Antiadhesive Copolymers at Solid/Liquid Interfaces: Complementary Characterization of Polymer Adsorption and Protein Fouling by Sum Frequency Generation Vibrational Spectroscopy and Quartz-Crystal Microbalance Measurements with Dissipation Monitoring.

Amphiphilic copolymers comprising hydrophilic segments of poly(ethylene glycol) and hydrophobic domains that are able to adhere to solid/liquid interfaces have proven to be versatile ingredients in formulated products for various types of applications. Recently, we have reported the successful synthesis of a copolymer designed for modifying the surface properties of polyesters as mimics for synthetic textiles. Using sum frequency generation (SFG) spectroscopy, it was shown that the newly developed copolymer adsorbs effectively on the targeted substrates even in the presence of surfactants as supplied by common detergents. In the present work, these studies were extended to evaluate the ability of the formed copolymer adlayers to passivate polyester surfaces against undesired deposition of bio(macro)molecules, as represented by fibrinogen as model protein foulants. In addition, SFG spectroscopy was used to elucidate the structure of fibrinogen at the interface between polyester and water. To complement the obtained data with an independent technique, analogous experiments were performed using quartz-crystal microbalance with dissipation monitoring for the detection of the relevant interfacial processes. Both methods give consistent results and deliver a holistic picture of brush copolymer adsorption on polyester surfaces and subsequent antiadhesive effects against proteins under different conditions representing the targeted application in home care products.

Hydration behaviors of nonfouling zwitterionic materials.

Zwitterionic materials have emerged as highly effective ultralow fouling materials for many applications, however the underlying mechanism of fouling resistance remains unclear. Using ab initio molecular dynamics simulations and surface-sensitive sum frequency generation vibrational spectroscopy, we studied the hydration behaviors of zwitterionic materials, including trimethylamine-N-oxide (TMAO) and carboxybetaines of different charge-separation distances, to understand their fouling-resistant mechanism and provide a design principle for improved performance. Our study reveals that the interplay among hydrogen bonding, net charge, and dipole moment is crucial to the fouling-resistant capabilities of zwitterionic materials. Shortening of the zwitterionic spacing strengthens hydrogen bonding with water against biomolecule attachment due to the increased electrostatic and induction interactions, charge transfer, and improved structural stability. Moreover, the shortened charge separation reduces the dipole moment of zwitterionic materials with an intrinsic near-neutral net charge, decreasing their electrostatic and dipole-dipole interactions with biofoulers, and increasing their resistance to fouling. Compared to carboxybetaine compounds, TMAO has the shortest zwitterionic spacing and exhibits the strongest hydrogen bonding, the smallest net charge, and the minimum dipole moment, making it an excellent nonfouling material.

Determination of protein conformation and orientation at buried solid/liquid interfaces.

Protein structures at solid/liquid interfaces mediate interfacial protein functions, which are important for many applications. It is difficult to probe interfacial protein structures at buried solid/liquid interfaces in situ at the molecular level. Here, a systematic methodology to determine protein molecular structures (orientation and conformation) at buried solid/liquid interfaces in situ was successfully developed with a combined approach using a nonlinear optical spectroscopic technique - sum frequency generation (SFG) vibrational spectroscopy, isotope labeling, spectra calculation, and computer simulation. With this approach, molecular structures of protein GB1 and its mutant (with two amino acids mutated) were investigated at the polymer/solution interface. Markedly different orientations and similar (but not identical) conformations of the wild-type protein GB1 and its mutant at the interface were detected, due to the varied molecular interfacial interactions. This systematic strategy is general and can be widely used to elucidate protein structures at buried interfaces in situ.

Monitoring the Molecular Structure of Fibrinogen during the Adsorption Process at the Buried Silicone Oil Interface In Situ in Real Time.

Interfacial proteins play important roles in many research fields and applications, such as biosensors, biomedical implants, nonfouling coatings, etc. Directly probing interfacial protein behavior at buried solid/liquid and liquid/liquid interfaces is challenging. We used sum frequency generation vibrational spectroscopy and a Hamiltonian data analysis method to monitor the molecular structure of fibrinogen on silicone oil during the adsorption process in situ in real time. The results showed that the adsorbed fibrinogen molecules tend to adopt a bent structure throughout the entire adsorption process with the same orientation. This is different from the case of adsorbed fibrinogen on CaF2 with a linear structure or on polystyrene with a bent structure but a different orientation. The method introduced herein is generally applicable for following time-dependent molecular structures of many other proteins and peptides at interfaces in situ in real time at the molecular level.

Probing Molecular Interactions of Antibody Drugs, Silicone Oil, and Surfactant at Buried Interfaces In Situ.

