Suspended Lipid Bilayers Research Articles

Evaluation of the passive permeability of antidepressants through pore-suspended lipid bilayer

HypothesisThe antidepressant drug imipramine, and its metabolite desipramine show different extents of interaction with, and passive permeation through, cellular membrane models, with the effects depending on the membrane composition. Through multimodal interrogation, we can observe that the drugs have a direct impact on the physicochemical properties of the membrane, that may play a role in their pharmacokinetics. ExperimentsMicrocavity pore-suspended lipid bilayers (MSLBs) of four different compositions, each with a different headgroup charge namely; zwitterionic dioleoylphosphatidylcholine (DOPC), mixed DOPC and negatively charged dioleoylphosphatidylglycerol (DOPG) (3:1), mixed DOPC and positively charged dioleoyltrimethylammoniumpropane (DOTAP) (3:1), and with increasing complex composition mimicking blood-brain-barrier (BBB) were prepared on gold and polydimethylsiloxane (PDMS) substrates using a Langmuir-Blodgett-vesicle fusion method. The molecular interaction and permeation of antidepressants, imipramine, and its metabolite desipramine with the lipid bilayers were evaluated using highly sensitive label-free electrochemical impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS). Drug-induced membrane packing/fluidity alterations were assessed using fluorescence lifetime imaging (FLIM) and fluorescence lifetime correlation spectroscopy (FLCS) of MSLB over microfluidic PDMS array. FindingsUsing EIS to evaluate in real-time membrane admittance changes, we found that imipramine greatly increases the ion permeability of negatively charged DOPC:DOPG (3:1) membranes. The effect was observed also at neutral (DOPC) and to a lesser extent at positively charged DOPC:DOTAP(3:1) membranes. In contrast, desipramine had a much weaker impact on ion permeability across all bilayer compositions. Temporal capacitance data show that desipramine intercalates at negatively charged membrane thereby increasing the thickness of the membrane. The overall kinetics of the imipramine permeation is higher than that of desipramine. This was confirmed using SERS, which also provides an evaluation of drug passive permeation based on arrival time across the membrane. Using FLCS, we found that imipramine increases the lipid membrane fluidity, whereas desipramine lowers it, with the exception of the negatively charged membrane. A translocation rate pharmacokinetics model was established for the first time at the MSLB platform by real-time monitoring of the variation in membrane resistance of pristine DOPC and blood-brain-barrier (BBB) membrane.

A Versatile Suspended Lipid Membrane System for Probing Membrane Remodeling and Disruption.

Artificial membrane systems can serve as models to investigate molecular mechanisms of different cellular processes, including transport, pore formation, and viral fusion. However, the current, such as SUVs, GUVs, and the supported lipid bilayers suffer from issues, namely high curvature, heterogeneity, and surface artefacts, respectively. Freestanding membranes provide a facile solution to these issues, but current systems developed by various groups use silicon or aluminum oxide wafers for fabrication that involves access to a dedicated nanolithography facility and high cost while conferring poor membrane stability. Here, we report the development, characterization and applications of an easy-to-fabricate suspended lipid bilayer (SULB) membrane platform leveraging commercial track-etched porous filters (PCTE) with defined microwell size. Our SULB system offers a platform to study the lipid composition-dependent structural and functional properties of membranes with exceptional stability. With dye entrapped in PCTE microwells by SULB, we show that sphingomyelin significantly augments the activity of pore-forming toxin, Cytolysin A (ClyA) and the pore formation induces lipid exchange between the bilayer leaflets. Further, we demonstrate high efficiency and rapid kinetics of membrane fusion by dengue virus in our SULB platform. Our suspended bilayer membrane mimetic offers a novel platform to investigate a large class of biomembrane interactions and processes.

Suspended Lipid Bilayer: A Versatile Platform for Nextgen Drug Discovery and Biomedical Applications

Suspended Lipid Bilayer: A Versatile Platform for Nextgen Drug Discovery and Biomedical Applications

Protein-coated nanoparticles exhibit Lévy flights on a suspended lipid bilayer.

