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

2019 ◽  
Vol 19 (11) ◽  
pp. 7149-7155
Author(s):  
Shengwei Tan ◽  
Ling Zhang ◽  
Lijuan Yu ◽  
Lei Xu
Keyword(s):  
Silicon Nitride ◽  
Ion Channel ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Protein Function ◽  
Lipid Membranes ◽  
Membrane Resistance ◽  
Label Free ◽  
Free Standing ◽  
Membrane Lifetime

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.

scholarly journals Correlated diffusion in lipid bilayers

2021 ◽  
Vol 118 (48) ◽  
pp. e2113202118
Author(s):  
Rafael L. Schoch ◽  
Frank L. H. Brown ◽  
Gilad Haran
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Lipid Membranes ◽  
Supported Lipid Bilayers ◽  
Single Molecule Tracking ◽  
Black Lipid Membranes ◽  
Molecular Tracking ◽  
Physical Materials ◽  
Multiple Molecules ◽  
Major Target

Lipid membranes are complex quasi–two-dimensional fluids, whose importance in biology and unique physical/materials properties have made them a major target for biophysical research. Recent single-molecule tracking experiments in membranes have caused some controversy, calling the venerable Saffman–Delbrück model into question and suggesting that, perhaps, current understanding of membrane hydrodynamics is imperfect. However, single-molecule tracking is not well suited to resolving the details of hydrodynamic flows; observations involving correlations between multiple molecules are superior for this purpose. Here dual-color molecular tracking with submillisecond time resolution and submicron spatial resolution is employed to reveal correlations in the Brownian motion of pairs of fluorescently labeled lipids in membranes. These correlations extend hundreds of nanometers in freely floating bilayers (black lipid membranes) but are severely suppressed in supported lipid bilayers. The measurements are consistent with hydrodynamic predictions based on an extended Saffman–Delbrück theory that explicitly accounts for the two-leaflet bilayer structure of lipid membranes.

scholarly journals Real-Time Monitoring of Interactions between Solid-Supported Lipid Vesicle Layers and Short- and Medium-Chain Length Alcohols: Ethanol and 1-Pentanol

Biomimetics ◽  
2019 ◽  
Vol 4 (1) ◽  
pp. 8 ◽  
Author(s):  
Shova Neupane ◽  
George Cordoyiannis ◽  
Frank Uwe Renner ◽  
Patricia Losada-Pérez
Keyword(s):  
Real Time ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Model Systems ◽  
Lipid Vesicle ◽  
Label Free ◽  
General Anesthetic ◽  
Medium Chain Length ◽  
Time Interaction ◽  
High Concentrations

Lipid bilayers represent the interface between the cell and its environment, serving as model systems for the study of various biological processes. For instance, the addition of small molecules such as alcohols is a well-known process that modulates lipid bilayer properties, being considered as a reference for general anesthetic molecules. A plethora of experimental and simulation studies have focused on alcohol’s effect on lipid bilayers. Nevertheless, most studies have focused on lipid membranes formed in the presence of alcohols, while the effect of n-alcohols on preformed lipid membranes has received much less research interest. Here, we monitor the real-time interaction of short-chain alcohols with solid-supported vesicles of dipalmitoylphosphatidylcholine (DPPC) using quartz crystal microbalance with dissipation monitoring (QCM-D) as a label-free method. Results indicate that the addition of ethanol at different concentrations induces changes in the bilayer organization but preserves the stability of the supported vesicle layer. In turn, the addition of 1-pentanol induces not only changes in the bilayer organization, but also promotes vesicle rupture and inhomogeneous lipid layers at very high concentrations.

scholarly journals Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers

2011 ◽  
Vol 137 (2) ◽  
pp. 217-223 ◽  
Author(s):  
Sourabh Banerjee ◽  
Crina M. Nimigean
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Lipid Membranes ◽  
Single Channel ◽  
Black Lipid Membranes ◽  
Potassium Channel Kcsa ◽  
Lipid Environment ◽  
Biochemical Preparation

Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.

