pH-Modulated Nanoarchitectonics for Enhancement of Multivalency-Induced Vesicle Shape Deformation at Receptor-Presenting Lipid Membrane Interfaces.

Multivalent ligand-receptor interactions between receptor-presenting lipid membranes and ligand-modified biological and biomimetic nanoparticles influence cellular entry and fusion processes. Environmental pH changes can drive these membrane-related interactions by affecting membrane nanomechanical properties. Quantitatively, however, the corresponding effects on high-curvature, sub-100 nm lipid vesicles are scarcely understood, especially in the multivalent binding context. Herein, we employed the label-free localized surface plasmon resonance (LSPR) sensing technique to track the multivalent attachment kinetics, shape deformation, and surface coverage of biotin ligand-functionalized, zwitterionic lipid vesicles with different ligand densities on a streptavidin receptor-coated supported lipid bilayer under varying pH conditions (4.5, 6, 7.5). Our results demonstrate that more extensive multivalent interactions caused greater vesicle shape deformation across the tested pH conditions, which affected vesicle surface packing as well. Notably, there were also pH-specific differences, i.e., a higher degree of vesicle shape deformation was triggered at a lower multivalent binding energy in pH 4.5 than in pH 6 and 7.5 conditions. These findings support that the nanomechanical properties of high-curvature lipid membranes, especially the membrane bending energy and the corresponding responsiveness to multivalent binding interactions, are sensitive to solution pH, and indicate that multivalency-induced vesicle shape deformation occurs slightly more readily in acidic pH conditions relevant to biological environments.

Unraveling How Cholesterol Affects Multivalency-Induced Membrane Deformation of Sub-100 nm Lipid Vesicles.

Cholesterol plays a critical role in modulating the lipid membrane properties of biological and biomimetic systems and recent attention has focused on its role in the functions of sub-100 nm lipid vesicles and lipid nanoparticles. These functions often rely on multivalent ligand-receptor interactions involving membrane attachment and dynamic shape transformations while the extent to which cholesterol can influence such interaction processes is largely unknown. To address this question, herein, we investigated the attachment of sub-100 nm lipid vesicles containing varying cholesterol fractions (0-45 mol %) to membrane-mimicking supported lipid bilayer (SLB) platforms. Biotinylated lipids and streptavidin proteins were used as model ligands and receptors, respectively, while the localized surface plasmon resonance sensing technique was employed to track vesicle attachment kinetics in combination with analytical modeling of vesicle shape changes. Across various conditions mimicking low and high multivalency, our findings revealed that cholesterol-containing vesicles could bind to receptor-functionalized membranes but underwent appreciably less multivalency-induced shape deformation than vesicles without cholesterol, which can be explained by a cholesterol-mediated increase in membrane bending rigidity. Interestingly, the extent of vesicle deformation that occurred in response to increasingly strong multivalent interactions was less pronounced for vesicles with greater cholesterol fraction. The latter trend was rationalized by taking into account the strong dependence of the membrane bending energy on the area of the vesicle-SLB contact region and such insights can aid the engineering of membrane-enveloped nanoparticles with tailored biophysical properties.

Distinct Binding Properties of Neutravidin and Streptavidin Proteins to Biotinylated Supported Lipid Bilayers: Implications for Sensor Functionalization.

The exceptional strength and stability of noncovalent avidin-biotin binding is widely utilized as an effective bioconjugation strategy in various biosensing applications, and neutravidin and streptavidin proteins are two commonly used avidin analogues. It is often regarded that the biotin-binding abilities of neutravidin and streptavidin are similar, and hence their use is interchangeable; however, a deeper examination of how these two proteins attach to sensor surfaces is needed to develop reliable surface functionalization options. Herein, we conducted quartz crystal microbalance-dissipation (QCM-D) biosensing experiments to investigate neutravidin and streptavidin binding to biotinylated supported lipid bilayers (SLBs) in different pH conditions. While streptavidin binding to biotinylated lipid receptors was stable and robust across the tested pH conditions, neutravidin binding strongly depended on the solution pH and was greater with increasingly acidic pH conditions. These findings led us to propose a two-step mechanistic model, whereby streptavidin and neutravidin binding to biotinylated sensing interfaces first involves nonspecific protein adsorption that is mainly influenced by electrostatic interactions, followed by structural rearrangement of adsorbed proteins to specifically bind to biotin functional groups. Practically, our findings demonstrate that streptavidin is preferable to neutravidin for constructing SLB-based sensing platforms and can improve sensing performance for detecting antibody-antigen interactions.

