Lowering protein fouling by rational processing of fluorine-free hydrophobic coatings
Graphical abstract
Introduction
Protein accumulation at liquid-solid interfaces has impacted a variety of applications in food-processing industries [1], implantable bioelectronics [2], implants (e.g. dental, hip replacement, coronary stents) [3], heat exchangers [4], filtration membranes [5], and industrial equipment [6], to name only a few [7]. Strategies devoted to mitigate irreversible adherence of bio-foulants predominantly include protective coatings. In general, many coating materials of inorganic [8], [9], organic [10], [11], [12] and also hybrid [13] nature that provided surfaces with mainly hydrophilic [14] character proved unfavorable to bioaccumulations. A large volume of literature supports these findings [15] but lately contradicting reports showed that hydrophobic surfaces may also reduce protein adherence [16].
Due to their low surface energy, hydrophobic and superhydrophobic materials [17], [18], [19] support the formation of air pockets at the surface that stabilize a Cassie-Baxter wetting state [20], associated with water repellent behavior (θ ≥ 150°). One key to achieve such wetting behavior is to rationally tune the dual-level nano- and micro- morphologies of the coating layer [21]. This feature was recently exploited to design coatings with antifouling properties. For example, Zheng et al. fabricated a polyurethane/Pluronic F-127 surface designed with Lotus leaf-like topology that significantly reduced protein adsorption when compared to substrates lacking such morphology [22]. Chapman and Regan have used a galvanic reaction to create architectural copper surfaces with superhydrophobic behavior that were efficient to lower protein adhesion, feature beneficial for sensor applications [23]. Reports underlining that hydrophobic surfaces have the ability to resist protein fouling also include that of Stallard et al. [16] The authors have studied in-situ adsorption of serum proteins on plasma deposited polymer surfaces [16]. The investigation found that, indeed, hydrophilic siloxane substrates were more unfavorable to protein adhesion than their homologues of intermediate hydrophobicity. Surprisingly, the analysis performed on superhydrophobic surfaces revealed a significant reduction in protein fouling. The authors attributed these findings to the specific surface chemistry that influences protein interaction with the coated substrates [16]. Wong et al. have explored the potential of layer-by-layer films for use as antifouling coatings [24]. Their study revealed that neutral charge and molecular-level heterogeneities created by rationally tuning a surface topology with fine hydrophobic/hydrophilic regions exhibited good resistance to protein adsorption [24]. These examples suggest that materials and innovative surface modification processes used in super/hydrophobic coating technologies may also prove useful in preventing protein fouling.
Silica is a popular inorganic material used in designing coatings because it has low toxicity to living bodies, is environmentally friendly and relatively cheap [25]. Mainly prepared from tetraethylorthosilicate (TEOS) [26], silica particles as coating material were used exclusively in modification of textiles [27], [28], [29], [30]. Despite its fire retardant [31], UV resistant [32] and especially antibacterial [33] properties silica has been less explored as a protein antifouling agent. This property was studied but it was inherently assigned to the organic material, used in tandem with silica. For example, Hu et al. prepared silica particles coated with a zwitterionic organosiloxane shell that proved stable in high salt concentrations and reduced adsorption of fetal bovine serum (FBS) [34]. Note that one of the paramount challenges in designing antifouling nano- and micro- particulate systems is their stability in saline buffer solutions.
Another silane agent that attracted attention lately in coating technology is methyltrimethoxysilane (MTMS). MTMS has an alkyl (CH3) inactive arm in comparison with its homologue TEOS. This structural feature allows easier preparation through a sol-gel process of hydrophobic surfaces than is possible with TEOS which tends to yield hydrophilic coatings that necessitate post-modifications to repel water. Hydrolysis and condensation of MTMS yield patchy species that carry hydrophobic (CH3) and hydrophilic (OH) sites [35], [36], [37]. Fewer studies have taken advantage of MTMS alone to achieve surface hydrophobicity [38], [39], [40]; rather it was used in combination with other silane agents [41], [42]. Furthermore, to date no investigation has been performed on the possible use of MTMS as a protective hydrophobic coating to reduce protein fouling.
