSA)was selected as a typical doping agent for the preparation of nitrogen and sulfur co-doped 3D graphene foa...     

The effect of surface microtopography of poly(dimethylsiloxane) on protein adsorption, platelet and cell adhesion

Chemical homogeneous poly(dimethylsiloxane) (PDMS) surface with dot-like protrusion pattern was used to investigate the individual effect of surface microtopography on protein adsorption and subsequent biological responses. Fibrinogen (Fg) and fibronectin (Fn) were chosen as model proteins due to their effect on platelet and cell adhesion, respectively. Fg labeled with ¹²⁵I and fluorescein isothiocyanate (FITC) was used to study its adsorption on flat and patterned surfaces. Patterned surface has a 46% increase in the adsorption of Fg when compared with flat surface. However, the surface area of the patterned surface was only 8% larger than that of the flat surface. Therefore, the increase in the surface area was not the only factor responsible for the increase in protein adsorption. Clear fluorescent pattern was visualized on patterned surface, indicating that adsorbed Fg regularly distributed and adsorbed most on the flanks and valleys of the protrusions. Such distribution and local enrichment of Fg presumably caused the specific location of platelets adhered from platelet-rich plasma (PRP) and flowing whole blood (FWB) on patterned surface. Furthermore, the different combination of surface topography and pre-adsorbed Fn could influence the adhesion of L929 cells. The flat surface with pre-adsorbed Fn was the optimum substrate while the virgin patterned surface was the poor substrate in terms of L929 cells spread.     

The effect of froth stability and wettability on the flotation of a xerographic toner

The Separation of ink and pulp fibers in recycled paper is primarily achieved by flotation methods. Xerographic toners from photocopiers and laser printers are known to cause problems in flotation deinking. Wettability and froth stability are two important factors which determine the floatability of xerographic toners. The floatability is investigated for a selected toner using a cationic, a nonionic, and an anionic surfactant. At low surfactant concentrations the froth is too unstable to support flotation, whereas at high surfactant concentrations the toner is rendered hydrophilic by adsorbed surfactant molecules and does not stick to air bubbles. Consequently, a maximum in flotation response is found at an intermediate surfactant concentration near the critical micelle concentration. Cationic, nonionic, and anionic surfactants all adsorb with their hydrocarbon tails on the toner surface. By choosing appropriate froth-stabilizing additives it is possible to enhance the flotation performance.     

The role of ligands on protein retention in adsorption chromatography: A surface energetics approach

Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650‐C, and Toyopearl Butyl 650‐C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.     

Detection of surface bound complement at increasing serum anticoagulant concentrations

Surface mediated immune complement activation can be detected by a variety of antibody utilizing methods such as ELISA, fluorescence- or radiolabelling techniques, QCM, and ellipsometry. In the present work we investigated how the common anticoagulants heparin, dalteparin, fondaparinux and sodium citrate affected the binding of anti-complement factor 3c (anti-C3c) on a model complement activator surface, immobilised IgG, after incubation in human blood serum.

The results show, as expected, that different anticoagulants affect the antibody binding differently. Increasing amounts of heparin, dalteparin and sodium citrate in normal serum resulted in a decreasing anti-C3c binding. The antibody deposition was not sensitive for the fondaparinux concentration. Surprisingly high concentrations of anti-coagulantia were needed to completely eradicate the antibody binding. Experiments in EGTA–serum showed that anticoagulants interfered directly with both the classical and alternative pathways. Control C3a-des arg ELISA measurements show that the lowered antibody surface binding was not a result of complement depletion in serum. Kallikrein generation by hydrophilic glass surfaces was not affected by high anticoagulant concentrations.     

Simultaneous monitoring of protein adsorption at the solid–liquid interface from sessile solution droplets by ellipsometry and axisymmetric drop shape analysis by profile

In this paper two in situ techniques are combined to simultaneously examine protein adsorption at the solid–liquid interface from sessile solution droplets. With axisymmetric drop shape analysis by profile (ADSA-P) the change in solid–liquid interfacial tension is determined, while ellipsometry is employed to measure the amount of protein adsorbed from the same solution droplet at the solid–liquid interface. Three proteins (human serum albumin (HSA), immunoglobulin G (IgG) and fibrinogen (Fb)) were dissolved to a concentration of 0.05 mg ml⁻¹ in PBS (pH 7) and sessile droplets were placed for 2 h on a 47.8 nm thick gold coating on glass. The gold coated glass was positioned onto a quartz prism with immersion oil. The prism was aligned in a rotating analyser ellipsometer and the optical beam was thus allowed to be reflected at the hydrophobic gold surface. The ADSA-P set-up was built in 90° cross-beamed set-up around the prism. By combining the results for the adsorbed amounts and the interfacial tension changes over the two hour adsorption period, two stages in the adsorption process could be distinguished. In the first stage, the adsorbed amounts increase in correspondence with the interfacial tension changes, indicating that the interfacial tension changes are caused by adsorption, whereas in the second stage interfacial tension changes continue despite the adsorbed amounts being constant. Consequently, the second stage must be associated with conformational changes of the adsorbed proteins. For HSA and Fb, the conformational contribution to the interfacial tension changes (7.8 and 5.3 mJ m⁻², respectively) were approximately 2-fold the adsorption contribution, while for IgG both were equal around 3 mJ m⁻².     

