Antibody-independent surface plasmon resonance assays for influenza vaccine quality control.

Surface plasmon resonance (SPR)-based biosensors have emerged as a powerful platform for bioprocess monitoring due to their ability to detect biointeractions in real time, without the need for labeling. Paramount for the development of a robust detection platform is the immobilization of a ligand with high specificity and affinity for the in-solution species of interest. Following the 2009 H1N1 pandemic, much effort has been made toward the development of quality control platforms for influenza A vaccine productions, many of which have employed SPR for detection. Due to the rapid antigenic drift of influenza's principal surface protein, hemagglutinin, antibodies used for immunoassays need to be produced seasonally. The production of these antibodies represents a 6-8-week delay in immunoassay and, thus, vaccine availability. This review focuses on SPR-based assays that do not rely on anti-HA antibodies for the detection, characterization, and quantification of influenza A in bioproductions and biological samples. KEY POINTS: • The single radial immunodiffusion assay (SRID) has been the gold standard for the quantification of influenza vaccines since 1979. Due to antigenic drift of influenza's hemagglutinin protein, new antibody reagents for the SRID assay must be produced each year, requiring 6-8 weeks. The resulting delay in immunoassay availability is a major bottleneck in the influenza vaccine pipeline. This review highlights ligand options for the detection and quantification of influenza viruses using surface plasmon resonance biosensors.

A Biosensor Assay Based on Coiled-Coil-Mediated Human ACE2 Receptor Capture for the Analysis of Its Interactions with the SARS-CoV-2 Receptor Binding Domain.

Surface plasmon resonance (SPR)-based biosensing enables the characterization of protein-protein interactions. Several SPR-based approaches have been designed to evaluate the binding mechanism between the angiotensin-converting enzyme 2 (ACE2) receptor and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein leading to a large range of kinetic and thermodynamic constants. This chapter describes a robust SPR assay based on the K5/E5 coiled-coil capture strategy that reduces artifacts. In this method, ACE2 receptors were produced with an E5-tag and immobilized as ligands in the SPR assay. This chapter details methods for high-yield production and purification of the studied proteins, functionalization of the sensor chip, conduction of the SPR assay, and data analysis.

A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

Grafting Strategies of Oxidation-Prone Coiled-Coil Peptides for Protein Capture in Bioassays: Impact of Orientation and the Oxidation State.

Many biomedical and biosensing applications require functionalization of surfaces with proteins. To this end, the E/K coiled-coil peptide heterodimeric system has been shown to be advantageous. First, Kcoil peptides are covalently grafted onto a given surface. Ecoil-tagged proteins can then be non-covalently captured via a specific interaction with their Kcoil partners. Previously, oriented Kcoil grafting was achieved via thiol coupling, using a unique Kcoil with a terminal cysteine residue. However, cysteine-terminated Kcoil peptides are hard to produce, purify, and oxidize during storage. Indeed, they tend to homodimerize and form disulfide bonds via oxidation of their terminal thiol group, making it impossible to later graft them on thiol-reactive surfaces. Kcoil peptides also contain multiple free amine groups, available for covalent coupling through carbodiimide chemistry. Grafting Kcoil peptides on surfaces via amine coupling would thus guarantee their immobilization regardless of their terminal cysteine's oxidation state, at the expense of the control over their orientation. In this work, we compare Kcoil grafting strategies for the subsequent capture of Ecoil-tagged proteins, for applications such as surface plasmon resonance (SPR) biosensing and cell culture onto protein-decorated substrates. We compare the "classic" thiol coupling of cysteine-terminated Kcoil peptides to the amine coupling of (i) monomeric Kcoil and (ii) dimeric Kcoil-Kcoil linked by a disulfide bond. We have observed that SPR biosensing performances relying on captured Ecoil-tagged proteins were similar for amine-coupled dimeric Kcoil-Kcoil and thiol-coupled Kcoil peptides, at the expense of higher Ecoil-tagged protein consumption. For cell culture applications, Ecoil-tagged growth factors captured on amine-coupled monomeric Kcoil signaled through cell receptors similarly to those captured on thiol-coupled Kcoil peptides. Altogether, while oriented thiol coupling of cysteine-terminated Kcoil peptides remains the most reliable and versatile platform for Ecoil-tagged protein capture, amine coupling of Kcoil peptides, either monomeric or dimerized through a cysteine bond, can offer a good alternative when the challenges and costs associated with the production of monomeric cysteine-tagged Kcoil are too dissuasive for the application.

