Stability Of Lipid Bilayers Research Articles

Extracellular vesicle biomarkers in ocular fluids associated with ophthalmic diseases

Extracellular vesicles (EVs) are released as highly stable lipid bilayer particles carrying proteins, lipids, glycans and miRNAs. The contents of EVs vary based on the cellular origin, biogenesis route and the functional state of the cell suggesting certain diseased conditions. A growing body of evidence show that EVs carry important molecules implicated in the development and progression of ophthalmic diseases. EVs associated with ophthalmic diseases are mainly carried by one of the three ocular biofluids which include tears, aqueous humor and vitreous humor. This review summarizes the list of EV derived biomarkers identified thus far in ocular fluids for ophthalmic disease diagnosis. Further, the methods used for sample collection, sample volume and the sample numbers used in these studies have been highlighted. Emphasis has been given to describe the EV isolation and the characterization methods used, EV size profiled and the EV concentrations analyzed by these studies, thus providing a roadmap for future EV biomarker studies in ocular fluids.

Harnessing genetically engineered cell membrane-derived vesicles as biotherapeutics

Cell membrane-derived vesicles (CMVs) are particles generated from living cells, including extracellular vesicles (EVs) and artificial extracellular vesicles (aEVs) prepared from cell membranes. CMVs possess considerable potential in drug delivery, regenerative medicine, immunomodulation, disease diagnosis, etc . owing to their stable lipid bilayer structure, favorable biocompatibility, and low toxicity. Although the majority of CMVs inherit certain attributes from the original cells, it is still difficult to execute distinct therapeutic functions, such as organ targeting, signal regulation, and exogenous biotherapeutic supplementation. Hence, engineering CMVs by genetic engineering, chemical modification, and hybridization is a promising way to endow CMVs with specific functions and open up novel vistas for applications. In particular, there is a growing interest in genetically engineered CMVs harnessed to exhibit biotherapeutics. Herein, we outline the preparation strategies and their characteristics for purifying CMVs. Additionally, we review the advances of genetically engineered CMVs utilized to target organs, regulate signal transduction, and deliver biomacromolecules and chemical drugs. Furthermore, we also summarize the emerging therapeutic applications of genetically engineered CMVs in addressing tumors, diabetes, systemic lupus erythematosus, and cardiovascular diseases.

Elucidating the Influence of Lipid Composition on Bilayer Perturbations Induced by the N-terminal Region of the Huntingtin Protein.

Understanding the membrane interactions of the N-terminal 17 residues of the huntingtin protein (HttN) is essential for unraveling its role in cellular processes and its impact on huntingtin misfolding. In this study, we used atomic force microscopy (AFM) to examine the effects of lipid specificity in mediating bilayer perturbations induced by HttN. Across various lipid environments, the peptide consistently induced bilayer disruptions in the form of holes. Notably, our results unveiled that cholesterol enhanced bilayer perturbation induced by HttN, while phosphatidylethanolamine (PE) lipids suppressed hole formation. Furthermore, anionic phosphatidylglycerol (PG) and cardiolipin lipids, along with cholesterol at high concentrations, promoted the formation of double-bilayer patches. This unique structure suggests that the synergy among HttN, anionic lipids, and cholesterol can enhance bilayer fusion, potentially by facilitating lipid intermixing between adjacent bilayers. Additionally, our AFM-based force spectroscopy revealed that HttN enhanced the mechanical stability of lipid bilayers, as evidenced by an elevated bilayer puncture force. These findings illuminate the complex interplay between HttN and lipid membranes and provide useful insights into the role of lipid composition in modulating membrane interactions with the huntingtin protein.