Biophysical Characterization of LTX-315 Anticancer Peptide Interactions with Model Membrane Platforms: Effect of Membrane Surface Charge.

LTX-315 is a clinical-stage, anticancer peptide therapeutic that disrupts cancer cell membranes. Existing mechanistic knowledge about LTX-315 has been obtained from cell-based biological assays, and there is an outstanding need to directly characterize the corresponding membrane-peptide interactions from a biophysical perspective. Herein, we investigated the membrane-disruptive properties of the LTX-315 peptide using three cell-membrane-mimicking membrane platforms on solid supports, namely the supported lipid bilayer, intact vesicle adlayer, and tethered lipid bilayer, in combination with quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. The results showed that the cationic LTX-315 peptide selectively disrupted negatively charged phospholipid membranes to a greater extent than zwitterionic or positively charged phospholipid membranes, whereby electrostatic interactions were the main factor to influence peptide attachment and membrane curvature was a secondary factor. Of note, the EIS measurements showed that the LTX-315 peptide extensively and irreversibly permeabilized negatively charged, tethered lipid bilayers that contained high phosphatidylserine lipid levels representative of the outer leaflet of cancer cell membranes, while circular dichroism (CD) spectroscopy experiments indicated that the LTX-315 peptide was structureless and the corresponding membrane-disruptive interactions did not involve peptide conformational changes. Dynamic light scattering (DLS) measurements further verified that the LTX-315 peptide selectively caused irreversible disruption of negatively charged lipid vesicles. Together, our findings demonstrate that the LTX-315 peptide preferentially disrupts negatively charged phospholipid membranes in an irreversible manner, which reinforces its potential as an emerging cancer immunotherapy and offers a biophysical framework to guide future peptide engineering efforts.

Mechanistic Aspects of the Evolution of 3D Cholesterol Crystallites in a Supported Lipid Membrane via a Quartz Crystal Microbalance with Dissipation Monitoring.

The irreversible formation of cholesterol monohydrate crystals within biological membranes is the leading cause of various diseases, including atherosclerosis. Understanding the process of cholesterol crystallization is fundamentally important and could also lead to the development of improved therapeutic strategies. This has driven several studies investigating the effect of the environmental parameters on the induction of cholesterol crystallite growth and the structure of the cholesterol crystallites, while the kinetics and mechanistic aspects of the crystallite formation process within lipid membranes remain poorly understood. Herein, we fabricated cholesterol crystallites within a supported lipid bilayer (SLB) by adsorbing a cholesterol-rich bicellar mixture onto a glass and silica surface and investigated the real-time kinetics of cholesterol crystallite nucleation and growth using epifluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring. Microscopic imaging showed the evolution of the morphology of cholesterol crystallites from nanorod- and plate-shaped habits during the initial stage to mostly large, micron-sized three-dimensional (3D) plate-shaped crystallites in the end, which was likened to Ostwald ripening. QCM-D kinetics revealed unique signal responses during the later stage of the growth process, characterized by simultaneous positive frequency shifts, nonmonotonous energy dissipation shifts, and significant overtone dependence. Based on the optically observed changes in crystallite morphology, we discussed the physical background of these unique QCM-D signal responses and the mechanistic aspects of Ostwald ripening in this system. Together, our findings revealed mechanistic details of the cholesterol crystallite growth kinetics, which may be useful in biointerfacial sensing and bioanalytical applications.

Pentacyanoammineferrate-Based Non-Enzymatic Electrochemical Biosensing Platform for Selective Uric Acid Measurement.

The electrochemical-based detection of uric acid (UA) is widely used for diagnostic purposes. However, various interfering species such as ascorbic acid, dopamine, and glucose can affect electrochemical signals, and hence there is an outstanding need to develop improved sensing platforms to detect UA with high selectivity. Herein, we report a pentagonal mediator-based non-enzymatic electrochemical biosensing platform to selectively measure UA in the presence of interfering species. The working electrode was fabricated by electrodepositing polymerized 1-vinylimidazole (PVI), which has an imidazole ligand, onto indium tin oxide (ITO), and then conjugating nickel ions to the PVI-coated ITO electrode. Electrode performance was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements and integrated together with pentacyanoammineferrate, which can bind to the amine groups of UA and function as an electron transferring mediator. The experimental results showed a wide linear range of UA concentration-dependent responses and the multi-potential step (MPS) technique facilitated selective detection of UA in the presence of physiologically relevant interfering species. Altogether, these findings support that pentacyanoammineferrate-based non-enzymatic electrodes are suitable biosensing platforms for the selective measurement of UA, and such approaches could potentially be extended to other bioanalytes as well.