Coiled-Coil-Based Biofunctionalization of 100 nm Gold Nanoparticles with the Trastuzumab Antibody for the Detection of HER2-Positive Cancer Cells.

We compared different biofunctionalization strategies for immobilizing trastuzumab, an IgG targeting the HER2 biomarker, onto 100 nm spherical gold nanoparticles because of the E/K coiled-coil peptide heterodimer. First, Kcoil peptides were grafted onto the gold surface while their Ecoil partners were genetically encoded at the C-terminus of trastuzumab's Fc region, allowing for a strong and specific interaction between the antibodies and the nanoparticles. Gold nanoparticles with no Kcoil peptides on their surface were also produced to immobilize Ecoil-tagged trastuzumab antibodies via the specific adsorption of their negatively charged Ecoil tags on the positively charged gold surface. Finally, the nonspecific adsorption of wild-type trastuzumab on the gold surface was also assessed, with and without Kcoil peptides grafted on it beforehand. We developed a thorough workflow to systematically compare the immobilization strategies regarding the stability of nanoparticles, antibody coverage, and ability to specifically bind to HER2-positive breast cancer cells. All nanoparticles were highly monodisperse and retained their localized surface plasmon resonance properties after biofunctionalization. A significant increase in the amount of immobilized antibodies was observed with the two oriented coil-based strategies compared to nonspecific adsorption. Finally, all biofunctionalization strategies allowed for the detection of HER2-positive breast cancer cells, but among the investigated approaches, we recommend using the E/K coiled-coil-based strategy for gold nanoparticle biofunctionalization because it allows for the qualitative and quantitative detection of HER2-positive cells with a higher contrast compared to HER2-negative cells.

Injectable Lyophilized Chitosan-Thrombin-Platelet-Rich Plasma (CS-FIIa-PRP) Implant to Promote Tissue Regeneration: In Vitro and Ex Vivo Solidification Properties.

Freeze-dried chitosan formulations solubilized in platelet-rich plasma (PRP) are currently evaluated as injectable implants with the potential for augmenting the standard of care for tissue repair in different orthopedic conditions. The present study aimed to shorten the solidification time of such implants, leading to an easier application and a facilitated solidification in a wet environment, which were direct demands from orthopedic surgeons. The addition of thrombin to the formulation before lyophilization was explored. The challenge was to find a formulation that coagulated fast enough to be applied in a wet environment but not too fast, which would make handling/injection difficult. Four thrombin concentrations were analyzed (0.0, 0.25, 0.5, and 1.0 NIH/mL) in vitro (using thromboelastography, rheology, indentation, syringe injectability, and thrombin activity tests) as well as ex vivo (by assessing the implant's adherence to tendon tissue in a wet environment). The biomaterial containing 0.5 NIH/mL of thrombin significantly increased the coagulation speed while being easy to handle up to 6 min after solubilization. Furthermore, the adherence of the biomaterial to tendon tissues was impacted by the biomaterial-tendon contact duration and increased faster when thrombin was present. These results suggest that our biomaterial has great potential for use in regenerative medicine applications.

A simple method for poly-D-lysine coating to enhance adhesion and maturation of primary cortical neuron cultures in vitro.

Introduction: Glass coverslips are used as a substrate since Harrison's initial nerve cell culture experiments in 1910. In 1974, the first study of brain cells seeded onto polylysine (PL) coated substrate was published. Usually, neurons adhere quickly to PL coating. However, maintaining cortical neurons in culture on PL coating for a prolonged time is challenging. Methods: A collaborative study between chemical engineers and neurobiologists was conducted to find a simple method to enhance neuronal maturation on poly-D-lysine (PDL). In this work, a simple protocol to coat PDL efficiently on coverslips is presented, characterized, and compared to a conventional adsorption method. We studied the adhesion and maturation of primary cortical neurons with various morphological and functional approaches, including phase contrast microscopy, immunocytochemistry, scanning electron microscopy, patch clamp recordings, and calcium imaging. Results: We observed that several parameters of neuronal maturation are influenced by the substrate: neurons develop more dense and extended networks and synaptic activity is enhanced, when seeded on covalently bound PDL compared to adsorbed PDL. Discussion: Hence, we established reproducible and optimal conditions enhancing maturation of primary cortical neurons in vitro. Our method allows higher reliability and yield of results and could also be profitable for laboratories using PL with other cell types.