Lateral diffusion of nano-objects on lipid membranes is a crucial process in cell biology. Recent studies indicate that nanoparticle lateral diffusion is affected by the presence of membrane proteins and deviates from Brownian motion. Gold nanoparticles (Au NPs) stabilized by short thiol ligands were dispersed near a free-standing bilayer formed in a 3D microfluidic chip. Using dark-field microscopy, the position of single NPs at the bilayer surface was tracked over time. Numerical analysis of the NP trajectories shows that NP diffusion on the bilayer surface corresponds to Brownian motion. The addition of bovine serum albumin (BSA) protein to the solution led to the formation of a protein corona on the NP surface. We found that protein-coated NPs show anomalous superdiffusion and that the distribution of their relative displacement obeys Lévy flight statistics. This superdiffusive motion is attributed to a drastic reduction in adhesive energies between the NPs and the bilayer in the presence of the protein corona. This hypothesis was confirmed by numerical simulations mimicking the random walk of a single particle near a weakly adhesive surface. These results may be generalized to other classes of nano-objects that experience adsorption-desorption behaviour with a weakly adhesive surface.

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

Nascent fusion pore opening monitored at single-SNAREpin resolution

Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.

Anionic nanoparticle-induced perturbation to phospholipid membranes affects ion channel function

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.

Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy

AbstractMiltefosine an alkylphosphocholine analogue, is the only drug taken orally for the treatment of leishmaniasis‐a parasitic disease caused by sandflies. Although it is believed that Miltefosine exerts its activity by acting at the lipid membrane, detailed understanding of the interaction of this drug with eukaryotic membranes is still lacking. Herein, we exploit microcavity pore suspended lipid bilayers (MSLBs) as a biomimetic platform in combination with a highly sensitive label‐free electrochemical impedance spectroscopy (EIS) technique to gain biophysical insight into the interaction of Miltefosine with host cell membrane as a function of lipid membranes composition. Four membrane compositions with increasing complexity were evaluated; DOPC, DOPC : Chol (75 : 25), domain forming DOPC : SM : Chol (40 : 40 : 20) and mammalian plasma membrane (MPM) mimetic DOPC:DOPE:Chol:SM:DOPS (32 : 25 : 20 : 15 : 8) and used to study the interaction of Miltefosine in a concentration‐dependent manner using EIS. The membrane resistance changes in response to Miltefosine were modelled by an empirical Langmuir isotherm binding model to provide estimates of binding saturation and equilibrium association constant. Miltefosine was found to have greatest impact on electrochemical properties of the simpler membrane systems; DOPC and DOPC : Chol, where these membranes were found to be more susceptible to membrane thinning, attributed to strong permeation/penetration of the drug whilst, compositions that included both Chol and SM, expected to contain large liquid‐ordered domains exhibited weaker changes to membrane resistance but strongest drug association. In contrast, at the MPM membrane, Miltefosine exerts weakest association, which is tentatively attributed to electrostatic effects from the anionic DOPS but some membrane thinning is observed reflected in change in resistance and capacitance values attributed to some weak permeation.

The Impact of Membrane Composition and Co‐Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy

AbstractThe interaction of the antibacterial drug, vancomycin with microcavity suspended lipid bilayers (MSLB) was investigated using non‐Faradaic electrochemical impedance spectroscopy (EIS). Five MSLB compositions were prepared with increasing complexity at gold substrates: DOPC, DOPC:Chol, DOPC:SM:Chol, mammalian plasma membrane mimetic (MPM), and E. coli polar lipid extract. The latter two, are intended to mimic eukaryotic and bacterial inner membrane compositions respectively. The extent of vancomycin association and the impact drug association has on the electrochemical properties of the membrane depended on biomembrane composition. Trends were similar across all membranes but the E. coli membrane composition. Vancomycin increased membrane resistance and decreased capacitance in a saturable, concentration dependent manner, but for E. coli membrane resistance change was negligible and capacitance increased. Membrane resistance data was fit to the Langmuir‐Freundlich model to give quantitative insight into the relative extent of association of drug with membrane as a function of membrane composition. Overall, electrochemical data indicates that vancomycin associates at the interface of lipid membranes rather than penetrates the layer and this is promoted by the presence of anionic phospholipid. Interestingly, co‐incubation of the E. coli extract bilayer with both rifampicin and vancomycin, known to act synergistically, significantly promotes vancomycin association to E. coli membrane.