Crystallization of Cholesterol in Phospholipid Membranes Follows Ostwald's Rule of Stages.

Crystallization of membrane-embedded components within phospholipid bilayers represents a distinct class of phase transformation that occurs in structurally organized, molecularly crowded, and dimensionally constrained amphiphilic fluids. Using unstable supported lipid bilayers-transiently assembled via surface-mediated fusion and spreading of bicellar precursors containing supersaturating concentrations of cholesterol-we monitor here the morphological evolution and dynamics of cholesterol crystallization within the membrane media. We find that the three-dimensional (3D) crystallization of cholesterol from an unstable two-dimensional (2D) in-membrane state proceeds via well-defined sequence of intermediates, including filaments, rods, helices, and 2D rectangular plates, before transforming into three-dimensional quadrilateral crystals-characteristic triclinic habit of cholesterol monohydrate. Our observations thus demonstrate that these structurally distinct cholesterol polymorphs are related to one another, contrasting with the notion that they represent disparate crystal habits stabilized by differences in lipid environments. Moreover, these observations indicate that cholesterol crystallization within the membrane media follows nonclassical multistep crystallization governed by the heuristic "Ostwald's rule of stages", which predicts that the crystallization kinetics proceed down the free energy landscape in a multistage process where each successive phase transition incurs the smallest loss of free energy relative to its predecessor. Furthermore, we find that the well-known cholesterol extracting agent, ß-cyclodextrin, acts by catalytically tipping the equilibrium in favor of crystal growth adding cholesterol from the membrane phase to the crystal in a layer-by-layer manner. Taken together, our results provide a new description of in-membrane cholesterol crystallization and may pave for a screening tool for identifying molecular candidates that target cholesterol crystals.

Supported Lipid Bilayer Formation from Phospholipid-Fatty Acid Bicellar Mixtures.

Supported lipid bilayers (SLBs) are versatile cell membrane-mimicking biointerfaces for various applications such as biosensors and drug delivery systems, and there is broad interest in developing simple, cost-effective methods to achieve SLB fabrication. One promising approach involves the deposition of quasi-two-dimensional bicelle nanostructures that are composed of long-chain phospholipids and either short-chain phospholipids or detergent molecules. While a variety of long-chain phospholipids have been used to prepare bicelles for SLB fabrication applications, only two short-chain phospholipids, 1,2-dihexanoyl-sn-glycero-3-phosphocholine and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (collectively referred to as DHPC), have been investigated. There remains an outstanding need to identify natural alternatives to DHPC, especially ones that are more affordable, to improve fabrication prospects and application opportunities. Herein, we explored the potential to fabricate SLBs from bicellar mixtures composed of long-chain phospholipids and lauric acid (LA), which is a low-cost, naturally abundant fatty acid that is widely used in soapmaking and various industrial applications. Quartz crystal microbalance-dissipation (QCM-D) experiments were conducted to track bicelle adsorption onto silica surfaces as a function of bicelle composition and lipid concentration, along with time-lapse fluorescence microscopy imaging and fluorescence recovery after photobleaching (FRAP) experiments to further characterize lipid adlayer properties. The results identified optimal conditions where it is possible to efficiently form SLBs from LA-containing bicelles at low lipid concentrations while also unraveling mechanistic insights into the bicelle-mediated SLB formation process and verifying that LA-containing bicelles are biocompatible with human cells for surface coating applications.

Unraveling How Ethanol-Induced Conformational Changes Affect BSA Protein Adsorption onto Silica Surfaces.

