Quartz Crystal Microbalance-dissipation Measurements Research Articles

Effect of Membrane Curvature Nanoarchitectonics on Membrane-Disruptive Interactions of Antimicrobial Lipids and Surfactants.

Single-chain lipid amphiphiles such as fatty acids and monoglycerides along with structurally related surfactants have received significant attention as membrane-disrupting antimicrobials to inhibit bacteria and viruses. Such promise has motivated deeper exploration of how these compounds disrupt phospholipid membranes, and the membrane-mimicking, supported lipid bilayer (SLB) platform has provided a useful model system to evaluate corresponding mechanisms of action and potency levels. Even so, it remains largely unknown how biologically relevant membrane properties, such as sub-100 nm membrane curvature, might affect these membrane-disruptive interactions, especially from a nanoarchitectonics perspective. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we fabricated intact vesicle adlayers composed of different-size vesicles (70 or 120 nm diameter) with varying degrees of membrane curvature on a titanium oxide surface and tracked changes in vesicle adlayer properties upon adding lauric acid (LA), glycerol monolaurate (GML), or sodium dodecyl sulfate (SDS). Above their critical micelle concentration (CMC) values, LA and GML caused QCM-D measurement shifts associated with tubule- and bud-like formation, respectively, and both compounds interacted similarly with small (high curvature) and large (low curvature) vesicles. In marked contrast, SDS exhibited distinct interactions with small and large vesicles. For large vesicles, SDS caused nearly complete membrane solubilization in a CMC-independent manner, whereas SDS was largely ineffective at solubilizing small vesicles at all tested concentrations. We rationalize these experimental observations by taking into account the interplay of the headgroup properties of LA, GML, and SDS and curvature-induced membrane geometry, and our findings demonstrate that membrane curvature nanoarchitectonics can strongly influence the membrane interaction profiles of antimicrobial lipids and surfactants.

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.

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.

Lipid Bicelle Micropatterning Using Chemical Lift-Off Lithography.

Supported lipid membranes are versatile biomimetic coatings for the chemical functionalization of inorganic surfaces. Developing simple and effective fabrication strategies to form supported lipid membranes with micropatterned geometries is a long-standing challenge. Herein, we demonstrate how the combination of chemical lift-off lithography (CLL) and easily prepared lipid bicelle nanostructures can yield micropatterned, supported lipid membranes on gold surfaces with high pattern resolution, conformal character, and biofunctionality. Using CLL, we functionalized gold surfaces with patterned arrays of hydrophilic and hydrophobic self-assembled monolayers (SAMs). Time-lapse fluorescence microscopy imaging revealed that lipid bicelles adsorbed preferentially onto the hydrophilic SAM regions, while there was negligible lipid adsorption onto the hydrophobic SAM regions. Functional receptors could be embedded within the lipid bicelles, which facilitated selective detection of receptor-ligand binding interactions in a model streptavidin-biotin system. Quartz crystal microbalance-dissipation measurements further identified that lipid bicelles adsorb irreversibly and remain intact on top of the hydrophilic SAM regions. Taken together, our findings indicate that lipid bicelles are useful lipid nanostructures for reproducibly assembling micropatterned, supported lipid membranes with precise pattern fidelity.

Probing the influence of tether density on tethered bilayer lipid membrane (tBLM)-peptide interactions

Tethered bilayer lipid membranes (tBLMs) represent a promising model membrane system that can host transmembrane proteins to serve as biosensors with exceptional detection performance. Herein, using the quartz crystal microbalance-dissipation (QCM-D) technique, we systematically investigated the influence of tether density on tBLM-peptide interactions and characterized the membrane binding dynamics of membrane-active peptides on tBLMs using AH peptide as a model. To achieve both physical stability and nanoscale separation from the support substrate, the tBLMs were fabricated on self-assembled monolayers (SAMs) obtained using a mixture of tether and spacer molecules with controlled tether-to-spacer ratio from 1:99 (T1) to 100:0 (T100). The solvent-assisted lipid bilayer (SALB) formation method was then employed to form the tBLMs, before the introduction of AH peptide. The QCM-D measurement responses indicated that the interactions between AH peptide and tBLMs involved peptide adsorption on the membrane layer followed by peptide translocation across the membrane. With increasing tether density, the abundance of hydrophobic groups within the tether chain led to stronger interactions and greater amount of translocated peptide. Depending on the tether density, this could result in significant structural transformation within the tBLM. Taken together, our work highlights the prospect of modulating membrane-peptide interactions by means of controlling the tBLM architecture, which will facilitate the creation of model membrane systems with highly tailored functionalities.

Quantitative Profiling of Nanoscale Liposome Deformation by a Localized Surface Plasmon Resonance Sensor.

