Nanoarchitectured air-stable supported lipid bilayer incorporating sucrose-bicelle complex system.

Cell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose-bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration-rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose-bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications.

Solvent-induced conformational tuning of lysozyme protein adlayers on silica surfaces: A QCM-D and LSPR study.

There is broad interest in functionalizing solid surfaces with lysozyme, which is a widely studied antimicrobial protein. To date, most efforts have focused on developing more effective immobilization schemes to promote lysozyme attachment in fully aqueous conditions, while there remains an outstanding need to understand how tuning the solution-phase conformational stability of lysozyme proteins can modulate adsorption behavior and resulting adlayer properties. Inspired by the unique conformational behavior of lysozyme proteins in water-ethanol mixtures, we conducted quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) measurements to systematically investigate the adsorption behavior of lysozyme proteins onto silica surfaces across a wide range of water-ethanol mixtures. Our findings revealed that lysozyme adsorption behavior strongly depended on the ethanol fraction in a non-monotonic fashion and this trend could be rationalized by taking into account how competing effects of water and ethanol solvation influence solution-phase protein size and conformational stability. Integrated analysis of the QCM-D and LSPR measurement trends enabled quantitative determination of the solvent mass within lysozyme adlayers, which tended to decrease at higher ethanol fractions and supported that the hydrodynamic properties of lysozyme adlayers are mainly influenced by the degree of protein conformational flexibility as opposed to solvation effects alone.

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.

Comparing Protein Adsorption onto Alumina and Silica Nanomaterial Surfaces: Clues for Vaccine Adjuvant Development.

Protein adsorption onto nanomaterial surfaces is important for various nanobiotechnology applications such as biosensors and drug delivery. Within this scope, there is growing interest to develop alumina- and silica-based nanomaterial vaccine adjuvants and an outstanding need to compare protein adsorption onto alumina- and silica-based nanomaterial surfaces. Herein, using alumina- and silica-coated arrays of silver nanodisks with plasmonic properties, we conducted localized surface plasmon resonance (LSPR) experiments to evaluate real-time adsorption of bovine serum albumin (BSA) protein onto alumina and silica surfaces. BSA monomers and oligomers were prepared in different water-ethanol mixtures and both adsorbing species consistently showed quicker adsorption kinetics and more extensive adsorption-related spreading on alumina surfaces as compared to on silica surfaces. We rationalized these experimental observations in terms of the electrostatic forces governing protein-surface interactions on the two nanomaterial surfaces and the results support that more rigidly attached BSA protein-based coatings can be formed on alumina-based nanomaterial surfaces. Collectively, the findings in this study provide fundamental insight into protein-surface interactions at nanomaterial interfaces and can help to guide the development of protein-based coatings for medical and biotechnology applications such as vaccines.

Conformational stability as a quality attribute for the cell therapy raw material human serum albumin.

Although human serum albumin (HSA) has been used for many decades, there is still a lack of suitable quality control (QC) attributes. Its current use as a raw material in gene-, cell- and tissue-therapies requires more appropriate functionally-relevant quality attributes and methods. This study investigated the conformational stability of serum albumin using circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) for evaluating the thermal sensitivity, and quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) for assessing the adsorption behavior. Different serum albumin samples were used, encompassing plasma-derived HSA (pHSA), recombinant octanoate-stabilized HSA (rHSA) and bovine serum albumin (BSA). The melting temperature (T m) as well as the onset temperature (T onset) were obtained from the derivative curves of the temperature gradient CD data at 222 nm. The results from DLS, as well as from real-time QCM-D and LSPR silica-adsorption kinetic profiles confirmed the relatively higher conformational stability of the octanoate (fatty acid) containing rHSA, while the additional negative charge resulted in a lower amount adsorbed to the silica surface compared to the non-stabilized HSA and BSA. Adsorption studies further revealed that BSA has a lower conformational stability and undergoes more extensive adsorption-induced spreading compared to the non-stabilized HSA. Collectively, the temperature-based (CD and DLS) as well as adsorption-based biosensor (QCM-D and LSPR) approaches gave congruent and discriminatory information about the conformational stability of different serum albumins, indicating that these techniques provide information on valuable QC attributes.

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.

Elucidating How Different Amphipathic Stabilizers Affect BSA Protein Conformational Properties and Adsorption Behavior.