Affinity-controlled capture and release of engineered monoclonal antibodies by macroporous dextran hydrogels using coiled-coil interactions.

Long-term delivery is a successful strategy used to reduce the adverse effects of monoclonal antibody (mAb)-based treatments. Macroporous hydrogels and affinity-based strategies have shown promising results in sustained and localized delivery of the mAbs. Among the potential tools for affinity-based delivery systems, the de novo designed Ecoil and Kcoil peptides are engineered to form a high-affinity, heterodimeric coiled-coil complex under physiological conditions. In this study, we created a set of trastuzumab molecules tagged with various Ecoil peptides and evaluated their manufacturability and characteristics. Our data show that addition of an Ecoil tag at the C-termini of the antibody chains (light chains, heavy chains, or both) does not hinder the production of chimeric trastuzumab in CHO cells or affect antibody binding to its antigen. We also evaluated the influence of the number, length, and position of the Ecoil tags on the capture and release of Ecoil-tagged trastuzumab from macroporous dextran hydrogels functionalized with Kcoil peptide (the Ecoil peptide-binding partner). Notably, our data show that antibodies are released from the macroporous hydrogels in a biphasic manner; the first phase corresponding to the rapid release of residual, unbound trastuzumab from the macropores, followed by the affinity-controlled, slow-rate release of antibodies from the Kcoil-functionalized macropore surface.

Coiled-coil peptide-based assembly of a plasmonic core-satellite polymer-metal nanocomposite as an efficient photothermal agent for drug delivery applications.

Polymer-metal nanocomposites have widespread applications in biomedical fields such as imaging, catalysis, and drug delivery. These particles are characterized by combined organic and inorganic properties. Specifically, photothermal nanocomposites incorporating polymeric and plasmonic nanoparticles (NPs) have been designed for both triggered drug release and as imaging agents. However, the usual design of nanocomposites confers characteristic issues, among which are the decrease of optical properties and resulting low photothermal efficiency, as well as interactions with loaded drugs. Herein, we report the design of a core-satellite polymer-metal nanocomposite assembled by coiled-coil peptides and its superior photothermal efficiency compared to electrostatic-driven nanocomposites which is the standard design. We also found that the orientation of gold nanorods on the surface of polymeric NPs is of importance in the final photothermal efficiency and could be exploited for various applications. Our findings provide an alternative to current wrapping and electrostatic assembly of nanocomposites with the help of coiled-coil peptides and an improvement of the control over core-satellite assemblies with plasmonic NPs. It paves the way to highly versatile assemblies due to the nature of coiled-coil peptides to be easily modified and sensitive to pH or temperature.

Macroporous dextran hydrogels for controlled growth factor capture and delivery using coiled-coil interactions.

Macroporous hydrogels possess a vast potential for various applications in the biomedical field. However, due to their large pore size allowing for unrestricted diffusion in the macropore network, macroporous hydrogels alone are not able to efficiently capture and release biomolecules in a controlled manner. There is thus a need for biofunctionalized, affinity-based gels that can efficiently load and release biomolecules in a sustained and controlled manner. For this purpose, we report here the use of a E/K coiled-coil affinity pair for the controlled capture and delivery of growth factors from highly interconnected, macroporous dextran hydrogels. By conjugating the Kcoil peptide to the dextran backbone, we achieved controlled loading and release of Ecoil-tagged Epidermal and Vascular Endothelial Growth Factors. To finely tune the behavior of the gels, we propose four control parameters: (i) macropore size, (ii) Kcoil grafting density, (iii) Ecoil valency and (iv) E/K affinity. We demonstrate that Kcoil grafting can produce a 20-fold increase in passive growth factor capture by macroporous dextran gels. Furthermore, we demonstrate that our gels can release as little as 20% of the loaded growth factors over one week, while retaining bioactivity. Altogether, we propose a versatile, highly tunable platform for the controlled delivery of growth factors in biomedical applications. STATEMENT OF SIGNIFICANCE: This work presents a highly tunable platform for growth factor capture and sustained delivery using affinity peptides in macroporous, fully interconnected dextran hydrogels. It addresses several ongoing challenges by presenting: (i) a versatile platform for the delivery of a wide range of stable, bioactive molecules, (ii) a passive, affinity-based loading of growth factors in the platform, paving the way for in situ (re)loading of the device and (iii) four different control parameters to finely tune growth factor capture and release. Altogether, our macroporous dextran hydrogels have a vast potential for applications in controlled delivery, tissue engineering and regenerative medicine.