Placental Trophoblast-Inspired Lipid Bilayers for Cell-Free Investigation of Molecular Interactions.

The placenta plays a key role in regulating the maternal-fetal transport but it is a difficult organ to study due to a lack of existing in vitro models. Lipid bilayers inspired by the placenta can provide a facile new in vitro tool with promise for screening molecular transport across this important organ. Here we developed lipid bilayers that mimic the composition of human placental trophoblast cells at different times during the course of pregnancy. Mass spectrometry identified five major lipid classes (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin) present at varying concentrations in trophoblasts representative of the first and third trimesters and full-term placenta. We successfully developed supported and suspended lipid bilayers mimicking these trophoblast lipid compositions and then demonstrated the utility of these synthetic placenta models for investigating molecular interactions. Specifically, we investigated the interactions with di(2-ethylhexyl) phthalate (DEHP), a common plasticizer and environmental toxicant, and amphotericin B, a common yet toxic, antifungal therapeutic. Overall, we observed that DEHP adsorbs and potentially embeds itself within all placental lipid bilayers, with varying levels of interaction. For both amphotericin B and a liposomal formulation of amphotericin B, AmBisome, we noted lower levels of permeation in transport studies with bilayers and trophoblast cells compared with DEHP, likely driven by differences in size. AmBisome interacted less with both the supported and suspended placental lipid bilayers in comparison to amphotericin B, suggesting that drug delivery carriers can vary the impact of a pharmaceutical agent on these lipid structures. We found that the apparent permeability observed in suspended bilayers was approximately an order of magnitude less than those observed for trophoblast monolayers, which is typical of lipid bilayers. Ultimately, these placenta mimetic lipid bilayers can serve as a platform for the rapid initial screening of molecular interactions with the maternal-fetal interface to better inform future testing.

Investigating interactions of phthalate environmental toxicants with lipid structures

Di(2-ethylhexyl) phthalate (DEHP) is one of the most abundant plasticizers in common household products. It leaches from materials, resulting in exposure associated with detrimental health effects. The main objective of this study was to investigate how DEHP and its metabolite, mono(2-ethylhexyl) phthalate (MEHP), interact with and permeate lipid structures, namely vesicles and planar bilayers. Using dynamic light scattering, we observed significant changes in the size and polydispersity of L-α-phosphatidylcholine (egg PC) vesicles when incubated with DEHP but not MEHP at the same concentrations (100 and 200 μM). We demonstrated that these effects are mitigated by pre-treatment with chitosan nanoparticles which adsorb the phthalates. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we observed a concentration dependence on the interaction of DEHP with egg PC supported lipid bilayers (SLBs). QCM-D results suggested lipid removal for 5 and 100 μM DEHP, and adsorption and potential embedment in the bilayer at 50 and 200 μM DEHP. SLB mass decrease was observed for all concentrations of MEHP (5, 50, 100, and 200 μM), suggesting lipid removal. We also investigated the permeability of DEHP and MEHP as well as several small molecules across a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) suspended lipid bilayer. We found that DEHP and MEHP both had low permeabilities, but only DEHP remained associated with the bilayer. Exposure to DEHP and MEHP influenced how several common small molecules interacted with DOPC bilayers. Ultimately, this work provides insight into mechanisms of phthalate interactions with lipid structures, having implications for human health.

Free-Standing Lipid Bilayers Based on Nanopore Array and Ion Channel Formation.

Integrated nanopores are novel and versatile single-molecule sensors for individual label-free biopolymer detection and characterization. However, their studies and application requires a stable lipid bilayer to maintain protein function. Herein, we describe a method for producing lipid bilayers across a nanopore array on a silicon nitride substrate. We used a painting technique commonly used with Teflon films to embed α-hemolysin (α-HL) into bilayer lipid membranes (BLMs) to form an ion channel. This was carried out in nanofluid developed in our lab. The membrane formation process, stability of BLMs and ion channel recordings were monitored by patch clamp in real-time. BLM formation was demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore in the range of ±100 mV. Membrane resistance (Rm) and capacitance (Cm) of the device with the bilayer were assessed by membrane test as above 1.0 GΩ and ~20±2 pF, respectively. The silicon nitride surface and aperture edge were smooth at the nanometer lever leading to remarkable membrane stability. The membrane lifetime was 5-24 h. A single α-HL channel inserted in 30-60 min applied a potential of +100 mV. The α-HL channel currents were recorded at ~100±10 pA. Such integrated nanopores enable analysis of channel functions under various solution conditions from the same BLM. This will open up a variety of applications for ion channels including high-throughput medical screening and diagnosis.

