Insight into the Mechanisms Driving the Self-Assembly of Functional Interfaces: Moving from Lipids to Charged Amphiphilic Oligomers.

Polymer-stabilized liquid/liquid interfaces are an important and growing class of bioinspired materials that combine the structural and functional capabilities of advanced synthetic materials with naturally evolved biophysical systems. These platforms have the potential to serve as selective membranes for chemical separations and molecular sequencers and to even mimic neuromorphic computing elements. Despite the diversity in function, basic insight into the assembly of well-defined amphiphilic polymers to form functional structures remains elusive, which hinders the continued development of these technologies. In this work, we provide new mechanistic insight into the assembly of an amphiphilic polymer-stabilized oil/aqueous interface, in which the headgroups consist of positively charged methylimidazolium ionic liquids, and the tails are short, monodisperse oligodimethylsiloxanes covalently attached to the headgroups. We demonstrate using vibrational sum frequency generation spectroscopy and pendant drop tensiometery that the composition of the bulk aqueous phase, particularly the ionic strength, dictates the kinetics and structures of the amphiphiles in the organic phase as they decorate the interface. These results show that H-bonding and electrostatic interactions taking place in the aqueous phase bias the grafted oligomer conformations that are adopted in the neighboring oil phase. The kinetics of self-assembly were ionic strength dependent and found to be surprisingly slow, being composed of distinct regimes where molecules adsorb and reorient on relatively fast time scales, but where conformational sampling and frustrated packing takes place over longer time scales. These results set the stage for understanding related chemical phenomena of bioinspired materials in diverse technological and fundamental scientific fields and provide a solid physical foundation on which to design new functional interfaces.

Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes.

In-plane lipid organization and phase separation in natural membranes play key roles in regulating many cellular processes. Highly cooperative, first-order phase transitions in model membranes consisting of few lipid components are well understood and readily detectable via calorimetry, densitometry, and fluorescence. However, far less is known about natural membranes containing numerous lipid species and high concentrations of cholesterol, for which thermotropic transitions are undetectable by the above-mentioned techniques. We demonstrate that membrane capacitance is highly sensitive to low-enthalpy thermotropic transitions taking place in complex lipid membranes. Specifically, we measured the electrical capacitance as a function of temperature for droplet interface bilayer model membranes of increasing compositional complexity, namely, (a) a single lipid species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some ternary compositions, capacitance exhibited an abrupt, temperature-dependent change that coincided with the transition detected by other techniques. In addition, capacitance measurements revealed transitions in mixed-lipid membranes that were not detected by the other techniques. Most notably, capacitance measurements of bTLE bilayers indicated a transition at ∼38 °C not seen with any other method. Likewise, capacitance measurements detected transitions in some well-studied ternary mixtures that, while known to yield coexisting lipid phases, are not detected with calorimetry or densitometry. These results indicate that capacitance is exquisitely sensitive to low-enthalpy membrane transitions because of its sensitivity to changes in bilayer thickness that occur when lipids and excess solvent undergo subtle rearrangements near a phase transition. Our findings also suggest that heterogeneity confers stability to natural membranes that function near transition temperatures by preventing unwanted defects and macroscopic demixing associated with high-enthalpy transitions commonly found in simpler mixtures.

Heating-enabled formation of droplet interface bilayers using Escherichia coli total lipid extract.