Effect of Chitosan on Alginate-Based Macroporous Hydrogels for the Capture of Glioblastoma Cancer Cells.

Glioblastoma multiforme is a type of brain cancer associated with a very low survival rate since a large number of cancer cells remain infiltrated in the brain despite the treatments currently available. This work presents a macroporous hydrogel trap, destined to be implanted in the surgical cavity following tumor resection and designed to attract and retain cancer cells, in order to eliminate them afterward with a lethal dose of stereotactic radiotherapy. The biocompatible hydrogel formulation comprises sodium alginate (SA) and chitosan (CHI) bearing complementary electrostatic charges and stabilizing the gels in saline and cell culture media, as compared to pristine SA gels. The highly controlled and interconnected porosity, characterized by X-ray microCT, yields mechanical properties comparable to those of brain tissues and allows F98 glioblastoma cells to penetrate the gels within the entire volume, as confirmed by fluorescence microscopy. The addition of a grafted -RGD peptide on SA, combined with CHI, significantly enhances the adhesion and retention of F98 cells within the gels. Overall, the best compromise between low proliferation and a high level of accumulation and retention of F98 cells was obtained with the hydrogel formulated with 1% SA and 0.2% CHI, without the -RGD adhesion peptide.

Multi-temperature experiments to ease analysis of heterogeneous binder solutions by surface plasmon resonance biosensing.

Surface Plasmon Resonance (SPR) biosensing is a well-established tool for the investigation of binding kinetics between a soluble species and an immobilized (bio)molecule. While robust and accurate data analysis techniques are readily available for single species, methods to exploit data collected with a solution containing multiple interactants are scarce. In a previous study, our group proposed two data analysis algorithms for (1) the precise and reliable identification of the kinetic parameters of N interactants present at different ratios in N mixtures and (2) the estimation of the composition of a given mixture, assuming that the kinetic parameters and the total concentration of all interactants are known. Here, we extend the first algorithm by reducing the number of necessary mixtures. This is achieved by conducting experiments at different temperatures. Through the Van't Hoff and Eyring equations, identifying the kinetic and thermodynamic parameters of N binders becomes possible with M mixtures with M comprised between 2 and N and at least N/M temperatures. The second algorithm is improved by adding the total analyte concentration as a supplementary variable to be identified in an optimization routine. We validated our analysis framework experimentally with a system consisting of mixtures of low molecular weight drugs, each competing to bind to an immobilized protein. We believe that the analysis of mixtures and composition estimation could pave the way for SPR biosensing to become a bioprocess monitoring tool, on top of expanding its already substantial role in drug discovery and development.

Impact of the temperature on the interactions between common variants of the SARS-CoV-2 receptor binding domain and the human ACE2.

Several key mutations in the Spike protein receptor binding domain (RBD) have been identified to influence its affinity for the human Angiotensin-Converting Enzyme 2 (ACE2). Here, we perform a comparative study of the ACE2 binding to the wild type (Wuhan) RBD and some of its variants: Alpha B.1.1.7, Beta B.1.351, Delta B.1.617.2, Kappa B.1.617.1, B.1.1.7 + L452R and Omicron B.1.1.529. Using a coiled-coil mediated tethering approach of ACE2 in a novel surface plasmon resonance (SPR)-based assay, we measured interactions at different temperatures. Binding experiments at 10 °C enhanced the kinetic dissimilarities between the RBD variants and allowed a proper fit to a Langmuir 1:1 model with high accuracy and reproducibility, thus unraveling subtle differences within RBD mutants and ACE2 glycovariants. Our study emphasizes the importance of SPR-based assay parameters in the acquisition of biologically relevant data and offers a powerful tool to deepen our understanding of the role of the various RBD mutations in ACE2 interaction binding parameters.