A novel bimetallic MXene derivative QD-based ECL sensor for miRNA-27a-3p detection

In this work, a novel ECL biosensor has been developed based on bimetallic MXene derivative QDs (Mo2TiC2 QDs) and SnS2 nanosheets/lipid bilayer to detect the gastric cancer marker miRNA-27a-3p. On the one hand, the inter-band excitation of Mo2TiC2 QDs can inject the additional carriers, which were less suppressed by boundary effects and made a significant contribution to the luminescence process. Semiconductor Mo2TiC2 further inhibited the formation of internal electric field and potential oxidation. Therefore, Mo2TiC2 QDs processed superior luminous intensity and better stability. On the other hand, SnS2 nanosheets coated with phospholipid bilayer were designed as sensing interface. SnS2 nanosheets not only enhanced the luminous intensity of Mo2TiC2 QDs by virtue of their large surface area and low dielectric constant, but also improved the stability of lipid bilayer. Due to the excellent properties and synergy work of Mo2TiC2 QDs and the lipid bilayer-modified SnS2 nanosheets, the sensing system displayed high sensitivity and good reproducibility in the analysis application. As a result, the biosensor displayed good linear correlation between the ECL intensity and the concentration of miRNA-27a-3p over a wide range from 1 fM to 10 nM with the detection limit as low as 1 fM. The sensing system including the joint contribution of Mo2TiC2 QDs, SnS2 nanosheets and lipid bilayers had great potential for clinical applications.

Inkjet-Printed Phospholipid Bilayers on Titanium Oxide Surfaces: Towards Functional Membrane Biointerfaces.

Functional biointerfaces hold broad significance for designing cell-responsive medical implants and sensor devices. Solid-supported phospholipid bilayers are a promising class of biological materials to build bioinspired thin-film coatings, as they can facilitate interactions with cell membranes. However, it remains challenging to fabricate lipid bilayers on medically relevant materials such as titanium oxide surfaces. There are also limitations in existing bilayer printing capabilities since most approaches are restricted to either deposition alone or to fixed microarray patterning. By combining advances in lipid surface chemistry and on-demand inkjet printing, we demonstrate the direct deposition and patterning of covalently tethered lipid bilayer membranes on titanium oxide surfaces, in ambient conditions and without any surface pretreatment process. The deposition conditions were evaluated by quartz crystal microbalance-dissipation (QCM-D) measurements, with corresponding resonance frequency (Δf) and energy dissipation (ΔD) shifts of around −25 Hz and <1 × 10−6, respectively, that indicated successful bilayer printing. The resulting printed phospholipid bilayers are stable in air and do not collapse following dehydration; through rehydration, the bilayers regain their functional properties, such as lateral mobility (>1 µm2/s diffusion coefficient), according to fluorescence recovery after photobleaching (FRAP) measurements. By taking advantage of the lipid bilayer patterned architectures and the unique features of titanium oxide’s photoactivity, we further show how patterned cell culture arrays can be fabricated. Looking forward, this work presents new capabilities to achieve stable lipid bilayer patterns that can potentially be translated into implantable biomedical devices.

Photo-triggerable liposomes based on lipid-porphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Does Sucrose Change Its Mechanism of Stabilization of Lipid Bilayers during Desiccation? Influences of Hydration and Concentration.

Sugar-membrane interactions are believed to be responsible for cell preservation during desiccation and freezing, but the molecular mechanism by which they achieve this is still not well understood. The associated decrease of the main phase transition temperature of phospholipid bilayers is explained by two opposing views on the matter: the direct sugar-phospholipid interaction at the bilayer interface (water replacement hypothesis) and an entropy-driven phase transition with sugar molecules concentrating away from the lipid interface (hydration forces explanation). Both mechanisms are supported by experiments but molecular dynamics (MD) simulations have overwhelmingly shown the occurrence of direct sugar-phospholipid interactions. We have performed MD simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers at different water and sucrose contents. The behavior of sucrose was found to depend on both the sucrose and water contents: at high sucrose concentration and at low hydration, it is best described by the hydration forces explanation model, whereas at low sucrose concentration, it is consistent with the water replacement hypothesis model. These simulations reveal that at low concentration, sucrose molecules preferentially interact directly with the membrane interface while at high concentration, they preferentially accumulate in the intermembrane solution. The transition between the two modes of interaction is revealed for the first time as being governed by the saturation of the lipid bilayer interface with sucrose molecules, and this occurs more rapidly as the level of hydration decreases.

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.