Droplet interface bilayers (DIBs) serve as a convenient platform to study interactions between synthetic lipid membranes and proteins. However, a majority of DIBs have been assembled using a single lipid type, diphytanoylphosphatidylcholine (DPhPC). The work described herein establishes a new method to assemble DIBs using total lipid extract from Escherichia coli (eTLE); it is found that incubating oil-submerged aqueous droplets containing eTLE liposomes at a temperature above the gel-fluid phase transition temperature (Tg) promotes monolayer self-assembly that does not occur below Tg. Once monolayers are properly assembled via heating, droplets can be directly connected or cooled below Tg and then connected to initiate bilayer formation. This outcome contrasts immediate droplet coalescence observed upon contact between nonheated eTLE-infused droplets. Specific capacitance measurements confirm that the interface between droplets containing eTLE lipids is a lipid bilayer with thickness of 29.6 Å at 25 °C in hexadecane. We observe that bilayers formed from eTLE or DPhPC survive cooling and heating between 25 and 50 °C and demonstrate gigaohm (GΩ) membrane resistances at all temperatures tested. Additionally, we study the insertion of alamethicin peptides into both eTLE and DPhPC membranes to understand how lipid composition, temperature, and membrane phase influence ion channel formation. Like in DPhPC bilayers, alamethicin peptides in eTLE exhibit discrete, voltage-dependent gating characterized by multiple open channel conductance levels, though at significantly lower applied voltages. Cyclic voltammetry measurements of macroscopic channel currents confirm that the voltage-dependent conductance of alamethicin channels in eTLE bilayers occurs at lower voltages than in DPhPC bilayers at equivalent peptide concentrations. This result suggests that eTLE membranes, via composition, fluidity, or the presence of subdomains, offer an environment that enhances alamethicin insertion. For both membrane compositions, increasing temperature reduces the lifetimes of single channel gating events and increases the voltage required to cause an exponential increase in channel current. However, the fact that alamethicin insertion in eTLE exhibits significantly greater sensitivity to temperature changes through its Tg suggests that membrane phase plays an important role in channel formation. These effects are much less severe in DPhPC, where heating from 25 to 50 °C does not induce a phase change. The described technique for heating-assisted monolayer formation permits the use of other high transition temperature lipids in aqueous droplets for DIB formation, thereby increasing the types of lipids that can be considered for assembling model membranes.

Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer.

Thickness and tension are important physical parameters of model cell membranes. However, traditional methods to measure these quantities require multiple experiments using separate equipment. This work introduces a new multi-step procedure for directly accessing in situ multiple physical properties of droplet interface bilayers (DIB), including specific capacitance (related to thickness), lipid monolayer tension in the Plateau-Gibbs border, and bilayer tension. The procedure employs a combination of mechanical manipulation of bilayer area followed by electrowetting of the capacitive interface to examine the sensitivities of bilayer capacitance to area and contact angle to voltage, respectively. These data allow for determining the specific capacitance of the membrane and surface tension of the lipid monolayer, which are then used to compute bilayer thickness and tension, respectively. The use of DIBs affords accurate optical imaging of the connected droplets in addition to electrical measurements of bilayer capacitance, and it allows for reversibly varying bilayer area. After validating the accuracy of the technique with diphytanoyl phosphatidylcholine (DPhPC) DIBs in hexadecane, the method is applied herein to quantify separately the effects on membrane thickness and tension caused by varying the solvent in which the DIB is formed and introducing cholesterol into the bilayer. Because the technique relies only on capacitance measurements and optical images to determine both thickness and tension, this approach is specifically well-suited for studying the effects of peptides, biomolecules, natural and synthetic nanoparticles, and other species that accumulate within membranes without altering bilayer conductance.

Macromolecular Crowding Affects Voltage-Dependent Alamethicin Pore Formation in Lipid Bilayer Membranes.

Macromolecular crowding is known to modulate chemical equilibria, reaction rates, and molecular binding events, both in aqueous solutions and at lipid bilayer membranes, natural barriers that enclose the crowded environments of cells and their subcellular compartments. Previous studies on the effects of macromolecular crowding in aqueous compartments on conduction through membranes have focused on single-channel ionic conduction through previously formed pores at thermodynamic equilibrium. Here, the effects of macromolecular crowding on the mechanism of pore formation itself were studied using the droplet interface bilayer (DIB) technique with the voltage-dependent pore-forming peptide alamethicin (alm). Macromolecular crowding was varied using 8 kDa molecular weight polyethylene glycol (PEG8k) or 500 kDa dextran (DEX500k) in two aqueous droplets on both sides of the bilayer membrane. In general, voltage thresholds for pore formation in the presence of crowders in the droplets decreased compared to their values in the absence of crowders, due to excluded volume effects, water binding by PEG, and changes in the ordering of water molecules and hydrogen-bonding interactions involving the polar lipid headgroups. In addition, asymmetric crowder loading (e.g., PEG8k-DEX500k on either side of the membrane) resulted in transmembrane osmotic pressure gradients that either enhanced or degraded the ionic conduction through the pores.