Surface Modification of Glass/PDMS Microfluidic Valve Assemblies Enhances Valve Electrical Resistance.

Microfluidic instrumentation offers unique advantages in biotechnology applications including reduced sample and reagent consumption, rapid mixing and reaction times, and a high degree of process automation. As dimensions decrease, the ratio of surface area to volume within a fluidic architecture increases, which gives rise to some of the unique advantages inherent to microfluidics. Thus, manipulation of surface characteristics presents a promising approach to tailor the performance of microfluidic systems. Microfluidic valves are essential components in a number of small volume applications and for automated microfluidic platforms, but rigorous evaluation of the sealing quality of these valves is often overlooked. In this work, the glass valve seat of hybrid glass/PDMS microfluidic valves was surface modified with hydrophobic silanes, octyldimethylchlorosilane (ODCS) or (tridecafluoro-1,1,2,2-tetrahydrooctyl)dimethylchlorosilane (PFDCS), to investigate the effect of surface energy on electrical resistance of valves. Valves with ODCS- or PFDCS-modified valve seats both exhibited >70-fold increases in electrical resistance (>500 GΩ) when compared to the same valve design with unmodified glass valve seats (7 ± 3 GΩ), indicative of higher sealing capacity. The opening times for valves with ODCS- or PFDCS-modified valve seats was ca. 5× shorter compared to unmodified valve seats, whereas the closing time was up to 8× longer for modified valve seats, although the total closing time was ≤1.5 s, compatible with numerous microfluidic valving applications. Surface modified valve assemblies offered sufficient electrical resistance to isolate sub-pA current signals resulting from electrophysiology measurement of α-hemolysin conductance in a suspended lipid bilayer. This approach is well-suited for the design of novel microfluidic architectures that integrate fluidic manipulations with electrophysiological or electrochemical measurements.

Array of Freestanding Planar Lipid Bilayers for Parallel Optical and Electrical Recordings

Here we report on the design and validation of an array platform for parallel high-resolution electrophysiological and optical recordings. The recording chip is based on a SU-8 coated glass slide (ca. 200 μm thickness) containing 4 cavities with individual ring-shaped Ag/AgCl-microelectrodes as well as a common ground electrode. Four suspended lipid bilayers can be self-assembled on the chip surface over the cavities. The electrodes are connected to a multichannel amplifier capable of simultaneous electrical recording with high-bandwidth and low noise (<1pA [email protected]). The ring shape of the electrodes creates an optical window allowing access with a high-NA oil or water-immersion objective on an inverted microscope. This allows for e.g. single-molecule fluorescence measurements from one of the four bilayers using time-correlated-single-photon-counting (TCSPC). Fluorescently labeled alpha-hemolysin for optical and electrical measurements was expressed in-vitro and purified. BODIPY at position 131 was incorporated during the translation in a site-directed manner via stop or four-base codon suppression using pre-charged tRNAs. A series of characterization and validation experiments performed with the system include: (1)Studies of diffusion rates of fluorescently labelled Hemolysin and lipid molecules in the bilayer using fluorescence correlation spectroscopy (FCS); (2)Monitoring of alpha-hemolysin membrane insertion using fluorescence lifetime imaging (FLIM) and (3)Visualization of fusion of labeled proteoliposomes resulting in membrane channel insertion. In the two latter cases, changes in fluorescence intensity were monitored simultaneously with alterations in membrane conductance. This system allows for rapid membrane array formation and by providing ready combination of fluorescence microscopy with high-resolution electrophysiology, enables correlated optical and electrical characterization and monitoring of the interactions of channel forming proteins in artificial lipid bilayers on a single-molecule level. The technology can be further implemented for studies of membrane protein folding and mobility as well as of membrane transport.

