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While high-throughput planar patch-clamp instruments are now established to perform whole-cell recordings for drug screening, the conventional micropipette-based approach remains the gold standard for performing cell-attached single-channel recordings. Generally, planar platforms are not well-suited for such studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms tend to use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, reducing the seal resistance. Here, femtosecond laser ablation was used to fabricate dual-pore glass chips optimized for use in cell-attached single-channel recordings that circumvent this problem by using different pores to position a cell and to establish a seal. This dual-pore design also permitted the use of a relatively small patch aperture (D ~ 150 to 300 nm) that is better-suited for establishing high-resistance seals than the micropores used typically in planar patch-clamp setups (D ~ 1 to 2 μm) without compromising the ability of the device to position a cell. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (rate of gigaseal formation = 61%, mean seal resistance = 53 GΩ), maintained gigaseals for prolonged durations (up to 6 hours), achieved RMS noise values as low as 0.46 pA at 5 kHz bandwidth, and enabled single-channel recordings in the cell-attached configuration that are comparable to those obtained by conventional patch-clamp.

Characterization of resistive pulses from the translocation of particles through lipid-coated nanopores has previously been used to determine the size, charge, and ligand affinity of proteins. Here, we coat the nanopores with a lipid bilayer to prevent non-specific adsorption, concentrate a protein of interest on the bilayer surface, and slow the translocation times of proteins through the pore so that events may be effectively time-resolved and detected. This study aims to characterize cholera toxin and the binding kinetics of the multivalent interaction with GM1 gangliosides embedded in the lipid coating. Understanding the parameters of the GM1 and cholera toxin interaction on a single molecule basis may provide additional information about the pathophysiology of vibrio cholerae, which is known to attack GM1 dense cells in the intestinal epithelia. By monitoring the frequency of translocation events of the protein-ligand complex as a function of time, it is possible to measure the binding kinetics of the interaction between cholera toxin and the GM1 lipid anchors. Thus far, we have determined the shape, volume, and charge of the membrane-associated protein in addition to the effective on-rate of binding under varying concentrations of the GM1 ligand.

Proteins represent the most diverse class of biomolecules in both structure and function and are involved in nearly every physiological process; their quantification, identification, and biophysical characterization is therefore of fundamental and practical importance. This work used electrolyte-filled nanopores that were coated with a lipid bilayer to characterize single, native proteins that were anchored to mobile ligands in the coating. This technique has previously been used to determine a protein's volume, charge, and ligand affinity by measuring the change in ionic current, ΔI, through the nanopore as proteins travel from one side to the other. Here, we took advantage of the dependence of ΔI on the orientation of a non-spherical particle in the nanopore to determine the shape and volume of eight different non-spherical proteins from the resulting distributions of ΔI values. Additionally, we present quantitative methods for determining the dipole moment and rotational diffusion coefficient of non-spherical proteins from single events and compare results from established techniques as well as simulations based on theory.

Despite the importance of proteins, nanopore sensing has so far been mostly focused on single molecule DNA and RNA characterization. One factor that limited experiments with proteins were nonspecific interactions of proteins with the walls of synthetic nanopores. We showed recently, that nanopores with fluid coatings of phospholipid bilayers circumvented this problem. In addition, anchoring proteins to lipid anchors slowed down protein translocation through nanopores and enabled determination of parameters such as the shape, volume, and dipole moment of individual non-spherical proteins. To slow down the translocation time of lipid-anchored proteins further, we have recently formed highly viscous coatings from archaea-inspired lipids. These synthetic lipids are composed of two hydrophilic head groups connected by a long hydrophobic chain; each head group is also attached to an acyl chain that spans half of the membrane thickness. As these lipids contain -in one molecule- the components of a typical lipid bilayer, a single layer is sufficient to form a stable membrane. Experimentation with many lipid coatings, including a variety of archaea-inspired lipids, has revealed differences in translocation of protein, bilayer stability and event frequency. Every bilayer composition tested has been designed to optimize each of these characteristics.

