Protein-resistant Properties Research Articles

Resistance of Galactoside-Terminated Alkanethiol Self-Assembled Monolayers to Marine Fouling Organisms

Self-assembled monolayers (SAMs) of galactoside-terminated alkanethiols have protein-resistance properties which can be tuned via the degree of methylation [Langmuir 2005, 21, 2971-2980]. Specifically, a partially methylated compound was more resistant to nonspecific protein adsorption than the hydroxylated or fully methylated counterparts. We investigate whether this also holds true for resistance to the attachment and adhesion of a range of marine species, in order to clarify to what extent resistance to protein adsorption correlates with the more complex adhesion of fouling organisms. The partially methylated galactoside-terminated SAM was further compared to a mixed monolayer of ω-substituted methyl- and hydroxyl-terminated alkanethiols with wetting properties and surface ratio of hydroxyl to methyl groups matching that of the galactoside. The settlement (initial attachment) and adhesion strength of four model marine fouling organisms were investigated, representing both micro- and macrofoulers; two bacteria (Cobetia marina and Marinobacter hydrocarbonoclasticus), barnacle cypris larvae (Balanus amphitrite), and algal zoospores (Ulva linza). The minimum in protein adsorption onto the partially methylated galactoside surface was partly reproduced in the marine fouling assays, providing some support for a relationship between protein resistance and adhesion of marine fouling organisms. The mixed alkanethiol SAM, which was matched in wettability to the partially methylated galactoside SAM, consistently showed higher settlement (initial attachment) of test organisms than the galactoside, implying that both wettability and surface chemistry are insufficient to explain differences in fouling resistance. We suggest that differences in the structure of interfacial water may explain the variation in adhesion to these SAMs.

Computational entropy estimation of linear polyether-modified surfaces and correlation with protein resistant properties of such surfaces

The non-specific adsorption of proteins on surfaces is a well-known and mostly undesirable phenomena, which is reduced by a surface coating with the linear polyether poly(ethylene glycol) (PEG) as the current benchmark material. However, the molecular mechanism of protein-resistant surfaces is still not fully understood. Two main hypotheses are generally applied. The first one is steric repulsion of the highly flexible tethered polymer chains, leading to an entropic penalty by adsorption of proteins due to the reduction in polymer chain mobility. The second one argues with well-hydrated polymer chains generating a repulsive interfacial water layer. In this article, we compare the three different protein-resistant polyether structures PEG, linear polyglycerol (LPG(OH)) and linear poly(methyl glycerol) (LPG(OMe)) to get new insights into the molecular mechanism behind protein resistance. In a theoretical approach, we apply an entropy estimator that assesses the conformational states of the tethered polyethers from MD simulations. It reveals the entropy differences between these polyethers to be in the order PEG>LPG(OH) > LPG(OMe). Moreover, experiments on fibrinogen adsorption of these surfaces via surface plasmon resonance spectroscopy are performed and correlated with the theoretical studies. We find that protein resistant properties of surfaces are likely to arise from an interplay of different factors.

Evaluation of photochemically immobilized poly(2-ethyl-2-oxazoline) thin films as protein-resistant surfaces.

Poly(2-ethyl-2-oxazoline) (PEOX) of various molecular weights were covalently immobilized on silicon wafers and gold slides to form protein-resistant surfaces via a fast and general photocoupling chemistry based on the CH insertion reaction of light-activated perfluorophenyl azide (PFPA). The thicknesses of the immobilized PEOX films ranged from 23 to 80 Å for molecular weight of 5000 to 500,000, and the grafting density reached 3.2 × 10(-3) Å(-2) for PEOX 5000. The protein-resistant property of the films was studied using bovine serum albumin (BSA) by fluorescence imaging, ellipsometry, and surface plasmon resonance imaging (SPRi). The fluorescence imaging and ellipsometry studies showed the largest amount of BSA adsorbed on PEOX 5000 and the smallest on PEOX 500,000. This was consistent with the kinetic analysis of BSA adsorption by SPRi showing that PEOX 5000 exhibited the fastest association rate and the slowest dissociation rate whereas PEOX 500,000 had the slowest association rate and the fastest dissociation rate. The PEOX film was then applied in the fabrication of carbohydrate microarrays to reduce the nonspecific adsorption of lectins and thus the background noises. Results showed that the microarray signals were significantly enhanced when the PEOX film was used.