Free energies of protein adsorption onto mineral particles — from the initial encounter to the onset of hysteresis

The kinetics and energetics of human serum albumin (HSA) adsorption onto silica and talc particles are compared, from the earliest moment of contact, up to 24 h exposure. The measured affinity constants are used to obtain the free energies of interaction (ΔG). The free energies of interaction are also obtained via the measured surface properties of HSA, water, silica and talc. Comparison between the ΔG-values obtained by the two different approaches makes it possible to deduct the manner and conformation of HSA adsorption at the earliest moment, as well as after onset of hysteresis. Two different modes of hysteresis are identified — one allows slow dissociation and the other approaches apparent irreversibility. The mechanisms of both are elucidated, as is the mechanism of desorption at high pH and by displacement with identical or quasi-identical molecules.     

PDMS微芯片吸附牛血清白蛋白的研究

材料表面的物理和化学性质对蛋白质的吸附具有很大的影响[1].对蛋白质吸附的研究是研制生物传感器、生物芯片和生物材料的基础.     

Kinetics and interaction constants of protein adsorption onto mineral microparticles — measurement of the constants at the onset of hysteresis

The kinetics of desorption of human serum albumin from silica and talc microparticles are presented. The evolution of hysteresis, i.e. of the progressive bond strengthening between protein molecules and solid substratum, was monitored by calculating the Langmuir adsorption isotherms, as well as the desorption rates and desorbable protein amounts after various particle–protein contact times. The effect of parameters such as temperature, ionic strength, presence of cation-complexing and protein denaturation agents, on the desorption rate and maximum desorbable amount were also examined. The results help elucidate the mechanism of protein adsorption and also the strength of the formed bonds before and after the onset of hysteresis.     

Competitive adsorption of -α-lysophosphatidylcholine/β-lactoglobulin mixtures at the interfaces of foams and foam lamellae

The stability of foams formed with the protein β-lactoglobulin as a function of increasing concentration of the lipid analogue -α-lysophosphatidylcholine were investigated using a microconductivity technique. The drainage, surface diffusion and thickness properties of thin liquid films (foam lamallae) were also studied using optical microscopy including epi-illumination, fluorescence recovery after photobleaching and film interferometry techniques. In addition, the surfactant binding properties of the protein were examined. The addition of small quantities of -α-lysophosphatidylcholine to β-lactoglobulin (molar ratio, R < 7:1) increased the foam stability, whereas a slightly higher concentration of surfactant in the mixture (R = 10) caused foam destabilisation. The explanation of these observations is based on changes in the composition and structure of the adsorbed interfacial layers of the thin films caused by competitive displacement of the protein by the surfactant.     

Protein adsorption on to low-temperature isotropic carbon 4. Competitive adsorption on carbon and silica studied by two-dimensional electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was applied to the study of competitive protein adsorption from diluted human plasma. We obtained the depletion (adsorption) of some 25 plasma proteins in the presence of low-temperature isotropic carbon (LTIC) or silica powders. The depletion data are used as a measure of protein adsorptivity. Generally, proteins of lowest abundance have the highest tendency to associate with the two solid surfaces studied. The adsorptivity of a protein is largely determined by its solubility. Most proteins detected exhibit similar depletion behavior on both adsorbents, suggesting a multilayer adsorption process. Three proteins, hemopexin, apolipoprotein A I, and apolipoprotein A II, are depleted differently in the presence of LTIC and silica powders.     