Lipid Corona Formation from Nanoparticle Interactions with Bilayers

Summary Although mixing nanoparticles with certain biological molecules can result in coronas that afford some control over how engineered nanomaterials interact with living systems, corona formation mechanisms remain enigmatic. Here, we report results from experiments and computer simulations that provide concrete lines of evidence for spontaneous lipid corona formation without active mixing upon attachment to stationary and suspended lipid bilayer membranes. Experiments show that polycation-wrapped particles disrupt the tails of zwitterionic lipids, increase bilayer fluidity, and leave the membrane with reduced ζ potentials. Computer simulations suggest that the contact ion pairing between the lipid head groups and the polycations' ammonium groups leads to the formation of stable, albeit fragmented, lipid bilayer coronas. The mechanistic insight regarding lipid corona formation can be used to improve control over nano-bio interactions and to help understand why some nanomaterial-ligand combinations are detrimental to organisms but others are not.

Nanoporous Hydrogels for the Observation of Anthrax Exotoxin Translocation Dynamics.

The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements. Here, we utilize nanoporous hydrogel membranes to assess the translocation of full-length anthrax lethal and edema factors through nanopores similar in diameter to protective antigen translocons. We find that, relative to globular serum and other proteins that do not translocate natively through nanopores, anthrax exotoxins demonstrate significantly reduced barriers to pore entry. Computed free-energy barriers to the unfolding of proteins and the dissociation of macromolecular complexes are generally found to coincide with translocation. Finally, a nanopore-blocking strategy is developed that utilizes nonspecific synthetic peptide constructs and effectively prevents LF translocation within the nanoporous hydrogel.

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels.

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

Voltage-Dependent Formation of Stable, Ion Conductive Pores in Suspended Lipid Bilayers from Oxidized Lipids

Oxidation of membrane lipids is a ubiquitous phenomenon in eukaryotic cells occurring as a consequence of the generation of reactive oxygen species by mitochondrial or peroxisomal activity and irradiation with photons or ions. Lipid hydroperoxidation, the primary step of the oxidative reactions, has been shown to induce molecular area increase, enhanced fluctuations and tube formation in giant unilamellar vesicles. Surprisingly, however, despite a 75% loss of elastic modulus, even fully hydroperoxidized membranes can remain effective barriers against small molecule diffusion1, contrary to membranes including other downstream oxidation products2,3,4. We were therefore interested to ascertain whether membrane permeability to ions of pure lipid bilayers is affected by lipid hydroperoxidation. Using a chip-based device for automated bilayer formation and parallel recording from suspended synthetic lipid bilayers (Orbit-16, Nanion), we compared responses to positive and negative voltages in the range of 50 to 200 mV of POPC- and 100% hydroperoxidized POPC (POPC-OOH)1 membranes formed on 50 µm-diam. microelectrode cavities of the MECA16 chip (Ionera). While control POPC membranes showed a stable conductance in the range of a few pS without evidence for pore-formation, POPC-OOH membranes responded with fluctuating conductance increases already at 50 mV. Conductance grew in a voltage and time-dependent manner, reaching values of several nS at +/-150 mV. Remarkably, the voltage induced conductance was stable over time and did not recover during a 1 s waiting period between pulses, showing cumulative behavior from one pulse to the next. Conductance fluctuations were characterized by multiple preferred levels separated by several tens of pS without evident equidistant periodicity. Taken together, these findings suggest that in response to voltage, hydroperoxidized POPC membranes form multiple, stable voltage-dependent, ion-permeant pores with conductances similar to protein ion channels.(1) Weber. et al. Lipid Oxidation Induces Structural Changes in Biomimetic Membranes. Soft Matter 2014, 10, 4241-4247.(2) Caetano et al. Photo-induced Destruction of Giant Vesicles in Methylene Blue Solutions. Langmuir, 2007, 23, 130-137.(3) Mertins et al. Physical Damage on Giant Vesicles Membrane as a Result of Methylene Blue Photoirradiation, Biophys. J. 2014, 106, 162-171.(4) Runas, K. A.; Malmstadt, N. Low Levels of Lipid Oxidation Radically Increase the Passive Permeability of Lipid Bilayers. Soft Matter 2015, 11, 499-505.