Protein adsorption at solid-liquid interfaces is highly relevant to a wide range of applications such as biosensors, drug delivery, and pharmaceuticals. Understanding how protein conformation in bulk solution impacts adsorption behavior is fundamentally important and could also lead to the development of improved protein-based coatings. To date, relevant studies have been conducted in aqueous solutions, while it remains largely unknown how organic solvents and more specifically solvent-induced conformational changes might influence protein adsorption. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) techniques, we systematically investigated the real-time adsorption behavior of bovine serum albumin (BSA) protein onto silica surfaces in different water-ethanol mixtures ranging from 0 to 60% (v/v) ethanol. The results showed that there was greater protein adsorption at higher ethanol fractions in the 10-30% range, while more complex adsorption profiles were observed in the 40-60% range. The combination of QCM-D and LSPR measurements led us to further identify specific cases in water-ethanol mixtures where washing steps caused densification of the adsorbed protein layer as opposed to typical desorption of weakly adsorbed molecules in aqueous conditions. We discuss mechanistic factors that drive these overall adsorption trends by taking into account how ethanol fraction affects BSA conformation in bulk solution. Together, our findings demonstrate that BSA proteins can adsorb onto silica surfaces across a wide range of water-ethanol mixture conditions, while specific adsorption profiles depended on the ethanol fraction in a manner closely linked to solution-phase conformational properties.

Understanding How Membrane Surface Charge Influences Lipid Bicelle Adsorption onto Oxide Surfaces.

The adsorption of two-dimensional bicellar disks onto solid supports is an emerging fabrication technique to form supported lipid bilayers (SLBs) that is efficient and requires minimal sample preparation. To date, nearly all relevant studies have focused on zwitterionic lipid compositions and silica-based surfaces, and extending the scope of investigation to other lipid compositions and surfaces would improve our understanding of application possibilities and underpinning formation processes. Herein, using the quartz crystal microbalance-dissipation technique, we systematically investigated the adsorption of charged lipid bicelles onto silicon dioxide, titanium oxide, and aluminum oxide surfaces. Depending on the lipid composition and substrate, we observed different adsorption pathways, including (i) SLB formation via one- or two-step adsorption kinetics, (ii) monotonic adsorption without SLB formation, and (iii) negligible adsorption. On each substrate, SLB formation could be achieved with particular lipid compositions, whereas the trend in adsorption pathways varied according to the substrate and could be controlled by adjusting the bicelle?substrate interaction strength. To rationalize these findings, we discuss how electrostatic and hydration forces affect bicelle?substrate interactions on different oxide surfaces. Collectively, our findings demonstrate the broad utility of lipid bicelles for SLB formation while revealing physicochemical insights into the role of interfacial forces in controlling bicelle adsorption pathways.

Characterizing the Supported Lipid Membrane Formation from Cholesterol-Rich Bicelles.

Supported lipid bilayers (SLBs) are simplified model membrane systems that mimic the fundamental properties of biological cell membranes and allow the surface-sensitive tools to be used in numerous sensing applications. SLBs can be prepared by various methods including vesicle fusion, solvent-assisted lipid bilayer (SALB), and bicelle adsorption and are generally composed of phospholipids. Incorporating other biologically relevant molecules, such as cholesterol (Chol), into SLBs has been reported with the vesicle fusion and SALB methods, whereas it remains unexplored with the bicelle absorption method. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy techniques, we explored the possibility of forming SLBs from Chol-containing bicelles and discovered that Chol-enriched SLBs can be fabricated with bicelles. We also compared the Chol-enriched SLB formation of the bicelle method to that of vesicle fusion and SALB and discussed how the differences in lipid assembly properties can cause the differences in the adsorption kinetics and final results of SLB formation. Collectively, our findings demonstrate that the vesicle fusion method is least favorable for forming Chol-enriched SLBs, whereas the SALB and bicelle methods are more favorable, highlighting the need to consider the application requirements when choosing a suitable method for the formation of Chol-enriched SLBs.

Influence of NaCl Concentration on Bicelle-Mediated SLB Formation.

The deposition of two-dimensional bicellar disks on hydrophilic surfaces is an emerging approach to fabricate supported lipid bilayers (SLBs) that requires minimal sample preparation, works at low lipid concentrations, and yields high-quality SLBs. While basic operating steps in the fabrication protocol mimic aspects of the conventional vesicle fusion method, lipid bicelles and vesicles have distinct architectural properties, and understanding how experimental parameters affect the efficiency of bicelle-mediated SLB formation remains to be investigated. Herein, using the quartz crystal microbalance-dissipation and localized surface plasmon resonance techniques, we investigated the effect of bulk NaCl concentration on bicelle-mediated SLB formation on silicon dioxide surfaces. For comparison, similar experiments were conducted with vesicles as well. In both cases, SLB formation was observed to occur rapidly provided that the NaCl concentration was sufficiently high (>50 mM). Under such conditions, the effect of NaCl concentration on SLB formation was minor in the case of bicelles and significant in the case of vesicles where it is expected to be related primarily to osmotic pressure. At lower NaCl concentrations, bicelles also formed SLBs but slowly, whereas adsorbed vesicles remained intact. These findings were complemented by time-lapsed fluorescence microscopy imaging and fluorescence recovery after photobleaching measurements that corroborated bicelle-mediated SLB formation across the range of tested NaCl concentrations. The results are discussed by comparing the architectural properties of bicelles and vesicles along with theoretical analysis of the corresponding adsorption kinetics.