Measurement of membrane tension of free standing lipid bilayers via laser-induced surface deformation spectroscopy

Soft Matter ◽  
2015 ◽  
Vol 11 (44) ◽  
pp. 8641-8647 ◽  
Author(s):  
Tomohiko Takei ◽  
Tatsuya Yaguchi ◽  
Takuya Fujii ◽  
Tomonori Nomoto ◽  
Taro Toyota ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Surface Deformation ◽  
Membrane Tension ◽  
Free Standing ◽  
Black Lipid Membranes ◽  
Non Invasive ◽  
Invasive Measurement

Non-invasive measurement of the membrane tension of free-standing black lipid membranes (BLMs), with sensitivity on the order of μN m−1, was achieved using laser-induced surface deformation (LISD) spectroscopy.

scholarly journals De novo design of transmembrane β-barrels

2020 ◽  
Author(s):  
Anastassia A. Vorobieva ◽  
Paul White ◽  
Binyong Liang ◽  
Jim E Horne ◽  
Asim K. Bera ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
First Principles ◽  
Lipid Membranes ◽  
De Novo ◽  
Computational Design ◽  
Protein Sequencing ◽  
Naturally Occurring ◽  
Transmembrane Pores ◽  
Almost All

AbstractThe ability of naturally occurring transmembrane β-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a “hypothesis, design and test” approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the β-turns and β-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history.One sentence summarySuccess in de novo design of transmembrane β-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

Microfluidic Emulsion Generation and Trapping Enabling Droplet-Interfaced Bilayer Lipid Membrane Arrays

2009 ◽  
Author(s):  
C. Shao ◽  
D. L. DeVoe
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Protein Interactions ◽  
Lipid Membranes ◽  
High Throughput Screening ◽  
Lipid Membrane ◽  
Bilayer Lipid Membrane ◽  
Single Molecule Level ◽  
Bilayer Lipid ◽  
Ion Translocation

Freestanding bilayer lipid membranes provide an exceptional platform for measurements of lipid/protein interactions and ion translocation events at the single molecule level. For drug screening applications, large arrays of individual bilayer supports are required. However, an effective method for generating, stabilizing, and monitoring arrays of lipid bilayers remains elusive. Here we investigate a novel approach towards the facile generation of bilayer arrays for high throughput screening. The approach takes advantage of fundamental microfluidic capabilities by combining an emulsion generator with droplet-interfaced membrane formation, allowing for fully-automated production of membrane arrays whose density is, in principle, unlimited.

Interferometric Scattering Microscopy

2019 ◽  
Vol 70 (1) ◽  
pp. 301-322 ◽  
Author(s):  
Gavin Young ◽  
Philipp Kukura
Keyword(s):  
Cross Section ◽  
Single Molecule ◽  
Protein Function ◽  
Mass Measurement ◽  
Single Particle Tracking ◽  
Imaging Method ◽  
Label Free ◽  
Mechanistic Studies ◽  
Efficient Detection ◽  
Biomolecular Dynamics

Interferometric scattering microscopy (iSCAT) is an extremely sensitive imaging method based on the efficient detection of light scattered by nanoscopic objects. The ability to, at least in principle, maintain high imaging contrast independent of the exposure time or the scattering cross section of the object allows for unique applications in single-particle tracking, label-free imaging of nanoscopic (dis)assembly, and quantitative single-molecule characterization. We illustrate these capabilities in areas as diverse as mechanistic studies of motor protein function, viral capsid assembly, and single-molecule mass measurement in solution. We anticipate that iSCAT will become a widely used approach to unravel previously hidden details of biomolecular dynamics and interactions.

scholarly journals A large size-selective DNA nanopore with sensing applications

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Rasmus P. Thomsen ◽  
Mette Galsgaard Malle ◽  
Anders Hauge Okholm ◽  
Swati Krishnan ◽  
Søren S.-R. Bohr ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Detection System ◽  
Optical Methods ◽  
Label Free ◽  
Sensing Applications ◽  
Large Size ◽  
Transmission Electron ◽  
Label Free Detection ◽  
External Signals