Despite the importance of proteins, nanopore sensing has so far been mostly focused on single molecule DNA and RNA characterization. One factor that limited experiments with proteins were nonspecific interactions of proteins with the walls of synthetic nanopores. We showed recently, that nanopores with fluid coatings of phospholipid bilayers circumvented this problem. In addition, anchoring proteins to lipid anchors slowed down protein translocation through nanopores and enabled determination of parameters such as the shape, volume, and dipole moment of individual non-spherical proteins. To slow down the translocation time of lipid-anchored proteins further, we have recently formed highly viscous coatings from archaea-inspired lipids. These synthetic lipids are composed of two hydrophilic head groups connected by a long hydrophobic chain; each head group is also attached to an acyl chain that spans half of the membrane thickness. As these lipids contain -in one molecule- the components of a typical lipid bilayer, a single layer is sufficient to form a stable membrane. Monolayers of these new lipids have a two to twenty-fold increased viscosity than typical phospholipid bilayers. The Archaea-inspired lipids discussed here show promise in terms of slowing translocation times and the resulting data made it possible to characterize individual proteins with increased accuracy than coatings from standard phospholipid bilayers.

The capability of plant cells to establish osmotic gradients allows them to actuate structures such as guard cells and the pulvini responsible for the swift nastic motion of mimosa pudica. The work presented here investigated the dynamics of osmotically-driven pressure generation in membrane-bound compartments, taking into account volume expansion, solute dilution, surface area to volume ratio, membrane hydraulic permeability, and changes in osmotic gradient and bulk modulus. We developed and experimentally validated a numerical simulation tool for characterizing and predicting the dynamics of pressure generators as a function of parameters that will be encountered in most systems that generate pressure osmotically. Finally, we demonstrate two applications of an osmosis-based pressure generator: actuation of a soft robot and continuous volume delivery over long periods of time.

Despite the widespread use of high-throughput planar patch-clamp instruments, the conventional pipette-based technique remains the method of choice for recording single-channel activity. Generally, planar platforms are not well suited for single-channel studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms typically use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, thereby reducing the seal resistance. Here, femtosecond laser ablation is used to fabricate dual-pore glass chips for use in low-noise, single-channel recordings that circumvent this problem. One pore positions a cell by suction while another nearby pore, the recording pore, avoids contamination by maintaining positive pressure until a cell is positioned and then establishes a seal. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (>10 GΩ), maintained gigaseals for prolonged durations (up to 6 hrs), and enabled single-channel recordings in cell-attached mode that are comparable to those obtained by conventional patch-clamp.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Despite the commercial success of automated high-throughput patch clamp technologies, instruments on the market typically cannot match the quality of single-channel recordings obtained by traditional patch clamp experiments with glass micropipettes. In automated approaches that use suction to position a cell to a micropore, the inner surface of the pore can be contaminated with proteins in solution prior to seal formation. Traditional patch clamping avoids this problem by applying a positive pressure to the micropipette prior to contacting the cell membrane, which enables significantly higher electrical seal resistances. This research aims to develop a universal platform for high-quality transport assays on glass chips that circumvents the problem of surface contamination and takes advantage of the high seal resistances and low capacitive current noise realized with glass substrates. Femtosecond laser ablation is used to fabricate a pair of micropores in close proximity to each other (distance < 5 µm) in glass, while the access channels to these pores are kept separate. This configuration makes it possible to use one pore for automated positioning of a cell by suction, while the other pore is kept clean by applying positive pressure. Once the cell is pre-positioned onto the first pore, suction may be applied to the second pore to establish a high quality seal that could be used to interrogate individual transmembrane transporter proteins and single ion channel proteins. In addition, this dual micropore configuration may enable the first automated gap junction recordings, bypassing the difficulties of manually positioning two micropipettes.

Recording ionic current through electrolyte-filled nanopores during the passage of proteins is an emerging technique for characterizing unmodified proteins in their native, aqueous environment. By measuring the reduction in current, ΔI, during the translocation of single proteins through an electrolyte-filled nanopore, this technique can characterize the size, charge, conformation, assembly and activity of hundreds of unlabeled proteins per second. For non-spherical proteins, however, broad distributions of ΔI values make estimates of protein size unreliable. This work employs lipid-bilayer coated nanopores and describes quantitative procedures for determining the shape and volume of single spherical and non-spherical proteins from distributions of ΔI values. Since the ΔI(t) signal is related to the orientation of non-spherical proteins in the nanopore, individual resistive-pulses can be used to determine the rotational diffusion coefficient and dipole moment of non-spherical proteins while in the nanopore. Moreover, this method has the potential to detect transient changes in the conformation of flexible proteins (e.g. an IgG antibody). This work extends the power of nanopores for characterizing proteins by adding the parameters of shape, volume, rotational diffusion coefficient, and dipole moment of non-spherical proteins to those that can already be determined in a single experiment such as the volume of spherical proteins, charge, and affinity for a ligand.