Molecular diffusion and nano-mechanical properties of multi-phase supported lipid bilayers.

Understanding the properties of cell membranes is important in the fields of fundamental and applied biology. While the characterization of simplified biological membrane mimics comprising liquid phase lipids has been routinely performed due to the ease of fabrication, the characterization of more realistic membrane mimics comprising multi-phase lipids remains challenging due to more complicated fabrication requirements. Herein, we report a convenient approach to fabricate and characterize multi-phase supported lipid bilayers (SLBs). We employed the solvent-assisted lipid bilayer (SALB) formation method to fabricate mixed lipid bilayers comprising liquid phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and gel phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids at room temperature. The fabrication procedure was performed inside a newly designed microfluidic chamber, which facilitated the subsequent characterization of the SLBs without exposure to air. The SLBs were then characterized via fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), atomic force microscopy (AFM) and AFM-based force-distance measurements. Interestingly, results from these characterization techniques revealed that regardless of the gel phase composition, the SALB formation method consistently yielded uniform SLBs at room temperature, even though the transition temperature of DPPC is considerably higher. Furthermore, the composition ratio of DOPC and DPPC in the precursor solution is well reproduced in the fabricated SLBs. We also identified from diffusivity measurements that a high ratio of gel phase lipid revitalizes lipid-lipid interactions, which led to reduced molecular fluidity and the suppression of thermal undulation within the SLBs. Taken together, our results highlight the robustness of the SALB formation method that allows the fabrication of complex lipid bilayers with a high degree of precision, which is suitable for functional studies of biological membranes.

Nanoplasmonic Ruler for Measuring Separation Distance between Supported Lipid Bilayers and Oxide Surfaces.

Unraveling the details of how supported lipid bilayers (SLBs) are coupled to oxide surfaces is experimentally challenging, and there is an outstanding need to develop highly surface-sensitive measurement strategies to determine SLB separation distances. Indeed, subtle variations in separation distance can be associated with significant differences in bilayer-substrate interaction energy. Herein, we report a nanoplasmonic ruler strategy to measure the absolute separation distance between SLBs and oxide surfaces. A localized surface plasmon resonance (LSPR) sensor was employed to track SLB formation onto titania- and silica-coated gold nanodisk arrays. To interpret measurement data, an analytical model relating the LSPR measurement response to bilayer-substrate separation distance was developed based on finite-difference time-domain (FDTD) simulations and theoretical calculations. The results indicate that there is a larger separation distance between SLBs and titania surfaces than silica surfaces, and the trend was consistent across three tested lipid compositions. We discuss these findings within the context of the interfacial forces underpinning bilayer-substrate interactions, and the nanoplasmonic ruler strategy provides the first direct experimental evidence comparing SLB separation distances on titania and silica surfaces.

Amyloid-ß Peptide Triggers Membrane Remodeling in Supported Lipid Bilayers Depending on Their Hydrophobic Thickness.

Amyloid-ß (Aß) peptide has been implicated in Alzheimer's disease, which is a leading cause of death worldwide. The interaction of Aß peptides with the lipid bilayers of neuronal cells is a critical step in disease pathogenesis. Recent evidence indicates that lipid bilayer thickness influences Aß membrane-associated aggregation, while understanding how Aß interacts with lipid bilayers remains elusive. To address this question, we employed supported lipid bilayer (SLB) platforms composed of different-length phosphatidylcholine (PC) lipids (C12:0 DLPC, C18:1 DOPC, C18:1-C16:0 POPC), and characterized the resulting interactions with soluble Aß monomers. Quartz crystal microbalance-dissipation (QCM-D) experiments identified concentration-dependent Aß peptide adsorption onto all tested SLBs, which was corroborated by fluorescence recovery after photobleaching (FRAP) experiments indicating that higher Aß concentrations led to decreased membrane fluidity. These commonalities pointed to strong Aß peptide-membrane interactions in all cases. Notably, time-lapsed fluorescence microscopy revealed major differences in Aß-induced membrane morphological responses depending on SLB hydrophobic thickness. For thicker DOPC and POPC SLBs, membrane remodeling involved the formation of elongated tubule and globular structures as a passive means to regulate membrane stress depending on Aß concentration. In marked contrast, thin DLPC SLBs were not able to accommodate extensive membrane remodeling. Taken together, our findings reveal that membrane thickness influences the membrane morphological response triggered upon Aß adsorption.