Photothermally-Assisted Lipid Bilayer Coating on a Sin Nanopore for High-Throughput Protein Channel Formation

In our recent study, we found that irradiation of a visible laser beam on a silicon nitride (SiN) membrane results in localized heating, which allows control over the temperature at a nanopore sensor with μs-scale kinetics. Here we demonstrate the formation of a planar lipid bilayer coating on a SiN pore using a photothermal heat shock. We first demonstrate that 100-nm-diameter liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) can be electroosmotically driven to a SiN aperture by showing their translocation through a 300 nm diameter pore. We then use this electroosmotic flow to reversibly trap/dock liposomes at the entrance of a 50 nm pore under applied voltage, as evidenced by a reduced ion current through the pore. Once a liposome is trapped, we use a laser pulse to rapidly heat the SiN pore, which causes a thermal shock that ruptures the liposome, forming a planar lipid bilayer with low leakage. We demonstrate that the resulting ruptured vesicle is a bilayer by testing its voltage stability, as well as by demonstrating DNA translocations through a a-hemolysin loaded liposome that was ruptured on the SiN aperture. Our lipid coating using SiN photothermal heating aids high throughput and highly stable lipid bilayers for studies of single-molecule transport and sensing.

The Mechanisms of Action of Triindolylmethane Derivatives on Lipid Membranes.

The effects of new synthetic antibacterial agents – tris(1-pentyl-1H-indol-3-yl)methylium chloride (LCTA-1975) and (1-(4-(dimethylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-3-yl)bis(1-propyl- 1H-indol-3-yl)methylium chloride (LCTA-2701 – on model lipid membranes were studied. The ability of the tested agents to form ion-conductive transmembrane pores, influence the electrical stability of lipid bilayers and the phase transition of membrane lipids, and cause the deformation and fusion of lipid vesicles was investigated. It was established that both compounds exert a strong detergent effect on model membranes. The results of differential scanning microcalorimetry and measuring of the threshold transmembrane voltage that caused membrane breakdown before and after adsorption of LCTA-1975 and LCTA-2701 indicated that both agents cause disordering of membrane lipids. Synergism of the uncoupling action of antibiotics and the alkaloid capsaicin on model lipid membranes was shown. The threshold concentration of the antibiotic that caused an increase in the ion permeability of the lipid bilayer depended on the membrane lipid composition. It was lower by an order of magnitude in the case of negatively charged lipid bilayers than for the uncharged membranes. This can be explained by the positive charge of the tested agents. At the same time, LCTA-2701 was characterized by greater efficiency than LCTA-1975. In addition to its detergent action, LCTA-2701 can induce ion-permeable transmembrane pores: step-like current fluctuations corresponding to the opening and closing of individual ion channels were observed. The difference in the mechanisms of action might be related to the structural features of the antibiotic molecules: in the LCTA-1975 molecule, all three substituents at the nitrogen atoms of the indole rings are identical and represent n-alkyl (pentyl) groups, while LCTA-2701 contains a maleimide group, along with two alkyl substituents (n-propyl). The obtained results might be relevant to our understanding of the mechanism of action of new antibacterial agents, explaining the difference in the selectivity of action of the tested agents on the target microorganisms and their toxicity to human cells. Model lipid membranes should be used in further studies of the trends in the modification and improvement of the structures of new antibacterial agents.

Emerging Roles of Air Gases in Lipid Bilayers.

Recent studies indicate that changing the physical properties of lipid bilayers may profoundly change the function of membrane proteins. Here, the effects of dissolved nitrogen and oxygen molecules on the mechanical properties and stability of lipid bilayers are investigated using differential confocal microscopy, atomic force microscopy, and molecular dynamics simulations. All experiments evidence the presence of dissolved air gas in lipid bilayers prepared without gas control. The lipid bilayers in degassed solutions are softer and less stable than those in ambient solutions. High concentrations of nitrogen increase the bending moduli and stability of the lipid bilayers and impede phase separation in ternary lipid bilayers. The effect of oxygen is less prominent. Molecular dynamics simulations indicate that higher nitrogen affinity accounts for increased rigidity. These findings have fundamental and wide implications for phenomena related to lipid bilayers and cell membranes, including the origin of life.