Antibody drugs have been rapidly developed to cure many diseases including COVID-19 infection. Silicone oil is commonly used as a lubricant coating material for devices used in the pharmaceutical industry to store and administer antibody drug formulations. However, the interaction between silicone oil and antibody molecules could lead to the adsorption, denaturation, and aggregation of antibody molecules, impacting the efficacy of antibody drugs. Here, we studied the molecular interactions between antibodies and silicone oil in situ in real time. The effect of the surfactant on such interactions was also investigated. Specifically, the adsorption dynamics of a bispecific antibody (BsAb) onto a silicone oil surface without and with different concentrations of the surfactant PS80 in antibody solutions were monitored. Also the possible lowest effective PS80 concentrations that can prevent the adsorption of BsAb as well as a monoclonal antibody (mAb) onto silicone oil were measured. It was found that different concentrations of PS80 are required for preventing the adsorption of different antibodies. Both BsAB and mAB denature on silicone oil without a surfactant. However, for a low surfactant concentration in the solution, although the surfactant could not completely prevent the antibody from adsorption, it could maintain the native structures of adsorbed BsAb and mAb antibodies on silicone oil. This is important knowledge, showing that to prevent antibody aggregation on silicone oil it is not necessary to add surfactant to a concentration high enough to completely minimize protein adsorption.

Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer-dimer mixture in situ.

Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g., by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules.

Probing Orientations and Conformations of Peptides and Proteins at Buried Interfaces.

Molecular structures of peptides/proteins at interfaces determine their interfacial properties, which play important roles in many applications. It is difficult to probe interfacial peptide/protein structures because of the lack of appropriate tools. Sum frequency generation (SFG) vibrational spectroscopy has been developed into a powerful technique to elucidate molecular structures of peptides/proteins at buried solid/liquid and liquid/liquid interfaces. SFG has been successfully applied to study molecular interactions between model cell membranes and antimicrobial peptides/membrane proteins, surface-immobilized peptides/enzymes, and physically adsorbed peptides/proteins on polymers and 2D materials. A variety of other analytical techniques and computational simulations provide supporting information to SFG studies, leading to more complete understanding of structure-function relationships of interfacial peptides/proteins. With the advance of SFG techniques and data analysis methods, along with newly developed supplemental tools and simulation methodology, SFG research on interfacial peptides/proteins will further impact research in fields like chemistry, biology, biophysics, engineering, and beyond.

Strong Surface Hydration and Salt Resistant Mechanism of a New Nonfouling Zwitterionic Polymer Based on Protein Stabilizer TMAO.

Zwitterionic polymers exhibit excellent nonfouling performance due to their strong surface hydrations. However, salt molecules may severely reduce the surface hydrations of typical zwitterionic polymers, making the application of these polymers in real biological and marine environments challenging. Recently, a new zwitterionic polymer brush based on the protein stabilizer trimethylamine N-oxide (TMAO) was developed as an outstanding nonfouling material. Using surface-sensitive sum frequency generation (SFG) vibrational spectroscopy, we investigated the surface hydration of TMAO polymer brushes (pTMAO) and the effects of salts and proteins on such surface hydration. It was discovered that exposure to highly concentrated salt solutions such as seawater only moderately reduced surface hydration. This superior resistance to salt effects compared to other zwitterionic polymers is due to the shorter distance between the positively and negatively charged groups, thus a smaller dipole in pTMAO and strong hydration around TMAO zwitterion. This results in strong bonding interactions between the O- in pTMAO and water, and weaker interaction between O- and metal cations due to the strong repulsion from the N+ and hydration water. Computer simulations at quantum and atomistic scales were performed to support SFG analyses. In addition to the salt effect, it was discovered that exposure to proteins in seawater exerted minimal influence on the pTMAO surface hydration, indicating complete exclusion of protein attachment. The excellent nonfouling performance of pTMAO originates from its extremely strong surface hydration that exhibits effective resistance to disruptions induced by salts and proteins.

Molecular Structure of the Surface-Immobilized Super Uranyl Binding Protein.

