Supported Lipid Bilayer Platform for Characterizing the Membrane-Disruptive Behaviors of Triton X-100 and Potential Detergent Replacements

Triton X-100 (TX-100) is a widely used detergent to prevent viral contamination of manufactured biologicals and biopharmaceuticals, and acts by disrupting membrane-enveloped virus particles. However, environmental concerns about ecotoxic byproducts are leading to TX-100 phase out and there is an outstanding need to identify functionally equivalent detergents that can potentially replace TX-100. To date, a few detergent candidates have been identified based on viral inactivation studies, while direct mechanistic comparison of TX-100 and potential replacements from a biophysical interaction perspective is warranted. Herein, we employed a supported lipid bilayer (SLB) platform to comparatively evaluate the membrane-disruptive properties of TX-100 and a potential replacement, Simulsol SL 11W (SL-11W), and identified key mechanistic differences in terms of how the two detergents interact with phospholipid membranes. Quartz crystal microbalance-dissipation (QCM-D) measurements revealed that TX-100 was more potent and induced rapid, irreversible, and complete membrane solubilization, whereas SL-11W caused more gradual, reversible membrane budding and did not induce extensive membrane solubilization. The results further demonstrated that TX-100 and SL-11W both exhibit concentration-dependent interaction behaviors and were only active at or above their respective critical micelle concentration (CMC) values. Collectively, our findings demonstrate that TX-100 and SL-11W have distinct membrane-disruptive effects in terms of potency, mechanism of action, and interaction kinetics, and the SLB platform approach can support the development of biophysical assays to efficiently test potential TX-100 replacements.

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.

pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopy's dipole spread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Green's tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14 degree orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

A Pillar-Free Diffusion Device for Studying Chemotaxis on Supported Lipid Bilayers

Chemotactic cell migration plays a crucial role in physiological and pathophysiological processes. In tissues, cells can migrate not only through extracellular matrix (ECM), but also along stromal cell surfaces via membrane-bound receptor–ligand interactions to fulfill critical functions. However, there remains a lack of models recapitulating chemotactic migration mediated through membrane-bound interactions. Here, using micro-milling, we engineered a multichannel diffusion device that incorporates a chemoattractant gradient and a supported lipid bilayer (SLB) tethered with membrane-bound factors that mimics stromal cell membranes. The chemoattractant channels are separated by hydrogel barriers from SLB in the cell loading channel, which enable precise control of timing and profile of the chemokine gradients applied on cells interacting with SLB. The hydrogel barriers are formed in pillar-free channels through a liquid pinning process, which eliminates complex cleanroom-based fabrications and distortion of chemoattractant gradient by pillars in typical microfluidic hydrogel barrier designs. As a proof-of-concept, we formed an SLB tethered with ICAM-1, and demonstrated its lateral mobility and different migratory behavior of Jurkat T cells on it from those on immobilized ICAM-1, under a gradient of chemokine CXCL12. Our platform can thus be widely used to investigate membrane-bound chemotaxis such as in cancer, immune, and stem cells.

Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-like Membrane

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6/m2 to ≃0.1/m2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

Ionic contrast across a lipid membrane for Debye length extension: towards an ultimate bioelectronic transducer

Despite technological advances in biomolecule detections, evaluation of molecular interactions via potentiometric devices under ion-enriched solutions has remained a long-standing problem. To avoid severe performance degradation of bioelectronics by ionic screening effects, we cover probe surfaces of field effect transistors with a single film of the supported lipid bilayer, and realize respectable potentiometric signals from receptor–ligand bindings irrespective of ionic strength of bulky solutions by placing an ion-free water layer underneath the supported lipid bilayer. High-energy X-ray reflectometry together with the circuit analysis and molecular dynamics simulation discovered biochemical findings that effective electrical signals dominantly originated from the sub-nanoscale conformational change of lipids in the course of receptor–ligand bindings. Beyond thorough analysis on the underlying mechanism at the molecular level, the proposed supported lipid bilayer-field effect transistor platform ensures the world-record level of sensitivity in molecular detection with excellent reproducibility regardless of molecular charges and environmental ionic conditions.