Characterizing the shape of sub-100 nm, biological soft-matter particulates (e.g., liposomes and exosomes) adsorbed at a solid-liquid interface remains a challenging task. Here, we introduce a localized surface plasmon resonance (LSPR) sensing approach to quantitatively profile the deformation of nanoscale, fluid-phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes contacting a titanium dioxide substrate. Experimental and theoretical results validate that, due to its high sensitivity to the spatial proximity of phospholipid molecules near the sensor surface, the LSPR sensor can discriminate fine differences in the extent of ionic strength-modulated liposome deformation at both low and high surface coverages. By contrast, quartz crystal microbalance-dissipation (QCM-D) measurements performed with equivalent samples were qualitatively sensitive to liposome deformation only at saturation coverage. Control experiments with stiffer, gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes verified that the LSPR measurement discrimination arises from the extent of liposome deformation, while the QCM-D measurements yield a more complex response that is also sensitive to the motion of adsorbed liposomes and coupled solvent along with lateral interactions between liposomes. Collectively, our findings demonstrate the unique measurement capabilities of LSPR sensors in the area of biological surface science, including competitive advantages for probing the shape properties of adsorbed, nanoscale biological particulates.

Real-time label-free detection and kinetic analysis of Etanercept—Protein A interactions using quartz crystal microbalance

A quartz crystal microbalance (QCM) was constructed to assess if such a biosensor has value as a complementary real-time label-free analysis platform for the biopharmaceutical industry. This was achieved through modifying QCM crystals with a low-fouling carboxymethyl-dextran layer bearing Protein A, and then injecting solutions containing Etanercept (i.e., Enbrel®) into the QCM chambers. The kinetics of Enbrel® – Protein A interactions was modeled using the Langmuir binding model and Enbrel® concentrations between 0.75–300ngmL−1. The resulting equilibrium dissociation and association constants (KD and KA) were 5.06×10−8M and 1.98×107M−1, respectively. The association and dissociation rate constants (kon and koff) decreased substantially as Enbrel® concentration, [C], increased, despite that the net binding rate, (kon[C]+koff), increased. The decrease in kon and koff was hypothesized to be a consequence of mass transport limitations. To verify this, QCM dissipation measurements were analyzed to provide insight on solution viscosity. As Enbrel® concentration increased, the net change in dissipation, ΔD, became larger. An augmentation of ΔD is associated with a higher solution viscosity, which would result in an increase in mass transport limitations. Therefore, the decrease in kon and koff for increasing Enbrel® concentration can be attributed to mass transport limitations. In conclusion, QCM is a valuable complementary real-time label-free biosensor analysis platform for the biopharmaceutical industry. Unlike the surface plasmon resonance (SPR) platform, QCM allows measuring dissipation, which can provide insight on how mass transport limitations impact interaction kinetics.

Solvent-assisted lipid bilayer formation on silicon dioxide and gold.

Planar lipid bilayers on solid supports mimic the fundamental structure of biological membranes and can be investigated using a wide range of surface-sensitive techniques. Despite these advantages, planar bilayer fabrication is challenging, and there are no simple universal methods to form such bilayers on diverse material substrates. One of the novel methods recently proposed and proven to form a planar bilayer on silicon dioxide involves lipid deposition in organic solvent and solvent exchange to influence the phase of adsorbed lipids. To scrutinize the specifics of this solvent-assisted lipid bilayer (SALB) formation method and clarify the limits of its applicability, we have developed a simplified, continuous solvent-exchange version to form planar bilayers on silicon dioxide, gold, and alkanethiol-coated gold (in the latter case, a lipid monolayer is formed to yield a hybrid bilayer) and varied the type of organic solvent and rate of solvent exchange. By tracking the SALB formation process with simultaneous quartz crystal microbalance-dissipation (QCM-D) and ellipsometry, it was determined that the acoustic, optical, and hydration masses along with the acoustic and optical thicknesses, measured at the end of the process, are comparable to those observed by employing conventional fabrication methods (e.g., vesicle fusion). As shown by QCM-D measurements, the obtained planar bilayers are highly resistant to protein adsorption, and several, but not all, water-miscible organic solvents could be successfully used in the SALB procedure, with isopropanol yielding particularly high-quality bilayers. In addition, fluorescence recovery after photobleaching (FRAP) measurements demonstrated that the coefficient of lateral lipid diffusion in the fabricated bilayers corresponds to that measured earlier in the planar bilayers formed by vesicle fusion. With increasing rate of solvent exchange, it was also observed that the bilayer became incomplete and a phenomenological model was developed in order to explain this feature. The results obtained allowed us to clarify and discriminate likely steps of the SALB formation process as well as determine the corresponding influence of organic solvent type and flow conditions on these steps. Taken together, the findings demonstrate that the SALB formation method can be adapted to a continuous solvent-exchange procedure that is technically minimal, quick, and efficient to form planar bilayers on solid supports.