Brush‐like copolymer as a physically adsorbed coating for protein separation by capillary electrophoresis

A brush-like copolymer consisting of poly(ethylene glycol) methyl ether methacrylate and N,N-dimethylacrylamide (PEGMA-DMA) was synthesized and used as a novel static physically adsorbed coating for protein separation by capillary electrophoresis for the first time, in order to stabilize electroosmotic flow (EOF) and suppress adsorption of proteins onto the capillary wall. Very stable and low EOF was obtained in PEGMA-DMA-coated capillary at pH 2.2-7.8. The effects of molar ratio of PEGMA to DMA, copolymer molecular mass, and pH on the separation of basic proteins were discussed. A comparative study of bare capillary with PEGMA-DMA-coated capillary for protein separation was also performed. The basic proteins could be well separated in PEGMA-DMA-coated capillary over the investigated pH range of 2.8-6.8 with good repeatability and high separation efficiency because the copolymer coating combines good protein-resistant property of PEG side chains with excellent coating ability of PDMA-contained backbone. Finally, the coating was successfully applied to the fast separation of other protein samples, such as protein mixture and egg white, which reveals that it is a potential coating for further proteomics analysis.

Methacrylate polymer layers bearing poly(ethylene oxide) and phosphorylcholine side chains as non-fouling surfaces: In vitro interactions with plasma proteins and platelets

Two methacrylate monomers, oligo(ethylene glycol) methyl ether methacrylate (OEGMA; MW = 300 g mol −1, poly(ethylene glycol) (PEG) side chains of average length n = 4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC; MW = 295 g mol −1), were grafted from silicon wafer surfaces via surface-initiated atom transfer radical polymerization. The grafted surfaces were used as model PEG and phosphorylcholine surface systems to allow comparison of the effectiveness of these two motifs in the prevention of plasma protein adsorption and platelet adhesion. It was found that at high graft density fibrinogen adsorption from plasma on the poly(MPC) and poly(OEGMA) surfaces for a given graft chain length was comparable and extremely low. At low graft density, poly(OEGMA) was slightly more effective than poly(MPC) in resisting fibrinogen adsorption from plasma. Flowing whole blood experiments showed that at low graft density the poly(OEGMA) surfaces were more resistant to fibrinogen adsorption and platelet adhesion than the poly(MPC) surfaces. At high graft density, both the poly(MPC) and poly(OEGMA) surfaces were highly resistant to fibrinogen and platelets. Immunoblots of proteins eluted from the surfaces after contact with human plasma were probed with antibodies against a range of proteins, including the contact phase clotting factors, fibrinogen, albumin, complement C3, IgG, vitronectin and apolipoprotein A-I. The blot responses were weak on the poly(MPC) and poly(OEGMA) surfaces at low graft density and zero at high graft density, again indicating strongly protein resistant properties for these surfaces. Since the side chains of the poly(OEGMA) are about 50% greater in size than those of poly(MPC), the difference in protein resistance between the poly(MPC) and poly(OEGMA) surfaces at low graft density may be due to the difference in surface coverage of the two graft types.