Characterizing How Acidic pH Conditions Affect the Membrane-Disruptive Activities of Lauric Acid and Glycerol Monolaurate.

Fatty acids and monoglycerides are single-chain lipid amphiphiles that interact with phospholipid membranes as part of various biological activities. For example, they can exhibit membrane-disruptive behavior against microbial pathogens on the human skin surface. Supported lipid bilayers (SLBs) provide a useful experimental platform to characterize these membrane-disruptive behaviors, although related studies have been limited to neutral pH conditions. Herein, we investigated how lauric acid (LA) and glycerol monolaurate (GML) interact with SLBs and cause membrane morphological changes under acidic pH conditions that are representative of the human skin surface. Although LA induces tubule formation under neutral pH conditions, we discovered that LA causes membrane phase separation under acidic pH conditions. By contrast, GML induced membrane budding in both pH environments, although there was more extensive membrane remodeling under acidic pH conditions. We discuss these findings in the context of how solution pH affects the ionization states and micellar aggregation properties of LA and GML as well as its effect on the bending stiffness of lipid bilayers. Collectively, the findings demonstrate that solution pH plays an important role in modulating the interaction of fatty acids and monoglycerides with phospholipid membranes, and hence influences the scope and potency of their membrane-disruptive activities.

Quantitative Comparison of Protein Adsorption and Conformational Changes on Dielectric-Coated Nanoplasmonic Sensing Arrays.

Nanoplasmonic sensors are a popular, surface-sensitive measurement tool to investigate biomacromolecular interactions at solid-liquid interfaces, opening the door to a wide range of applications. In addition to high surface sensitivity, nanoplasmonic sensors have versatile surface chemistry options as plasmonic metal nanoparticles can be coated with thin dielectric layers. Within this scope, nanoplasmonic sensors have demonstrated promise for tracking protein adsorption and substrate-induced conformational changes on oxide film-coated arrays, although existing studies have been limited to single substrates. Herein, we investigated human serum albumin (HSA) adsorption onto silica- and titania-coated arrays of plasmonic gold nanodisks by localized surface plasmon resonance (LSPR) measurements and established an analytical framework to compare responses across multiple substrates with different sensitivities. While similar responses were recorded on the two substrates for HSA adsorption under physiologically-relevant ionic strength conditions, distinct substrate-specific behavior was observed at lower ionic strength conditions. With decreasing ionic strength, larger measurement responses occurred for HSA adsorption onto silica surfaces, whereas HSA adsorption onto titania surfaces occurred independently of ionic strength condition. Complementary quartz crystal microbalance-dissipation (QCM-D) measurements were also performed, and the trend in adsorption behavior was similar. Of note, the magnitudes of the ionic strength-dependent LSPR and QCM-D measurement responses varied, and are discussed with respect to the measurement principle and surface sensitivity of each technique. Taken together, our findings demonstrate how the high surface sensitivity of nanoplasmonic sensors can be applied to quantitatively characterize protein adsorption across multiple surfaces, and outline broadly-applicable measurement strategies for biointerfacial science applications.

Correlating Membrane Morphological Responses with Micellar Aggregation Behavior of Capric Acid and Monocaprin.