Scratching the Surface of the Protein Corona: Challenging Measurements and Controversies.

This Review aims to provide a systematic analysis of the literature regarding ongoing debates in protein corona research. Our goal is to portray the current understanding of two fundamental and debated characteristics of the protein corona, namely, the formation of mono- or multilayers of proteins and their binding (ir)reversibility. The statistical analysis we perform reveals that these characterisitics are strongly correlated to some physicochemical factors of the NP-protein system (particle size, bulk material, protein type), whereas the technique of investigation or the type of measurement (in situ or ex situ) do not impact the results, unlike commonly assumed. Regarding the binding reversibility, the experimental design (either dilution or competition experiments) is also shown to be a key factor, probably due to nontrivial protein binding mechanisms, which could explain the paradoxical phenomena reported in the literature. Overall, we suggest that to truly predict and control the protein corona, future efforts should be directed toward the mechanistic aspects of protein adsorption.

On the Use of Surface Plasmon Resonance Biosensing to Understand IgG-FcγR Interactions.

Surface plasmon resonance (SPR)-based optical biosensors offer real-time and label-free analysis of protein interactions, which has extensively contributed to the discovery and development of therapeutic monoclonal antibodies (mAbs). As the biopharmaceutical market for these biologics and their biosimilars is rapidly growing, the role of SPR biosensors in drug discovery and quality assessment is becoming increasingly prominent. One of the critical quality attributes of mAbs is the N-glycosylation of their Fc region. Other than providing stability to the antibody, the Fc N-glycosylation influences immunoglobulin G (IgG) interactions with the Fcγ receptors (FcγRs), modulating the immune response. Over the past two decades, several studies have relied on SPR-based assays to characterize the influence of N-glycosylation upon the IgG-FcγR interactions. While these studies have unveiled key information, many conclusions are still debated in the literature. These discrepancies can be, in part, attributed to the design of the reported SPR-based assays as well as the methodology applied to SPR data analysis. In fact, the SPR biosensor best practices have evolved over the years, and several biases have been pointed out in the development of experimental SPR protocols. In parallel, newly developed algorithms and data analysis methods now allow taking into consideration complex biomolecular kinetics. In this review, we detail the use of different SPR biosensing approaches for characterizing the IgG-FcγR interactions, highlighting their merit and inherent experimental complexity. Furthermore, we review the latest SPR-derived conclusions on the influence of the N-glycosylation upon the IgG-FcγR interactions and underline the differences and similarities across the literature. Finally, we explore new avenues taking advantage of novel computational analysis of SPR results as well as the latest strategies to control the glycoprofile of mAbs during production, which could lead to a better understanding and modelling of the IgG-FcγRs interactions.

Robust Segmentation-Free Algorithm for Homogeneity Quantification in Images.

OBJECTIVE: Homogeneity is a notion used to describe images in various fields and is often linked to critical aspects of those fields. However, this term is rarely defined in the literature and no gold standard exists for its quantification. A few quantification algorithms have been proposed, but they lack both simplicity and robustness. As a result, the scientific community uses the notion of homogeneity in subjective analysis, preventing objective comparison of a large number of data or of different studies. The main objectives of this manuscript are to propose a definition of homogeneity and an algorithm for its quantification. METHOD: This algorithm, called MASQH, rely on a multi-scale, statistical and segmentation-free approach and outputs a single homogeneity index, which makes it robust and easy to use. RESULTS: The performance and reliability of the method are demonstrated through three case studies: Firstly, on synthetic images to study the behavior and assess the relevance of the algorithm in diverse situations and hence, in various potential fields. Secondly, on histological images derived from experimental chitosan-platelet-rich-plasma hybrid biomaterial, where the quantitative results are compared to a qualitative classification provided by an expert in the field. Thirdly, on experimental nanocomposites images for which results are compared to two other homogeneity quantification algorithms from the field of nanocomposites. CONCLUSION AND SIGNIFICANCE: By quantifying homogeneity, the MASQH method may help to compare disparate studies in the literature and quantitatively demonstrate the impact of homogeneity in various fields. The MASQH method is freely available online.