Implementation of Specific Bioconjugation in Polystyrene‐block‐poly(tert‐butyl acrylate)‐Based Bio‐Interfaces

AbstractSummary: Here we report on the expansion of a previously introduced biointerface platform based on thin spin‐coated films of polystyrene‐block‐poly(tert‐butyl acrylate) (PS‐b‐PtBA). Following the selective deprotection of the tert‐butyl‐ester groups in the PtBA skin layer by hydrolysis under acidic conditions, the activation with N‐hydroxysuccinimide and the subsequent derivatization with α,ω‐biotin‐amine end‐functionalized poly(ethylene glycol) (PEG), strepavidin was immobilized as anchor layer for specific immobilization of biotin‐tagged molecules. Based on contact angle, Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM) and confocal fluorescence microscopy data it was shown that the polymer films were efficiently modified. The complexation of a biotin‐tagged dye, as well as the non‐specific adsorption of proteins was determined by confocal fluorescence microscopy measurements. The surface coverage of the immobilized dye as a function of dye concentration in solution was found to be consistent with a high affinity‐type adsorption isotherm, while the protein resistant properties were found to be similar to non‐biotinylated PEG. The scope of micro‐ and nanostructured PS‐b‐PtBA biointerfaces is thereby considerably expanded to encompass the specific immobilization of biotin‐tagged biomolecules.

Preparation of Amphiphilic Polymer-Functionalized Carbon Nanotubes for Low-Protein-Adsorption Surfaces and Protein-Resistant Membranes

Multiwalled carbon nanotubes functionalized with poly(sulfone) (PSF) and poly(sulfobetaine methacrylate) (PSBMA) (MWNT-PSF/PSBMA) have been prepared through sequential atom transfer radical polymerization. The structure of MWNT-PSF/PSBMA hybrid has been characterized with FTIR, Raman spectroscopy, and high-resolution transmission electron microscopy. Incorporation of PSBMA chains to MWNTs introduces amphiphilic and protein-resistant properties to MWNT-PSF/PSBMA. Addition of 1 wt % MWNT-PSF/PSBMA to PSF films significantly improves their protein-resistant characteristic, as the composite films show a 4.4% of protein adsorption compared to poly(styrene) Petri dishes. The PSF/MWNT-PSF/PSBMA composite has been applied to prepare antifouling ultrafiltration membranes for protein separation. This work demonstrates an effective and convenient approach to prepare low-protein-adsorption surfaces and antifouling membranes.

Protein‐resistant polyurethane by sequential grafting of poly(2‐hydroxyethyl methacrylate) and poly(oligo(ethylene glycol) methacrylate) via surface‐initiated ATRP

Protein-resistant polyurethane (PU) surfaces were prepared by sequentially grafting poly(2-hydroxyethyl methacrylate) (poly(HEMA)) and poly(oligo(ethylene glycol) methacrylate) (poly(OEGMA)) via surface-initiated atom transfer radical polymerization (s-ATRP). The chain lengths of poly(HEMA) and poly(OEGMA) were regulated via the ratio of monomer to sacrificial initiator in solution. The surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). The protein resistant properties of the surfaces were assessed by single and binary adsorption experiments with fibrinogen (Fg), lysozyme (Lys), and lactalbumin (Lac). The adsorption of all three proteins on the sequentially grafted poly(HEMA)-poly(OEGMA) surfaces (PU/PH/PO) was greatly reduced compared with the unmodified PU. Adsorption decreased with increasing poly(OEGMA) chain length. On the PU/PH/PO surface with longest poly(OEGMA) chain length (∼100), the decrease in Lys adsorption was in the range of 95-98% and the decrease in Fg and Lac adsorption was >99% compared with the unmodified PU. Adsorption from binary protein solutions showed that the PU/PH/PO surfaces resisted these proteins more or less equally, that is, independent of protein size.

Poly(vinylpyrrolidone- b-styrene) block copolymers tethered surfaces for protein adsorption and cell adhesion regulation

Poly(vinylpyrrolidone- b-styrene) (PVP- b-PS) diblock copolymers tethered to glass surfaces were prepared, and the effects on protein adsorption and cellular behavior to the glass and the modified glass surfaces investigated. The PVP- b-PS grafting process was confirmed by water contact angle and XPS measurements. The results obtained for the water contact angles suggest that there are two phases that coexist on the PVP- b-PS block copolymer tethered surface, under aqueous conditions. Although the PVP- b-PS surface possessed, to some extent, a protein resistant property, following introduction of the PS segment to the end of tethered PVP, both fibrinogen and lysozyme adsorption were increased significantly. The PVP- b-PS modified surface, based on Western-blot analysis, appeared to have the greatest amount of surface bound vitronectin, however the conformation of the adsorbed vitronectin may have subsequently been affected by the surface tethered copolymer as was suggested by cell culture results. From these results, we proposed that protein adsorption and cell adhesion can be regulated by tuning the chemical compositions of diblock copolymers tethered to surfaces.