Author Correction: Dynamical nonlinear memory capacitance in biomimetic membranes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dynamical nonlinear memory capacitance in biomimetic membranes.

Two-terminal memory elements, or memelements, capable of co-locating signal processing and memory via history-dependent reconfigurability at the nanoscale are vital for next-generation computing materials striving to match the brain's efficiency and flexible cognitive capabilities. While memory resistors, or memristors, have been widely reported, other types of memelements remain underexplored or undiscovered. Here we report the first example of a volatile, voltage-controlled memcapacitor in which capacitive memory arises from reversible and hysteretic geometrical changes in a lipid bilayer that mimics the composition and structure of biomembranes. We demonstrate that the nonlinear dynamics and memory are governed by two implicitly-coupled, voltage-dependent state variables-membrane radius and thickness. Further, our system is capable of tuneable signal processing and learning via synapse-like, short-term capacitive plasticity. These findings will accelerate the development of low-energy, biomolecular neuromorphic memelements, which, in turn, could also serve as models to study capacitive memory and signal processing in neuronal membranes.

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Evaporation-induced monolayer compression improves droplet interface bilayer formation using unsaturated lipids.

In this article, we report on a new experimental methodology to enable reliable formation of droplet interface bilayer (DIB) model membranes with two types of unsaturated lipids that have proven difficult for creating stable DIBs. Through the implementation of a simple evaporation technique to condition the spontaneously assembled lipid monolayer around each droplet, we increased the success rates of DIB formation for two distinct unsaturated lipids, namely 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), from less than 10% to near 100%. Separately, using a pendant drop tensiometer, we learned that: (a) DOPC and POPC monolayers do not spontaneously assemble into their tightest possible configurations at an oil-water interface, and (b) reducing the surface area of a water droplet coated with a partially packed monolayer leads to a more tightly packed monolayer with an interfacial tension lower than that achieved by spontaneous assembly alone. We also estimated from Langmuir compression isotherms obtained for both lipids that the brief droplet evaporation procedure prior to DIB formation resulted in a 6%-16% reduction in area per lipid for DOPC and POPC, respectively. Finally, the increased success rates of formation for DOPC and POPC DIBs enabled quantitative characterization of unsaturated lipid membrane properties including electrical resistance, rupture potential, and specific capacitance.

Memristive Ion Channel-Doped Biomembranes as Synaptic Mimics.

Solid-state neuromorphic systems based on transistors or memristors have yet to achieve the interconnectivity, performance, and energy efficiency of the brain due to excessive noise, undesirable material properties, and nonbiological switching mechanisms. Here we demonstrate that an alamethicin-doped, synthetic biomembrane exhibits memristive behavior, emulates key synaptic functions including paired-pulse facilitation and depression, and enables learning and computing. Unlike state-of-the-art devices, our two-terminal, biomolecular memristor features similar structure (biomembrane), switching mechanism (ion channels), and ionic transport modality as biological synapses while operating at considerably lower power. The reversible and volatile voltage-driven insertion of alamethicin peptides into an insulating lipid bilayer creates conductive pathways that exhibit pinched current-voltage hysteresis at potentials above their insertion threshold. Moreover, the synapse-like dynamic properties of the biomolecular memristor allow for simplified learning circuit implementations. Low-power memristive devices based on stimuli-responsive biomolecules represent a major advance toward implementation of full synaptic functionality in neuromorphic hardware.