Natural proteins such as bovine serum albumin (BSA) are readily extracted from biological fluids and widely used in various applications such as drug delivery and surface coatings. It is standard practice to dope BSA proteins with an amphipathic stabilizer, most commonly fatty acids, during purification steps to maintain BSA conformational properties. There have been extensive studies investigating how fatty acids and related amphiphiles affect solution-phase BSA conformational properties, while it is far less understood how amphipathic stabilizers might influence noncovalent BSA adsorption onto solid supports, which is practically relevant to form surface coatings. Herein, we systematically investigated the binding interactions between BSA proteins and different molar ratios of caprylic acid (CA), monocaprylin (MC), and methyl caprylate (ME) amphiphiles-all of which have 8-carbon-long, saturated hydrocarbon chains with distinct headgroups-and resulting effects on BSA adsorption behavior on silica surfaces. Our findings revealed that anionic CA had the greatest binding affinity to BSA, which translated into greater solution-phase conformational stability and reduced adsorption-related conformational changes along with relatively low packing densities in fabricated BSA adlayers. On the other hand, nonionic MC had moderate binding affinity to BSA and could stabilize BSA conformational properties in the solution and adsorbed states while also enabling BSA adlayers to form with higher packing densities. We discuss physicochemical factors that contribute to these performance differences, and our findings demonstrate how rational selection of amphiphile type and amount can enable control over BSA adlayer properties, which could lead to improved BSA protein-based surface coatings.

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 natural sequence variation in serum albumin proteins affects conformational stability and protein adsorption.

Serum albumins are evolutionary conserved proteins that are found in many animal species, and purified forms are widely used in biotechnology applications, such as components within surface passivation coatings and drug delivery systems. As such, there has long been interest in studying how serum albumins adsorb onto solid supports, although existing studies are limited to one or two species. Herein, we comprehensively investigated three serum albumins of bovine (BSA), human (HSA), and rat (RSA) origin, and discovered striking differences in their conformational stabilities and adsorption properties. Together with bioinformatics analysis, dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy measurements revealed that the proteins form different types of macromolecular assemblies in solution. BSA and HSA existed as individual monomers while RSA formed multimers, and each protein exhibited sequence-dependent variations in conformational stability as well. Quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) experiments further showed that BSA and HSA proteins adsorb to form well-packed adlayers, and the extent of protein uptake and spreading depended on their unique conformational stabilities. Conversely, RSA adsorption resulted in sparse adlayers and appreciably less spreading of the adsorbed multimers, as confirmed by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy experiments. Together, our findings demonstrate that significant differences in conformational stability and adsorption behavior exist even between evolutionary conserved serum albumins with high sequence and structural similarity and illustrate how rational engineering of protein structures and stabilities, guided by insights from nature, might be useful to design protein-based coatings for various biointerfacial science applications.

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.

Temperature-Induced Denaturation of BSA Protein Molecules for Improved Surface Passivation Coatings.

Bovine serum albumin (BSA) is the most widely used protein for surface passivation applications, although it has relatively weak, nonsticky interactions with hydrophilic surfaces such as silica-based materials. Herein, we report a simple and versatile method to increase the stickiness of BSA protein molecules adsorbing onto silica surfaces, resulting in up to a 10-fold improvement in blocking efficiency against serum biofouling. Circular dichroism spectroscopy, dynamic light scattering, and nanoparticle tracking analysis showed that temperature-induced denaturation of BSA proteins in bulk solution resulted in irreversible unfolding and protein oligomerization, thereby converting weakly adhesive protein monomers into a more adhesive oligomeric form. The heat-treated, denatured BSA oligomers remained stable after cooling. Room-temperature quartz crystal microbalance-dissipation and localized surface plasmon resonance experiments revealed that denatured BSA oligomers adsorbed more quickly and in larger mass quantities onto silica surfaces than native BSA monomers. We also determined that the larger surface contact area of denatured BSA oligomers is an important factor contributing to their more adhesive character. Importantly, denatured BSA oligomers were a superior passivating agent to inhibit biofouling on silica surfaces and also improved Western blot application performance. Taken together, the findings demonstrate how temperature-induced denaturation of BSA protein molecules can lead to improved protein-based coatings for surface passivation applications.

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.