Self-Assembly of TMAO at Hydrophobic Interfaces and Its Effect on Protein Adsorption: Insights from Experiments and Simulations

We offer a novel process to render hydrophobic surfaces resistant to relatively small proteins during adsorption. This was accomplished by self-assembly of a well-known natural osmolyte, trimethylamine oxide (TMAO), a small amphiphilic molecule, on a hydrophobic alkanethiol surface. Measurements of lysozyme (LYS) adsorption on several homogeneous substrates formed from functionalized alkanethiol self-assembled monolayers (SAMs) in the presence and absence of TMAO, and direct interaction energy between the protein and functionalized surfaces, demonstrate the protein-resistant properties of a noncovalently adsorbed self-assembled TMAO layer. Molecular dynamics simulations clearly show that TMAO molecules concentrate near the CH(3)-SAM surface and are preferentially excluded from LYS. Interestingly, TMAO molecules adsorb strongly on a hydrophobic CH(3)-SAM surface, but a trade-off between hydrogen bonding with water, and hydrophobic interactions with the underlying substrate results in a nonintuitive orientation of TMAO molecules at the interface. Additionally, hydrophobic interactions, usually responsible for nonspecific adsorption of proteins, are weakly affected by TMAO. In addition to TMAO, other osmolytes (sucrose, taurine, and betaine) and a larger homologue of TMAO (N,N-dimethylheptylamine-N-oxide) were tested for protein resistance and only N,N-dimethylheptylamine-N-oxide exhibited resistance similar to TMAO. The principle of osmolyte exclusion from the protein backbone is responsible for the protein-resistant property of the surface. We speculate that this novel process of surface modification may have wide applications due to its simplicity, low cost, regenerability, and flexibility.

Protein adsorption onto CF3-terminated oligo(ethylene glycol) containing self-assembled monolayers (SAMs): the influence of ionic strength and electrostatic forces

Oligo(ethylene glycol) (OEG) containing self-assembled monolayers (SAMs) on gold are known for their protein resistant properties. The underlying molecular mechanisms and the contributions of the interactions involved, however, are still not completely understood. It is known that electrostatic, van der Waals, hydrophobic, and hydration forces all play a role in the interaction between proteins and surfaces, but it is difficult to study their influence separately and to quantify their contributions. In the present study we investigate five different OEG containing SAMs and the influence of the ionic strength and the electrostatic component on the amount of a negatively charged protein (fibrinogen) that adsorbs onto them. Atomic force microscopy (AFM) was employed to record force-distance curves with hydrophobic probes depending on the ion concentration, and the amount of the protein that adsorbs relative to a hydrophobic surface was quantified using ellipsometry. The findings suggest that electrostatic forces can create a very low energy barrier thus only slightly decreasing the number of negatively charged proteins in solution with sufficient energy to approach the surface closely, and have a rather small influence on the amount that adsorbs. The films we investigated were not protein resistant. This supports other studies, reporting that a strong short-range repulsion as for example caused by hydration forces is required to make these films resistant to the non-specific adsorption of proteins.

A surface decorated with diblock copolymer for biomolecular conjugation

A simple and attractive method was introduced to construct bioactive surfaces that exhibit non-specific protein resistant properties and high loading capacities for immobilizing various specific biomolecules. These bioactive surfaces may find wide potential biomedical applications.