Recently, a super uranyl binding protein (SUP) was developed, which exhibits excellent sensitivity/selectivity to bind uranyl ions. It can be immobilized onto a surface in sensing devices to detect uranyl ions. Here, sum frequency generation (SFG) vibrational spectroscopy was applied to probe the interfacial structures of surface-immobilized SUP. The collected SFG spectra were compared to the calculated orientation-dependent SUP SFG spectra using a one-excitonic Hamiltonian approach based on the SUP crystal structures to deduce the most likely surface-immobilized SUP orientation(s). Furthermore, discrete molecular dynamics (DMD) simulation was applied to refine the surface-immobilized SUP conformations and orientations. The immobilized SUP structures calculated from DMD simulations confirmed the SUP orientations obtained from SFG data analyzed based on the crystal structures and were then used for a new round of SFG orientation analysis to more accurately determine the interfacial orientations and conformations of immobilized SUP before and after uranyl ion binding, providing an in-depth understanding of molecular interactions between SUP and the surface and the effect of uranyl ion binding on the SUP interfacial structures. We believe that the developed method of combining SFG measurements, DMD simulation, and Hamiltonian data analysis approach is widely applicable to study biomolecules at solid/liquid interfaces.

Probing Molecular Interactions between Surface-Immobilized Antimicrobial Peptides and Lipopolysaccharides In Situ.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. Recently, a label-free immobilized antimicrobial peptide (AMP) surface plasmon resonance platform was developed to successfully distinguish LPS from multiple bacterial strains. Among the tested AMPs, SMAP29 exhibited excellent affinity with LPS and has two independent LPS-binding sites located at two termini of the peptide. In this study, sum frequency generation vibrational spectroscopy was applied to investigate molecular interactions between three LPS samples and surface-immobilized SMAP29 via the N-terminus, the C-terminus, and a middle site at the solid/liquid interface in situ in real-time, supplemented by circular dichroism spectroscopy. It was found that the conformations and orientations of surface-immobilized SMAP29 via different sites are different when interacting with the same LPS, with different interaction kinetics. The same SMAP29 sample also has different structures and interaction kinetics while interacting with different LPS samples with different charge densities and hydrophobicities. The observed results on molecular interactions between surface-immobilized peptides and LPS can well interpret the different adsorption amounts of various LPSs on different surface-immobilized peptides.

Probing the Interfacial Interactions of Monoclonal and Bispecific Antibodies at the Silicone Oil-Aqueous Solution Interface by Using Sum Frequency Generation Vibrational Spectroscopy.

Silicone oil has been widely utilized in the pharmaceutical industry especially as a lubricant coating commonly used in syringes for the smooth delivery of drugs. Protein structure perturbation and aggregation have been reported upon protein contacting silicone oil by using indirect methods and ex-situ techniques. The conclusions derived from such indirect and ex-situ methods may not truly reflect the exact nature of the protein-silicone oil interfacial interactions. Recently, we have successfully demonstrated that sum frequency generation (SFG) vibrational spectroscopy can be used as a powerful and direct method of studying the fusion protein-silicone oil interfacial interactions in situ and in real time. In this article, we studied monoclonal and bispecific antibody interactions with the silicone oil surface by using SFG spectroscopy. Being structurally and functionally different in the nature of fusion proteins and antibodies, this study is important in enhancing our current understanding of protein-silicone oil interfacial interactions. Both types of antibodies investigated here readily and strongly adsorb onto the silicone oil surface and remain stable at least for 10 h. SFG spectra in the amide I region for monoclonal and bispecific antibodies centered at 1660 and 1665 cm-1, respectively, suggest the difference in their molecular structures. The absence of the antibody signals in the amide I region of time-dependent and static SFG spectra obtained for preadsorbed antibodies onto silicone oil after contacting polysorbate 80 (PS-80) surfactant suggests that PS-80 can effectively remove both types of antibodies from the silicone oil surface. This study demonstrated the feasibility of using SFG spectroscopy as a powerful tool for probing the antibody-interfacial interactions in situ and in real time.

Chemically Immobilized Antimicrobial Peptide on Polymer and Self-Assembled Monolayer Substrates.

Surfaces with chemically immobilized antimicrobial peptides have been shown to have great potential in various applications such as biosensors and antimicrobial coatings. This research investigated the chemical immobilization of a cecropin-melittin hybrid antimicrobial peptide on two different surfaces, a polymer surface prepared by chemical vapor deposition (CVD) polymerization and a self-assembled monolayer surface. We probed the structure of immobilized peptides using spectroscopic methods and correlated such structural information to the measured antimicrobial activity. We found that the hybrid peptide adopts an α-helical structure after immobilization onto both surfaces. As we have shown previously for another α-helical peptide, MSI-78, immobilized on a SAM, we found that the α-helical hybrid peptide lies down when it contacts bacteria. This study shows that the antimicrobial activity of the surface-immobilized peptides on the two substrates can be well explained by the spectroscopically measured peptide structural data. In addition, it was found that the polymer-based antimicrobial peptide coating is more stable. This is likely due to the fact that the SAM prepared using silane may be degraded after several days whereas the polymer prepared by CVD polymerization is more stable than the SAM, leading to a more stable antimicrobial coating.