The interaction of single-chain lipid amphiphiles with phospholipid membranes is relevant to many scientific fields, including molecular evolution, medicine, and biofuels. Two widely studied compounds within this class are the medium-chain saturated fatty acid, capric acid, and its monoglyceride derivative, monocaprin. To date, most studies about these compounds have involved in vitro evaluation of their biological activities, while mechanistic details of how capric acid and monocaprin interact with phospholipid bilayers remain elusive. Herein, we investigated the effect of these two compounds on the morphological and fluidic properties of prefabricated, supported lipid bilayers (SLBs). The critical micelle concentration (CMC) of each compound was determined by fluorescence spectroscopy measurements. At or above its CMC, capric acid induced the formation of elongated tubules protruding from the SLB, as determined by quartz crystal microbalance-dissipation and fluorescence microscopy experiments. By contrast, monocaprin induced the formation of elongated tubules or membrane buds below and above its CMC, respectively. Fluorescence recovery after photobleaching (FRAP) experiments indicated that capric acid increased bilayer fluidity only above its CMC, whereas monocaprin increased bilayer fluidity both above and below its CMC. We discuss these findings in the context of the two compounds' structural properties, including net charge, molecular length and hydrogen-bonding capacity. Collectively, the findings demonstrate that capric acid and monocaprin differentially affect the morphological and fluidic properties of SLBs, and that the aggregation state of the compounds plays a critical role in modulating their interactions with phospholipid membranes.

Controlling adsorption and passivation properties of bovine serum albumin on silica surfaces by ionic strength modulation and cross-linking.

Understanding the physicochemical factors that influence protein adsorption onto solid supports holds wide relevance for fundamental insights into protein structure and function as well as for applications such as surface passivation. Ionic strength is a key parameter that influences protein adsorption, although how its modulation might be utilized to prepare well-coated protein adlayers remains to be explored. Herein, we investigated how ionic strength can be utilized to control the adsorption and passivation properties of bovine serum albumin (BSA) on silica surfaces. As protein stability in solution can influence adsorption kinetics, the size distribution and secondary structure of proteins in solution were first characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and circular dichroism (CD) spectroscopy. A non-monotonic correlation between ionic strength and protein aggregation was observed and attributed to colloidal agglomeration, while the primarily α-helical character of the protein in solution was maintained in all cases. Quartz crystal microbalance-dissipation (QCM-D) experiments were then conducted in order to track protein adsorption onto silica surfaces as a function of ionic strength, and the measurement responses indicated that total protein uptake at saturation coverage is lower with increasing ionic strength. In turn, the QCM-D data and the corresponding Voigt-Voinova model analysis support that the surface area per bound protein molecule is greater with increasing ionic strength. While higher protein uptake under lower ionic strengths by itself did not result in greater surface passivation under subsequent physiologically relevant conditions, the treatment of adsorbed protein layers with a gluteraldehyde cross-linking agent stabilized the bound protein in this case and significantly improved surface passivation. Collectively, our findings demonstrate that ionic strength modulation influences BSA adsorption uptake on account of protein spreading and can be utilized in conjunction with covalent cross-linking strategies to prepare well-coated protein adlayers for improved surface passivation.

Probing the Interaction of Dielectric Nanoparticles with Supported Lipid Membrane Coatings on Nanoplasmonic Arrays.

The integration of supported lipid membranes with surface-based nanoplasmonic arrays provides a powerful sensing approach to investigate biointerfacial phenomena at membrane interfaces. While a growing number of lipid vesicles, protein, and nucleic acid systems have been explored with nanoplasmonic sensors, there has been only very limited investigation of the interactions between solution-phase nanomaterials and supported lipid membranes. Herein, we established a surface-based localized surface plasmon resonance (LSPR) sensing platform for probing the interaction of dielectric nanoparticles with supported lipid bilayer (SLB)-coated, plasmonic nanodisk arrays. A key emphasis was placed on controlling membrane functionality by tuning the membrane surface charge vis-à-vis lipid composition. The optical sensing properties of the bare and SLB-coated sensor surfaces were quantitatively compared, and provided an experimental approach to evaluate nanoparticle-membrane interactions across different SLB platforms. While the interaction of negatively-charged silica nanoparticles (SiNPs) with a zwitterionic SLB resulted in monotonic adsorption, a stronger interaction with a positively-charged SLB resulted in adsorption and lipid transfer from the SLB to the SiNP surface, in turn influencing the LSPR measurement responses based on the changing spatial proximity of transferred lipids relative to the sensor surface. Precoating SiNPs with bovine serum albumin (BSA) suppressed lipid transfer, resulting in monotonic adsorption onto both zwitterionic and positively-charged SLBs. Collectively, our findings contribute a quantitative understanding of how supported lipid membrane coatings influence the sensing performance of nanoplasmonic arrays, and demonstrate how the high surface sensitivity of nanoplasmonic sensors is well-suited for detecting the complex interactions between nanoparticles and lipid membranes.