Protein-Resistant Materials via Surface-Initiated Atom Transfer Radical Polymerization of 2-Methacryloyloxyethyl Phosphorylcholine

Poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) was grafted from various polymeric substrates to prepare protein-resistant materials. The poly(MPC) chain length was adjusted via the ratio of monomer to sacrificial initiator in solution. The surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS). The protein-resistant properties of the poly(MPC)-grafted surfaces were evaluated by single adsorption experiments with fibrinogen and lysozyme. It was shown that the simple three-step grafting method could be applied to modify various biomaterial surfaces including polyurethane and silicones. The adsorption of fibrinogen and lysozyme to the modified surfaces was greatly reduced compared to the unmodified surfaces, and adsorption decreased with increasing poly(MPC) chain length. On polyurethane film grafted with poly(MPC) of chain length 100, the reduction in adsorption was approx. 96% for lysozyme and approx. 99% for fibrinogen.

Protein adsorption on poly( N-isopropylacrylamide)-modified silicon surfaces: Effects of grafted layer thickness and protein size

In this work, we investigated the protein adsorption on the end-tethered thermoresponsive poly( N-isopropylacrylamide) (PNIPAAm) brushes with varying grafted layer thickness prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) on initiator-immobilized silicon surfaces. The thickness of a grafted layer was modulated by adjusting reaction time and/or solvent composition. The surface properties as a function of thickness were investigated by water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscope (AFM). The influence of PNIPAAm-grafted layer thickness on human serum albumin (HSA) adsorption in phosphate-buffered saline (PBS) (pH 7.4) at different temperature was evaluated using a radiolabeling method. In a lower thickness range (<15 nm), protein adsorption on PNIPAAm-grafted layer shows a thermoresponsive change in a certain extent, but the variation is not remarkable. However, it is interesting to observe that these surfaces exhibit good protein-resistant property. For the surface with a PNIPAAm thickness of 13.4 nm, the HSA adsorption level measured at room temperature was ∼7 ng/cm 2, corresponding to a reduction of 97% compared to the unmodified silicon surface. For thicker PNIPAAm-grafted surface with thickness of 38.1 nm, the adsorption results of three proteins (HSA, fibrinogen, and lysozyme) with different sizes and charges indicate that the PNIPAAm-modified surface exhibits a size-sensitive property of protein adsorption.

Protein-Resistant Polymer Coatings Based on Surface-Adsorbed Poly(aminoethyl methacrylate)/Poly(ethylene glycol) Copolymers

We report on the protein-resistant properties of glass substrates coated with novel copolymers of 2-aminoethyl methacrylate hydrochloride and poly(ethylene glycol) methyl ether methacrylate (AEM-PEG). In comparison to currently available protein-blocking polymer systems, such as poly-l-lysine-poly(ethylene glycol), silane-based poly(ethylene glycol), and poly(ethylene glycol) brushes prepared by surface-initiated polymerization, the proposed AEM-PEG offers the combined advantages of low cost, simplicity of use, and applicability in aqueous solutions. We demonstrate the capability of AEM-PEG to block the surface binding of globular proteins (tubulin), their assemblies (microtubules), and functional motor proteins (kinesin-1). Moreover, we demonstrate the applicability of AEM-PEG for surface patterning of proteins in microfluidic devices.

Nonfouling Polyampholytes from an Ion-pair Comonomer with Biomimetic Adhesive Groups.

Nonfouling surfaces play a very important role for the development of biosensors,1 medical implants,2 and drug delivery vehicles.3 However, few materials and modification methods have been reported to improve the nonfouling properties of surfaces effectively and conveniently. Among these reported materials, zwitterionic polymers, with both positive and negative charges on the side chains, showed excellent nonfouling properties in our previous studies.4–7 Based on these studies, it was hypothesized that a nanometer-scale homogeneous mixture of balanced charge groups will present protein-resistant properties. Recently, this assumption has been proved on hydrogels via free radical polymerization,8 polymer thin films 9 via surface-initiated atom transfer radical polymerization (SI-ATRP) and polymer thin films via self-assembly. 10, 11 The ratios of positively to negatively charged monomers need to be optimized to achieve 1:1 on surfaces for most of these cases.