A rule governing the FtsH-mediated proteolysis of the MgtC virulence protein from Salmonella enterica serovar Typhimurium

A tightly controlled turnover of membrane proteins is required for lipid bilayer stability, cell metabolism, and cell viability. Among the energy-dependent AAA+ proteases in Salmonella, FtsH is the only membrane-bound protease that contributes to the quality control of membrane proteins. FtsH preferentially degrades the C-terminus or N-terminus of misfolded, misassembled, or damaged proteins to maintain physiological functions. We found that FtsH hydrolyzes the Salmonella MgtC virulence protein when we substitute the MgtC 226th Trp, which is well conserved in other intracellular pathogens and normally protects MgtC from the FtsH-mediated proteolysis. Here we investigate a rule determining the FtsH-mediated proteolysis of the MgtC protein at Trp226 residue. Substitution of MgtC tryptophan 226th residue to alanine, glycine, or tyrosine leads to MgtC proteolysis in a manner dependent on the FtsH protease whereas substitution to phenylalanine, methionine, isoleucine, leucine, or valine resists MgtC degradation by FtsH. These data indicate that a large and hydrophobic side chain at 226th residue is required for protection from the FtsH-mediated MgtC proteolysis.

Formation of MspA channel on Nanopore-Spanning Lipid Bilayer

Biological nanopores are novel and versatile single-molecule sensor for individual label-free biopolymer detection and characterization. However, their studies and applications require a stable lipid bilayer to maintain protein function. Herein, we provided a method for producing lipid bilayers across nanopore array on a silicon nitride substrate by vesicle technique and MspA was embedded into lipid bilayer membrane formed ion channel. The formation process and stability of lipid bilayer membrane and translocation of samples were monitored real-time by patch clamp. The hybrid nanopore system exhibit excellent electrical properties and enhanced stability, making this system ideal for high-throughput DNA sequencing.

Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels

The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.

Cholesterol Metabolism in T Cells.

Compartmentalization and spatial control of biochemical reactions is the foundation of cell-based life on earth. The lipid bilayer system employed by eukaryote cells not only keeps them separate from the environment but also provides a platform for key receptors to sense and interact with outside factors. Arguably one of the cell types most reliant on interactions of this kind, immune cells depend on their membrane to keep functioning properly. In this review, the influence of variation in cholesterol levels, a key component of lipid bilayer stability, on T cells will be discussed in detail. In comparison to other cells, T cells must be able to undergo rapid activation followed by proliferation. Furthermore, receptor colocalization is an important mechanism in this activation process. The impact of cholesterol availability on the processes of T cell proliferation and receptor sensitivity, as well as its potential for immunomodulation in disease treatment will be considered.

Diphytanoyl lipids as model systems for studying membrane-active peptides

The branched chains in diphytanoyl lipids provide membranes with unique properties, such as high chemical/physical stability, low water permeability, and no gel-to-fluid phase transition at ambient temperature. Synthetic diphytanoyl phospholipids are often used as model membranes for electrophysiological experiments. To evaluate whether these sturdy lipids are also suitable for solid-state NMR, we have examined their interactions with a typical amphiphilic peptide in comparison with straight-chain lipids. First, their phase properties were monitored using 31P NMR, and the structural behaviour of the antimicrobial peptide PGLa was studied by 19F NMR and circular dichroism in oriented membrane samples. Only lipids with choline headgroups (DPhPC) were found to form stable lipid bilayers in oriented samples, while DPhPG, DPhPE and DPhPS display non-lamellar structures. Hence, the experimental temperature and hydration are crucial factors when using supported diphytanoyl lipids, as both parameters must be maintained in an appropriate range to avoid the formation of non-bilayer structures. For the same reason, a high content of other diphytanoyl lipids besides DPhPC in mixed lipid systems is not favourable. Unlike the situation in straight-chain membranes, we found that the α-helical PGLa was not able to insert into the tightly packed fluid bilayer of DPhPC but remained in a surface-bound state even at very high peptide concentration. This behaviour can be explained by the high cohesivity and the negative spontaneous curvature of the diphytanoyl lipids. These characteristic features must therefore be taken into consideration, both, in electrophysiological studies, and when interpreting the structural behaviour of membrane-active peptides in such lipid environment.

Enhanced stability of freestanding lipid bilayer and its stability criteria.