Monomers, polymers and copolymers are the most used organic materials for production of antifouling surfaces but, except the fluorine-derived ones [43], [44], render surfaces hydrophilic [45]. Natural amino acids were found to prevent protein film formation despite the fact they share similar repeat units. For example, Lin et al. have employed thiol chemistry and modified gold-coated stainless steel surfaces, commonly used in bioelectronics, with zwitterionic cysteine [46]. The performance of cysteine-modified surfaces was significantly greater than that of pristine stainless steel and gold, respectively [46]. Ultra-low protein adsorption (<0.3 ng/cm2) was reported by Chen et al. by using polyampholytes made of alternating glutamic acid/lysine and aspartic acid/lysine [47]. Poly(vinyl pyrrolidone), poly(ethylene glycol), polyethylene oxide, polyurethane, polydimethylsiloxane, poly(2-methoxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine) along with their derived copolymers are a few examples of polymeric surface modifiers that enable protein repellency. Interaction of these macromolecules with body fluids was largely described in several review articles [48], [49], [50], [51].
Poly(methyl methacrylate) (PMMA) and its derivatives are another class of biocompatible polymers often involved in designing antifouling surfaces. As an example, Liu, Guo and Zhang reported that spin-cast films of PMMA alone on silicon wafers resulted in thicker adsorbed protein layers when compared to PMMA derivatives with longer side chains [52]. The PMMA film was hydrophilic while the other homologues were more hydrophobic [52]. Based on such studies, one can infer that a simple approach, not fully explored to date, is tuning the dual-morphology of PMMA protective films to create topological surfaces that will potentially limit protein adherence.
In this study, glass slides (GS) and stainless steel (SS) surfaces were modified with inorganic and organic functionalities. The purpose of the investigation was two-fold: 1) to rationally choose the preparation parameters and approaches leading to reproducible hydrophobic coating layers irrespective of the substrate nature and 2) to evaluate the potential in lowering protein adsorption of the two-step inorganic and multi-step organic coatings. Protection of surfaces with an inorganic silica-derived layer was realized by hydrolysis and condensation of MTMS catalyzed by ammonium hydroxide in anhydrous ethanol for various amounts of reaction time (R). Three methods were used to initially functionalize GS: soaking (S), dip coating and spin casting. The coating processing parameters were chosen after monitoring the hydrophobicity of GS surface via contact angle measurements. The organic coat was prepared by mixing polystyrene (PS) and polymethylmethacrylate (PMMA) in different ratios. Coating films were spin cast at three speeds. PS was selectively removed using cyclohexane to yield a hierarchical PMMA layer. SS surfaces were modified under the same conditions identified for GS. The functionalized GS and SS surfaces were incubated in a saline buffer solution of fetal bovine serum (FBS). Qualitative and quantitative investigations on the amount of protein adsorbed on the substrates revealed that the two coating approaches led to a significant reduction in FBS fouling. These investigations give new insights into rationally designing processes enabling the creation of surfaces able to exhibit target properties.
Section snippets
Materials
Fetal bovine serum (FBS, USA origin, sterile-filtered, suitable for cell culture, hemoglobin ≤ 25 mg/dL, endotoxin ≤ 10 EU/mL), Triton X-100 (laboratory grade), phosphate buffer saline (PBS, tablet), methyltrimethoxysilane (MTMS, 98%), poly(methyl methacrylate), (PMMA, Mw = 15,000 Da), polystyrene (PS, Mw = 35,000 Da), tetrahydrofuran (THF), cyclohexane and ammonium hydroxide (NH4OH, 28–30%) were purchased from Sigma-Aldrich. Pierce bicinchoninic acid (BCA) protein assay kit (catalog # 23,227)
Preparation of coated substrates
Basic pH conditions favor hydrolysis and condensation of MTMS yielding a multitude of active species (linear and cyclic oligomers, particles and particle aggregates) [37], [55]. These species reacted with the free hydroxyl (OH) groups of glass and covered GS surfaces more or less uniformly. The two constituents, PS and PMMA of the multi-step organic coating (Scheme 1) undergo phase separation upon surface deposition and THF evaporation. Selective removal of PS enabled an architectural surface
Conclusion
Preparation protocols involving two-step inorganic and multi-step organic coatings were designed to functionalize glass (GS) and stainless steel (SS) substrates, imparting them with hydrophobic properties. The inorganic coating was prepared by reacting methyltrimethoxysilane (MTMS) in the presence of a base catalyst, ammonium hydroxide (NH4OH), both dispersed in anhydrous ethanol. For the organic homologue, polymethylmethacrylate (PMMA) and polystyrene (PS) were used, followed by the selective
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgments
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors thank Prof. Elsa Reichmanis (School of Chemical and Biomolecular Engineering, Georgia Institute of Technology) and Prof. Paul Russo (School of Materials Science and Engineering, Georgia Institute of Technology) for access to their laboratory facilities. The authors also acknowledge the Institute of Electronics and Nanotechnology (Georgia Institute of
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These authors contributed equally
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Present address: Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109-2136, USA