Nanomechanics of Clathrin Protein Shells

The nanomechanical properties of protein shells, ranging from virus capsids to clathrin-coated vesicles, are functionally important and of notable recent interest. Using atomic force microscopy (AFM) and, more recently, quartz crystal microbalance-dissipation (QCM-D) instrumentation, we investigate mechanical properties of intact clathrin-coated vesicles (CCVs), reassembled pure clathrin cage baskets, and single triskelia. CCVs are purified subcellular trafficking organelles of about 100 nm diameter, consisting of an outer clathrin lattice shell over a receptor- and adaptor- loaded lipid vesicle. The clathrin cages formed by self-assembly of clathrin triskelia are structurally similar to the outer shell of CCVs. From AFM and complementary QCM-D measurements, we now provide an integrative mechanical model of the clathrin protein shell and of the coupling between the clathrin coat and the membrane vesicle, in biologically relevant fluid environments. Whereas the Young's modulus of the triskelia arm is as high as a few MPa, the effective shear moduli of clathrin triskelia, clathrin baskets, and CCVs are one to two orders of magnitude smaller and vary with the buffer pH and ionic strength, as well as their proximity to a given substrate support. Such modulation of the nanomechanical properties of the assembling/disassembling protein shells may be an essential aspect of cellular function.

Human Immunoglobulin Adsorption Investigated by Means of Quartz Crystal Microbalance Dissipation, Atomic Force Microscopy, Surface Acoustic Wave, and Surface Plasmon Resonance Techniques

Time-resolved adsorption behavior of a human immunoglobin G (hIgG) protein on a hydrophobized gold surface is investigated using multitechniques: quartz crystal microbalance/dissipation (QCM-D) technique; combined surface plasmon resonance (SPR) and Love mode surface acoustic wave (SAW) technique; combined QCM-D and atomic force microscopy (AFM) technique. The adsorbed hIgG forms interfacial structures varying in organization from a submonolayer to a multilayer. An "end-on" IgG orientation in the monolayer film, associated with the surface coverage results, does not corroborate with the effective protein thickness determined from SPR/SAW measurements. This inconsistence is interpreted by a deformation effect induced by conformation change. This conformation change is confirmed by QCM-D measurement. Combined SPR/SAW measurements suggest that the adsorbed protein barely contains water after extended contact with the hydrophobic surface. This limited interfacial hydration also contributed to a continuous conformation change in the adsorbed protein layer. The viscoelastic variation associated with interfacial conformation changes induces about 1.5 times overestimation of the mass uptake in the QCM-D measurements. The merit of combined multitechnique measurements is demonstrated.

Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly( l-lysine)/poly( l-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.

Characterization of heparin surfaces using photoelectron spectroscopy and quartz crystal microbalance

In order to gain insight into the molecular structure of a new heparin surface (Corline heparin surface, CHS), quartz crystal microbalance dissipation (QCM-D) and photoelectron spectroscopy (PES) were used. Two surfaces, with different surface thickness and surface concentration of heparin, were compared. QCM-D measurements provided information on the thickness of the two different heparin surfaces and confirmed that the process of formation of the two layers were virtually identical. PES showed that the thicker coating resulted in complete coverage of the substrate, whereas with the thinner coating Fe signals originating from the stainless steel substrate could be detected. Information of the molecular surface structure of the heparin coatings could be derived from the ratios at different photon energies between S2p signals from sulfonate groups in heparin and disulfide groups originating from a cross-linking unit in the heparin conjugate.

Cell adhesion on supported lipid bilayers.

The cell and protein repellent properties of supported phospholipid bilayer (SPB) membranes were investigated. The SPBs were prepared by vesicle adsorption on SiO(2) surfaces. The vesicles of phosphatidylcholine fuse and rupture, and form a supported bilayer covering the surface. We carried out cell culture experiments on several surfaces, including SPBs, using two types of epithelial cells to address the cell adhesional properties. The Quartz Crystal Microbalance Dissipation (QCM-D) technique was used to monitor the SPB formation and subsequent protein adsorption. Neither cell type adhered or proliferated on SiO(2) surfaces coated with SPBs, whereas both cell types adhered and proliferated on the three control surfaces of SiO(2), tissue culture glass, and TiO(2). The QCM-D measurements showed that about two orders of magnitude less mass adsorbed on a SPB surface compared to a TiO(2) surface, from serum-containing media (10% fetal bovine serum). The reduced adsorption on the SPB is a likely explanation for the nondetectable epithelial cell adhesion on the SPB surface. Biomembranes are therefore attractive candidate systems to achieve alternating cell-resistant and cell-interacting regions on surfaces, by including specific cell-binding proteins in the latter regions.

Adsorption and Frictional Properties of Gemini Surfactants at Solid Surfaces

The adsorption and frictional properties of gemini surfactants at hydrophilic gold surfaces were measured using QCM-D (quartz crystal microbalance dissipation) and AFM. The molecular packing of a series of gemini surfactants was determined from QCM-D measurements, and the frictional behaviors of the surfactant films were characterized by employing atomic force microscopy (AFM). The results show that by changing the length of the spacer group from 3 to 12 a systematic change in the molecular packing at the surface is obtained. Furthermore, the molecular packing is seen to correlate to the frictional behavior of the surfactant film. A linear relation between the spacer group length, the adsorbed amount, and the frictional properties of the layer at the solid surface is found. This is discussed in terms of the critical packing parameter (CPP) of the surfactant, and a relation between CPP and frictional behavior is proposed. No correlation between spacer length and viscoelasticity of the adsorbed surfactant layer was detected using QCM-D. This indicates that the resolution of the dissipation factor from QCM-D measurements is not sufficient to describe the viscoelastic character of the thin surfactant film.