Tunable Protein-Resistance of Polycation-Terminated Polyelectrolyte Multilayers

The prevention of nonspecific protein adsorption is a crucial prerequisite for many biomedical and biotechnological applications. Therefore, the design of robust and versatile methods conferring optimal protein-resistance properties to surfaces has become a challenging issue. Here we report the unexpected case of polycation-ending polyelectrolyte multilayers (PEM) that efficiently prevented the adsorption of a negatively charged model protein, glucose oxidase (GOX). PEM films were based on two typical weak poyelectrolytes: poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). No chemical modification of the polyelectrolytes was required and tunable GOX adsorption was possible by simply changing the buildup pH conditions. Protein-resistance properties are attributed to high film hydration becoming the predominant factor over electrostatic interactions. We explain this effect by oscillations of the internal PAA ionization state throughout the buildup, which results in an excess of carboxylic acid groups within the film. This excess acts as a reservoir of potential carboxylate groups compensating the outer PAH positive charges. Partial results indicated that the system was also resistant to the adsorption of a positively charged protein, lysozyme. Control of the internal ionization of weak polyelectrolyte multilayers might open a route toward simple tuning of protein adsorption. These results should help to rationalize the design of biomaterials, biosensors, or protein separation devices.

Amphiphilicity in homopolymer surfaces reduces nonspecific protein adsorption.

Amphiphilic homopolymer films have been immobilized onto substrates to study the interactions of these polymers with proteins. X-ray photoelectron spectroscopy (XPS) was utilized to measure the amount of protein adsorption. Amphiphilic homopolymers have been shown to reduce protein adsorption, despite the high affinity of the hydrophobic or hydrophilic functional groups by themselves toward proteins. This protein-resistant property seems to arise from the unique molecular-scale alternation of incompatible functionalities. The combination of incompatible functionalities with a predefined alternating pattern within a monomer could provide a potential design for nonfouling materials.

Construction of Protein-Resistant pOEGMA Films by Helicon Plasma-Enhanced Chemical Vapor Deposition

This paper describes the formation of protein-resistant, poly(ethylene glycol) methyl ether methacrylate (pOEGMA) thin films by helicon plasma-enhanced chemical vapor deposition (helicon-PECVD). pOEGMA was successfully grafted onto a silicon substrate, as a model substrate, without any additional surface initiators, by plasma polymerization of OEGMA. The resulting pOEGMA films were characterized by ellipsometry, FT-IR spectroscopy, X-ray photoelectron spectroscopy and contact angle goniometry. To investigate the protein-resistant property of the pOEGMA films, four different proteins, bovine serum albumin, fibrinogen, lysozyme and ribonuclease A, were tested as model proteins for ellipsometric measurements. The ellipsometric thickness change for all the model proteins was less than 3 Å, indicating that the formed pOEGMA films are protein-resistant.

Surfaces having dual fibrinolytic and protein resistant properties by immobilization of lysine on polyurethane through a PEG spacer

The objective of this work is to develop a blood contacting surface that possesses both resistance to nonspecific protein adsorption and clot lysing properties. Chemical modification of a polyurethane (PU) surface with poly(ethylene glycol) (PEG); and lysine was used to create a plasminogen-binding potentially fibrinolytic surface. The preparation involves modification of the PU surface with dihydroxy PEG, reaction of the unreacted distal OH with N,N'-disuccinimidyl carbonate (DSC) to produce a PU-PEG-NHS surface, followed by conjugation of epsilon-amino-protected lysine (H-Lys(t-BOC)-OH) by reaction of the alpha-amino group with the NHS and deprotection. The result is a lysine-derivatized surface in which the epsilon-amino groups of the lysine are free to participate in binding plasminogen and tissue plasminogen activator (t-PA). Surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Protein adsorption experiments showed that nonspecific protein adsorption was greatly reduced on these surfaces and that they adsorbed significant quantities of plasminogen from plasma. After incubation with plasma and treatment with t-PA the surfaces were able to dissolve nascent plasma clots formed around them.