skip to main content


The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Friday, September 29 until 11:59 PM ET on Saturday, September 30 due to maintenance. We apologize for the inconvenience.

Title: Rapid and Real-Time Measurement of Membrane Potential Through Intramembrane Field Compensation

Synthetic lipid membranes are self-assembled biomolecular double layers designed to approximate the properties of living cell membranes. These membranes are employed as model systems for studying the interactions of cellular envelopes with the surrounding environment in a controlled platform. They are constructed by dispersing amphiphilic lipids into a combination of immiscible fluids enabling the biomolecules to self-assemble into ordered sheets, or monolayers at the oil-water interface. The adhesion of two opposing monolayer sheets forms the membrane, or the double layer. The mechanical properties of these synthetic membranes often differ from biological ones mainly due to the presence of residual solvent in between the leaflets. In fact, the double layer compresses in response to externally applied electrical field with an intensity that varies depending on the solvent present. While typically viewed as a drawback associated with their assembly, in this work the elasticity of the double layer is utilized to further quantify complex biophysical phenomena. The adsorption of charged molecules on the surface of a lipid bilayer is a key property to decipher biomolecule interactions at the interface of the cell membrane, as well as to develop effective antimicrobial peptides and similar membrane-active molecules. This adsorption generates a difference in the boundary potentials on either side of the membrane which may be tracked through electrophysiology. The soft synthetic membranes produced in the laboratory compress when exposed to an electric field. Tracking the minimum membrane capacitance allows for quantifying when the intrinsic electric field produced by the asymmetry is properly compensated by the supplied transmembrane voltage. The technique adopted in this work is the intramembrane field compensation (IFC). This technique focuses on the current generated by the bilayer in response to a sinusoidal voltage with a DC component, VDC. Briefly, the output sinusoidal current is divided into its harmonics and the second harmonic equals zero when VDC compensates the internal electric field. In this work, we apply the IFC technique to droplet interface bilayers (DIB) enabling the development of a biological sensor. A certain membrane elasticity is needed for accurate measurements and is tuned through the solvent selection. The asymmetric DIBs are formed, and an automated PID-controlled IFC design is implemented to rapidly track and compensate the membrane asymmetry. The closed loop system continuously reads the current and generates the corresponding voltage until the second harmonic is abated. This research describes the development and optimization of a biological sensor and examines how varying the structure of the synthetic membrane influences its capabilities for detecting membrane-environment interactions. This platform may be applied towards studying the interactions of membrane-active molecules and developing models for the associated phenomena to enhance their design.

more » « less
Award ID(s):
Author(s) / Creator(s):
Date Published:
Journal Name:
Smart Materials, Adaptive Structures, and Intelligent Systems 2020
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. We describe a method to determine membrane bending rigidity from capacitance measurements on large area, free-standing, planar, biomembranes. The bending rigidity of lipid membranes is an important biological mechanical property that is commonly optically measured in vesicles, but difficult to quantify in a planar, unsupported system. To accomplish this, we simultaneously image and apply an electric potential to free-standing, millimeter area, planar lipid bilayers composed of DOPC and DOPG phospholipids to measure the membrane Young’s (elasticity) modulus. The bilayer is then modeled as two adjacent thin elastic films to calculate bending rigidity from the electromechanical response of the membrane to the applied field. Using DOPC, we show that bending rigidities determined by this approach are in good agreement with the existing work using neutron spin echo on vesicles, atomic force spectroscopy on supported lipid bilayers, and micropipette aspiration of giant unilamellar vesicles. We study the effect of asymmetric calcium concentration on symmetric DOPC and DOPG membranes and quantify the resulting changes in bending rigidity. This platform offers the ability to create planar bilayers of controlled lipid composition and aqueous ionic environment, with the ability to asymmetrically alter both. We aim to leverage this high degree of compositional and environmental control, along with the capacity to measure physical properties, in the study of various biological processes in the future. 
    more » « less
  2. Abstract

    The nonlinear optical phenomenon second harmonic light scattering (SHS) can be used for detecting molecules at the membrane surfaces of living biological cells. Over the last decade, SHS has been developed for quantitatively monitoring the adsorption and transport of small and medium size molecules (both neutral and ionic) across membranes in living cells. SHS can be operated with both time and spatial resolution and is even capable of isolating molecule‐membrane interactions at specific membrane surfaces in multi‐membrane cells, such as bacteria. In this review, we discuss select examples from our lab employing time‐resolved SHS to study real‐time molecular interactions at the plasma membranes of biological cells. We first demonstrate the utility of this method for determining the transport rates at each membrane/interface in a Gram‐negative bacterial cell. Next, we show how SHS can be used to characterize the molecular mechanism of the century old Gram stain protocol for classifying bacteria. Additionally, we examine how membrane structures and molecular charge and polarity affect adsorption and transport, as well as how antimicrobial compounds alter bacteria membrane permeability. Finally, we discuss adaptation of SHS as an imaging modality to quantify molecular adsorption and transport in sub‐cellular regions of individual living cells.