Poly(2-Propylacrylic Acid) Increases In Vitro Bioactivity of Chitosan/mRNA Nanoparticles.

Chitosan-based nanoparticles have been extensively studied for the delivery of nucleic acids. Previous results suggest that these nanoparticles have limited ability to escape the endosome, one of the main cellular barriers hindering nucleic acid delivery. Escape can be improved by the addition of endosomolytic agents during the formulation process or by developing delivery systems with intrinsic properties to disrupt endosomal membranes. In this study, Poly(2-Propylacrylic Acid) (PPAA), an anionic synthetic polymer with known membrane lytic activity was added to the binary chitosan/mRNA nanoparticles to improve bioactivity. The ionization behavior of PPAA was characterized to identify conditions in which PPAA is sufficiently charged to interact electrostatically with chitosan and thus form nanoparticles. The physicochemical characteristics (hydrodynamic diameter, polydispersity index, ζ-potential) and the in vitro transfection efficiency (bioactivity) of this new family of CS/mRNA/PPAA ternary nanoparticles were evaluated. The addition of PPAA to CS/mRNA nanoparticles was shown to be an efficient strategy to augment in vitro bioactivity. The optimal formulation reached an expression level  ~86% of the commercial lipid control at pH 6.5 without any signs of metabolic toxicity. In this paper, we report the effect of salt and pH on the ionization behavior of PPAA and demonstrate 1) successful incorporation of PPAA into/onto nanoparticles, 2) improved bioactivity with PPAA, and 3) that the kosmotropic effects of trehalose play a minimal role in the apparent increase in bioactivity in presence of trehalose.

Determination of the composition of heterogeneous binder solutions by surface plasmon resonance biosensing.

Surface plasmon resonance-based biosensors have been extensively applied to the characterization of the binding kinetics between purified (bio)molecules, thanks to robust data analysis techniques. However, data analysis for solutions containing multiple interactants is still at its infancy. We here present two algorithms for (1) the reliable and accurate determination of the kinetic parameters of N interactants present at different ratios in N mixtures and (2) the estimation of the ratios of each interactant in a given mixture, assuming that their kinetic parameters are known. Both algorithms assume that the interactants compete to bind to an immobilized ligand in a 1:1 fashion and necessitate prior knowledge of the total concentration of all interactants combined. The effectiveness of these two algorithms was experimentally validated with a model system corresponding to mixtures of four small molecular weight drugs binding to an immobilized protein. This approach enables the in-depth characterization of mixtures using SPR, which may be of considerable interest for many drug discovery or development applications, notably for protein glycovariant analysis.

Binding mechanism of a de novo coiled coil complex elucidated from surface forces measurements.

We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy. The binding energy of the coiled-coil complex increased in a stepwise fashion rather than monotonically with the overlapping distance, each step corresponding to the interaction between a quantized number of heptads. Surface forces data were corroborated to surface plasmon resonance measurements and molecular dynamics simulations and allowed the calculation of the energetic contribution of each heptad within the coiled-coil complex.

Superlubricity of Zwitterionic Bottlebrush Polymers in the Presence of Multivalent Ions.

In this study, we report lubrication properties of physisorbed zwitterionic bottlebrush polymers in the presence of multivalent ions using the surface force apparatus. Unlike polyelectrolyte brushes, the lubrication properties of which diminish drastically in the presence of multivalent ions at concentrations as low as 0.1 mM, zwitterionic bottlebrush polymers exhibit friction coefficients as low as ∼10-3 at such concentrations of multivalent ions up to intermediate normal loads. This lubrication ability persists until surface wear occurs at high normal loads. The surface wear is demonstrated to be triggered by the multivalent ions bridging the polymer chains and dehydrating the zwitterionic moieties. Finally, the analysis of the polymer film stability suggests that the partial desorption of polymers in the presence of the ions does not affect the lubrication performance. Therefore, even in the physisorbed state, zwitterionic brushes perform significantly better than covalently grafted polyelectrolyte brushes in the presence of multivalent ions.