AbstractTransmembrane nanostructures like ion channels and transporters perform key biological functions by controlling flow of molecules across lipid bilayers. Much work has gone into engineering artificial nanopores and applications in selective gating of molecules, label-free detection/sensing of biomolecules and DNA sequencing have shown promise. Here, we use DNA origami to create a synthetic 9 nm wide DNA nanopore, controlled by programmable, lipidated flaps and equipped with a size-selective gating system for the translocation of macromolecules. Successful assembly and insertion of the nanopore into lipid bilayers are validated by transmission electron microscopy (TEM), while selective translocation of cargo and the pore mechanosensitivity are studied using optical methods, including single-molecule, total internal reflection fluorescence (TIRF) microscopy. Size-specific cargo translocation and oligonucleotide-triggered opening of the pore are demonstrated showing that the DNA nanopore can function as a real-time detection system for external signals, offering potential for a variety of highly parallelized sensing applications.

scholarly journals Theoretical and computational modeling of the dynamics of multicellular and lipid membrane systems

2016 ◽  
Author(s):  
◽  
Matthew McCune
Keyword(s):  
Computational Modeling ◽  
Neutron Scattering ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Drug Testing ◽  
Lipid Membrane ◽  
Dynamic Properties ◽  
Practical Implication ◽  
Free Standing ◽  
Silica Substrate

This dissertation presents two research projects that apply theoretical and computational modeling to (1) describe and predict the formation and shape evolution of three-dimensional (3D) bioprinted tissue constructs, and (2) investigate the effect of a silica substrate on the structural and dynamic properties of a single fully hydrated lipid bilayer. (1) Bioprinting, a novel tissue engineering technique, has the ultimate goal of using 3D printers with bioink made from a person’s own cells to create tissues in the laboratory for transplantation or drug testing. The outcome of the post-bioprinting process, where the bioink particles fuse to form the desired 3D tissue construct, is difficult to predict and experimental techniques have generally been optimized through trial and error. To address this shortcoming, by employing theoretical modeling and computer simulations, we have developed and implemented an effective procedure that is capable of describing and predicting the shape dynamics during post-printing structure formation in 3D bioprinting. In particular, we have explained and demonstrated that the post-printing fusion process is considerably faster when using cylindrical instead of spheroidal bioink particles, a result that has considerable practical implication for extrusion bioprinting. (2) The study of lipid bilayers using neutron scattering experiments requires samples that contain a large stack of membranes. The analysis and computer simulation of such systems is challenging mainly due to the unknown amount of water separating the membranes. To overcome this difficulty, more recent experiments place single lipid membranes onto a support and stack about a hundred of them together. In this project we use molecular dynamics simulations of both free-standing and hydrated single-supported lipid bilayers to investigate the effect of the silica substrate on the structural and dynamical properties of the lipids and hydration waters. Our results may provide useful information in interpreting some recent neutron scattering experiments.

scholarly journals Nanopores: a versatile tool to study protein dynamics

2020 ◽  
Author(s):  
Sonja Schmid ◽  
Cees Dekker
Keyword(s):  
Protein Dynamics ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Label Free ◽  
Cellular Functions ◽  
Ensemble Averaging ◽  
Time Resolved ◽  
Wide Range ◽  
Versatile Tool

Abstract Proteins are the active workhorses in our body. These biomolecules perform all vital cellular functions from DNA replication and general biosynthesis to metabolic signaling and environmental sensing. While static 3D structures are now readily available, observing the functional cycle of proteins – involving conformational changes and interactions – remains very challenging, e.g., due to ensemble averaging. However, time-resolved information is crucial to gain a mechanistic understanding of protein function. Single-molecule techniques such as FRET and force spectroscopies provide answers but can be limited by the required labelling, a narrow time bandwidth, and more. Here, we describe electrical nanopore detection as a tool for probing protein dynamics. With a time bandwidth ranging from microseconds to hours, nanopore experiments cover an exceptionally wide range of timescales that is very relevant for protein function. First, we discuss the working principle of label-free nanopore experiments, various pore designs, instrumentation, and the characteristics of nanopore signals. In the second part, we review a few nanopore experiments that solved research questions in protein science, and we compare nanopores to other single-molecule techniques. We hope to make electrical nanopore sensing more accessible to the biochemical community, and to inspire new creative solutions to resolve a variety of protein dynamics – one molecule at a time.

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