We present a new strategy to dramatically enhance the stability of freestanding lipid bilayers. We found that an addition of a water in oil emulsion stabilizer, SPAN 80 to a solvent phase guarantees nearly millimeter-scale stable freestanding lipid bilayers. The water permeability, bilayer area, contact angle, and interfacial tension were measured as a function of time and SPAN 80-to-lipid weight ratio (ΦSPAN 80) with several different solvents. Surprisingly, the SPAN 80, instead of remaining in the bilayer, was moved out of the bilayer during the bilayer formation. Also we studied the effect of solvent on freestanding bilayer formation, and found that squalene was the only solvent that was not incorporated into the bilayer. The regime of stable bilayer formation was experimentally determined to be 3/1 < ΦSPAN 80 < 15/1, and we suggest general stability criteria for bilayer formation. This technique and the suggested stability criteria can be potentially helpful to many model membrane-based researches in life sciences, physical sciences and biomedical engineering fields.

Characterization of exosomal release in bovine endometrial intercaruncular stromal cells.

BackgroundCell-to-cell communication between the blastocyst and endometrium is critical for implantation. In recent years, evidence has emerged from studies in humans and several other animal species that exosomes are secreted from the endometrium and trophoblast cells and may play an important role in cell-to-cell communication maternal-fetal interface during early pregnancy. Exosomes are stable extracellular lipid bilayer vesicles that encapsulate proteins, miRNAs, and mRNAs, with the ability to deliver their cargo to near and distant sites, altering cellular function(s). Furthermore, the exosomal cargo can be altered in response to environmental cues (e.g. hypoxia). The current study aims to develop an in vitro system to evaluate maternal-embryo interactions via exosomes (and exosomal cargo) produced by bovine endometrial stromal cells (ICAR) using hypoxia as a known stimulus associated with the release of exosomes and alterations to biological responses (e.g. cell proliferation).MethodsICAR cells cultured under 8 % O2 or 1 % O2 for 48 h and changes in cell function (i.e. migration, proliferation and apoptosis) were evaluated. Exosome release was determined following the isolation (via differential centrifugation) and characterization of exosomes from ICAR cell-conditioned media. Exosomal proteomic content was evaluated by mass spectrometry.ResultsUnder hypoxic conditions (i.e. 1 % O2), ICAR cell migration and proliferation was decreased (~20 and ~32 %, respectively) and apoptotic protein caspase-3 activation was increased (∼1.6 fold). Hypoxia increased exosome number by ~3.6 fold compared with culture at 8 % O2. Mass spectrometry analysis identified 128 proteins unique to exosomes of ICAR cultured at 1 % O2 compared with only 46 proteins unique to those of ICAR cultured at 8 % O2. Differential production of proteins associated with specific biological processes and molecular functions were identified, most notably ADAM10, pantetheinase and kininogen 2.ConclusionsIn summary, we have shown that a stimulus such as hypoxia can alter both the cellular function and exosome release of ICAR cells. Alterations to exosome release and exosomal content in response to stimuli may play a crucial role in maternal-fetal crosstalk and could also affect placental development.

(Invited) Nanopore Sensing with a Stable Lipid Bilayer in Microfabricated Devices

Artificial cell membranes have emerged as a biomimetic tool in such areas as membrane protein study, synthetic biology, and drug discovery. Planar lipid bilayer is the planar membrane and it is used for measuring transmembrane current of ion channels, membrane peptides, or pore-forming proteins. However, the stability of the lipid bilayer and the reproducibility of bilayer formation are serious issues for the practical experiments. To improve these issues, we have tried to use microfabrication technologies based on photolithography, micro-milling, and 3D printing. Microfabrication and microfluidics have the potential to significantly change the way of recent chemical or biological experiments. Microfabricated devices offer the ability to work with smaller sample volumes, shorter reaction time, and the possibility of parallel operation. They also hold the promise of integrating an entire onto a single chip, as lab-on-a-chip. In this presentation, I introduce a stable and reproducible preparation procedure for the planar lipid bilayers using “droplet contact method” in microfabicated devices. In addition, I will show the recent applications, such as single molecule detection with biological nanopores and DNA computing in the droplet network.

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels.

We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.