Protein adsorption characteristics of calcium hydroxyapatites modified with pyrophosphoric acids

Protein adsorption characteristics of calcium hydroxyapatite (Hap) modified with pyrophosphoric acids (PP(a)) were examined. The PP(a) modified Hap particles (abbreviated as PP-Hap) possessed anchored polyphosphate (PP: P-{O-PO(OH)}(n)-OH) branches on their surfaces. The proteins of bovine serum albumin (BSA: isoelectric point (iep)=4.7, molecular mass (M(s))=67,200 Da, acidic protein), myoglobin (MGB: iep=7.0, M(s)=17,800 Da, neutral protein), and lysozyme (LSZ: iep=11.1, M(s)=14,600 Da, basic protein) were examined. The zeta potential (zp) of PP-Hap particles as a function of pH overlapped; zp-pH curves were independent of the concentration of pyrophosphoric acids (abbreviated as [PP(a)]) used for modifying Hap surface. The saturated amounts of adsorbed BSA (Delta n(ads)(BSA)) were increased three-fold by the surface modification with PP(a) though they were independent of the [PP(a)]. Furthermore, the fraction of BSA desorption was independent of the [PP(a)]. This enhancement of BSA adsorption onto the PP-Hap is due to the hydrogen bonding between oxygen and OH groups of the PP-branches and functional groups of BSA molecules. In the case of LSZ, a more higher adsorption enhancement was observed; the saturated amount of adsorbed LSZ (Delta n(ads)(LSZ)) for Hap modified at [PP(a)]=6 mmol/dm(3) was nine-fold than that for Hap unmodified. This remarkable adsorption enhancement was explained by a three-dimensional binding mechanism; LSZ molecules were trapped inside of the PP-branches. Hence, a fraction of LSZ desorption was decreased with an increase in the [PP(a)]; as more PP-branches are presented on the surface the higher retardation of LSZ desorption was induced. It was expected from their small size that MGB adsorb between the PP-branches as well as LSZ. However, the amounts of adsorbed MGB (Delta n(ads)(MGB)) did not vary and were independent of the [PP(a)] due to the small numbers of functional groups of MGB. In addition, no dependence of the fraction of MGB desorption on the [PP(a)] was observed. The results of zp for all the protein systems supported the mode of protein adsorption discussed. The anchored structure of the PP-branches developed on the Hap surface to provide three-dimensional protein adsorption spaces was proved by a comparative experiment that was elucidating the effect of pyrophosphate ions for BSA adsorption onto Hap.     

Dynamics of adsorption and desorption of proteins at an air/water interface

Adsorption and desorption dynamics of lysozyme and β-casein at the air/water interface were investigated through stress relaxation experiments. The resulting surface tension changes due to a step-type surface area disturbance, as a function of time, were measured through a capillary wave probe. The adsorption data, obtained after a surface area expansion, can be well fitted to a diffusion-controlled adsorption model. However, desorption relaxation following a surface compression is much slower and cannot be modeled by the diffusion theory. Characteristic diffusion frequency and high-frequency dilational elasticity for protein layers were also obtained and found to be consistent with data reported in the literature.     

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.     

Competitive adsorption of an anionic and a nonionic surfactant on polystyrene latex particles as monitored by small x-ray scattering

Adsorption of the anionic surfactant sodium dodecylsulfate (SDS) as well as competitive adsorption of SDS with the nonionic surfactant Triton X-405 on a polystyrene latex has been examined by the use of small-angle x-ray scattering (SAXS). Since the latex particles only have a low contrast towards water whereas the electron densities of the added surfactants are rather high, their gradual adsorption can be monitored with good sensitivity. The particles covered with SDS have been investigated by the contrast variation method. A distinct core-shell structure has been observed. After maximum coverage of the surface is reached, the additional surfactant forms free micelles. The analysis presented here allows the simultaneous investigation of the covered particles and the free micelles. This is due to their respective characteristic scattering contributions in different angular ranges. Information on the competitive adsorption could be obtained by analyzing the structure of the coated particles and the composition of the free mixed micelles. The adsorption is shown to be an equilibrium process where adsorption of the nonionic component is found to be much stronger than of the SDS molecules.     

The effect of thiolation on the mechanical and protein adsorption properties of polyurethanes

Segmented polyurethanes are important polymers for a number of industrial and technological applications. The purpose of this work was to synthesize polybutadiene-based polyurethanes and subsequently graft carboxylate and sulfonate side chains via thiol-ene reaction. Spectroscopic investigations showed that grafting yielded good conversion for the vinyl unsaturation of the polybutadiene soft segment. DSC and tensile testing revealed that grafted polyurethanes had a better segmental compatibility and superior mechanical properties than the control polyurethane without grafting. The carboxylic side chains of the soft segment were responsible for the observed improved mechanical properties. Initial protein adsorption tests on these polymers were found to be higher than the control surface. The polyurethanes of the current study could be used for biomedical applications where protein attachment to the surface is needed for specific cell adhesion and tissue repair.