    more » « less
  3. null (Ed.)
    A mechanistic understanding of the influence of the surface properties of engineered nanomaterials on their interactions with cells is essential for designing materials for applications such as bioimaging and drug delivery as well as for assessing nanomaterial safety. Ligand-coated gold nanoparticles have been widely investigated because their highly tunable surface properties enable investigations into the effect of ligand functionalization on interactions with biological systems. Lipophilic ligands have been linked to adverse biological outcomes through membrane disruption, but the relationship between ligand lipophilicity and membrane interactions is not well understood. Here, we use a library of cationic ligands coated on 2 nm gold nanoparticles to probe the impact of ligand end group lipophilicity on interactions with supported phosphatidylcholine lipid bilayers as a model for cytoplasmic membranes. Nanoparticle adsorption to and desorption from the model membranes were investigated by quartz crystal microbalance with dissipation monitoring. We find that nanoparticle adsorption to model membranes increases with ligand lipophilicity. The effects of ligand structure on gold nanoparticle attachment were further analyzed using atomistic molecular dynamics simulations, which showed that the increase in ligand lipophilicity promotes ligand intercalation into the lipid bilayer. Together, the experimental and simulation results could be described by a two-state model that accounts for the initial attachment and subsequent conversion to a quasi-irreversibly bound state. We find that only nanoparticles coated with the most lipophilic ligands in our nanoparticle library undergo conversion to the quasi-irreversible state. We propose that the initial attachment is governed by interaction between the ligands and phospholipid tail groups, whereas conversion into the quasi-irreversibly bound state reflects ligand intercalation between phospholipid tail groups and eventual lipid extraction from the bilayer. The systematic variation of ligand lipophilicity enabled us to demonstrate that the lipophilicity of cationic ligands correlates with nanoparticle-bilayer adsorption and suggested that changing the nonpolar ligand R group promotes a mechanism of ligand intercalation into the bilayer associated with irreversible adsorption. 
    more » « less
  4. Biological supramolecular assemblies, such as phospholipid bilayer membranes, have been used to demonstrate signal processing via short-term synaptic plasticity (STP) in the form of paired pulse facilitation and depression, emulating the brain’s efficiency and flexible cognitive capabilities. However, STP memory in lipid bilayers is volatile and cannot be stored or accessed over relevant periods of time, a key requirement for learning. Using droplet interface bilayers (DIBs) composed of lipids, water and hexadecane, and an electrical stimulation training protocol featuring repetitive sinusoidal voltage cycling, we show that DIBs displaying memcapacitive properties can also exhibit persistent synaptic plasticity in the form of long-term potentiation (LTP) associated with capacitive energy storage in the phospholipid bilayer. The time scales for the physical changes associated with the LTP range between minutes and hours, and are substantially longer than previous STP studies, where stored energy dissipated after only a few seconds. STP behavior is the result of reversible changes in bilayer area and thickness. On the other hand, LTP is the result of additional molecular and structural changes to the zwitterionic lipid headgroups and the dielectric properties of the lipid bilayer that result from the buildup of an increasingly asymmetric charge distribution at the bilayer interfaces. 
    more » « less
  5. Supported lipid bilayers are often used as model systems for studying interactions of biological membranes with protein or nanoparticles. A supported lipid bilayer is a phospholipid bilayer built on a solid substrate. The latter is typically made of silica or a metal oxide due to the ease of its formation and range of compatible measurement techniques. Recently, a solvent-assisted method involving supported lipid bilayer formation has allowed the extension of compatible substrate materials to include noble metals such as gold. Here, we examine the influence of substrate composition (SiO2 vs Au) on the interactions between anionic ligand-coated Au nanoparticles or cytochrome c and zwitterionic supported lipid bilayers using quartz crystal microbalance with dissipation monitoring. We find that anionic nanoparticles and cytochrome c have higher adsorption to bilayers formed on Au relative to those on SiO2 substrates. We examine the substrate-dependence of nanoparticle adsorption with DLVO theory and all-atom simulations, and find that the stronger attractive van der Waals and weaker repulsive electrostatic forces between anionic nanoparticles and Au substrates vs anionic nanoparticles and SiO2 substrates could be responsible for the change in adsorption observed. Our results also indicate that the underlying substrate material influences the degree to which nanoscale analytes interact with supported lipid bilayers; therefore, interpretation of the supported lipid bilayer model system should be conducted with understanding of support properties. 
    more » « less