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Bilayer vesicles that mimic a real biological cell can be tailored to carry out a specific function by manipulating the molecular composition of the amphiphiles. These bio-inspired and bio-mimetic structures are increasingly being employed for a number of applications from drug delivery to water purification and beyond. Complex hybrid bilayers are the key building blocks for fully synthetic vesicles that can mimic biological cell membranes, which often contain a wide variety of molecular species. While the assembly and morpholgy of pure phospholid bilayer vesicles is well understood, the functionality and structure dramaticlly changes when copolymer and/or carbon nanotube porins (CNTP) are added. The aim of this study is to understand how the collective molecular interactions within hybrid vesicles affect their nanoscale structure and properties. In situ small and wide angle X-ray scattering (SAXS/WAXS) and molecular dynamics simulations (MD) are used to investigate the morphological effect of molecular interactions between polybutadiene polyethylene oxide, lipids and carbon nanotubes (CNT) within the hybrid vesicle bilayer. Within the lipid/copolymer system, the hybrid bilayer morphology transitions from phase separated lipid and compressed copolymer at low copolymer loadings to a mixed bilayer where opposing lipids are mostly separated from the inner region. This transition begins between 60 wt% and 70 wt%, with full homogenization observed by 80 wt% copolymer. The incorporation of CNT into the hybrid vesicles increases the bilayer thickness and enhances the bilayer symmetry. Analysis of the WAXS and MD indicate that the CNT-dioleoyl interactions are much stronger than the CNT-polybutadiene.

Carbon nanotube porins (CNTPs) are a convenient membrane-based model system for studying nanofluidic transport that replicates a number of key structural features of biological membrane channels. We present a generalized approach for CNTP synthesis using sonochemistry-assisted segmenting of carbon nanotubes. Prolonged tip sonication in the presence of lipid molecules debundles and fragments long carbon nanotube aggregates into stable and water-soluble individual CNTPs with lengths in the range 5–20 nm. We discuss the main parameters that determine the efficiency and the yield of this process, describe the optimized conditions for high-yield CNTP synthesis, and demonstrate that this methodology can be adapted for synthesis of CNTPs of different diameters. We also present the optical properties of CNTPs and show that a combination of Raman and UV–vis–NIR spectroscopy can be used to monitor the quality of the CNTP synthesis. Overall, CNTPs represent a versatile nanopore building block for creating higher-ord...

Limited biocompatibility and fouling propensity can restrict real-world applications of a large variety of biosensors. Biological systems are adept at protecting and separating vital components of biological machinery with semipermeable membranes that often contain defined pores and gates to restrict transmembrane transport only to specific species. Here we use a similar approach for creating fouling-resistant pH sensors. We integrate silicon nanoribbon transistor sensors with an antifouling lipid bilayer coating that contains proton-permeable carbon nanotube porin (CNTP) channels and demonstrate robust pH detection in a variety of complex biological fluids.

Carbon nanotubes represent one of the most interesting examples of a nanofluidic channel that combines extremely small diameters with atomically smooth walls and well-defined chemical functionalities at the pore entrance. In the past, sub-1 nm diameter carbon nanotube porins (CNTPs) embedded in a lipid membrane matrix demonstrated extremely high water permeabilities and strong ion selectivities. In this work, we explore additional factors that can influence transport in these channels. Specifically, we use stopped-flow transport measurements to focus on the effect of chemical modifications of the CNT rims and chaotropic polyethyleneglycol (PEG) additives on CNTP water permeability and Arrhenius activation energy barriers for water transport. We show that PEG, especially in its more chaotropic coiled configuration, enhances the water transport and reduces the associated activation energy. Removal of the static charges on the CNTP rim by converting –COOH groups to neutral methylamide groups also reduces the activation energy barriers and enhances water transport rates.

Biological membranes provide a fascinating example of a separation system that is multifunctional, tunable, precise, and efficient. Biomimetic membranes, which mimic the architecture of cellular membranes, have the potential to deliver significant improvements in specificity and permeability. Here, a fully synthetic biomimetic membrane is reported that incorporates ultra-efficient 1.5 nm diameter carbon nanotube porin (CNTPs) channels in a block-copolymer matrix. It is demonstrated that CNTPs maintain high proton and water permeability in these membranes. CNTPs can also mimic the behavior of biological gap junctions by forming bridges between vesicular compartments that allow transport of small molecules.

Horner and Pohl argue that high water transport rates reported for carbon nanotube porins (CNTPs) originate from leakage at the nanotube-bilayer interface. Our results and new experimental evidence are consistent with transport through the nanotube pores and rule out a defect-mediated transport mechanism. Mechanistic origins of the high Arrhenius factor that we reported for narrow CNTPs at pH 8 require further investigation.

Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. In this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ∼30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.

In-plane mobility of proteins in lipid membranes is one of the fundamental mechanisms supporting biological functionality. Here we use high-speed atomic force microscopy (HS-AFM) to show that a novel type of biomimetic channel—carbon nanotube porins (CNTPs)—is also laterally mobile in supported lipid membranes, mimicking biological protein behaviour. HS-AFM can capture real-time dynamics of CNTP motion in the supported lipid bilayer membrane, build long-term trajectories of the CNTP motion and determine the diffusion coefficients associated with this motion. Our analysis shows that diffusion coefficients of CNTPs fall into the same range as those of proteins in supported lipid membranes. CNTPs in HS-AFM experiments often exhibit ‘directed’ diffusion behaviour, which is common for proteins in live cell membranes. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

Figures S1-S3

Carbon nanotube porins (CNTPs) are 10- to 20-nm-long segments of lipid-stabilized single-walled carbon nanotubes (CNTs) that can be inserted into phospholipid membranes to form nanometer-scale-diameter pores that approximate the geometry and many key transport characteristics of biological membrane channels. We describe protocols for CNTP synthesis by ultrasound-assisted cutting of long CNTs in the presence of lipid amphiphiles, and for validation of CNTP incorporation into a lipid membrane using a proton permeability assay. In addition, we describe protocols for measuring conductance of individual CNTPs in planar lipid bilayers and plasma membranes of live cells. The protocol for the preparation and testing of the CNTPs in vesicle systems takes 3 d, and single CNTP conductance measurements take 2-5 h. The CNTPs produced by this cutting protocol remain stable and active for at least 10-12 weeks.

Carbon nanotube porins (CNTPs), small segments of carbon nanotubes capable of forming defined pores in lipid membranes, are important future components for bionanoelectronic devices as they could provide a robust analog of biological membrane channels. In order to control the incorporation of these CNT channels into lipid bilayers, it is important to understand the structure of the CNTPs before and after insertion into the lipid bilayer as well as the impact of such insertion on the bilayer structure. Here we employed a noninvasive in situ probe, small-angle X-ray scattering, to study the integration of CNT porins into dioleoylphosphatidylcholine bilayers. Our results show that CNTPs in solution are stabilized by a monolayer of lipid molecules wrapped around their outer surface. We also demonstrate that insertion of CNTPs into the lipid bilayer results in decreased bilayer thickness with the magnitude of this effect increasing with the concentration of CNTPs.

Synthetic diblock and triblock copolymer membranes are semi-permeable barriers that are capable of self-assembly into tunable membranes, which have contrasting mechanical properties from biologically derived and synthetic lipid bilayers. These thick and “robust” polymer membranes are typically thicker and have significantly reduced water permeability compared to lipid bilayers. These properties make them valuable alternative membranes for applications requiring exposure to physical stresses such as biosensors, drug delivery, and filtration. Previous attempts to engineer the permeability of these membranes included insertion of protein channels to improve permeably, (eg. Aquaporins), changing the polymer constituent to an inherently leaky polymer (eg. polystyrene-b-polyisocyanoalanine(2-thiophene-3-yl-ethyl)amide (PS-PIAT)), and removal of lipids from a mixed lipid-polymer vesicle following cross-linking and hydrolysis. We report a new strategy for improving permeability of polymer-membranes, based on incorporation of carbon nanotube porins into the polymer matrix. These short CNT pores exhibit the excellent transport properties inherent to carbon nanotubes, but are small enough that they can be easily incorporated into the membrane essentially acting as protein channel mimics. Here, we examine the permeability of diblock co-polymer membranes with and without these CNT pores and report the enhancement of permeability and ion transport characteristics of these novel membrane constructs.

Carbon nanotube porins (CNTPs), ultra-short carbon nanotubes (5-20nm) that can self-insert into lipid bilayers, allow single-channel planar lipid bilayer measurements of ionic transport through carbon nanotube (CNT) pores. CNTs with sub-nanometer diameters have been predicted to display ultra-fast water transport and high levels of ionic exclusion resulting from one-dimensional water wire transport through their hydrophobic cores. Here, we show that ultra-short 0.8-nm-diameter CNTPs, can spontaneously insert into lipid bilayers to form highly cation selective channels that exhibit low ionic conductance. Using reversal potential in various asymmetric salt conditions, we observe that these CNTPs exhibit large permselectivity values, corresponding to a high selectivity for both potassium and sodium over chloride. We also show evidence that neutralization of charges on the CNTs enhances ion selectivity. We also show that by using a solid-state nanopore to act as a support and form a completely solvent-free bilayer system, we can observe extremely stable ionic currents through these nanotubes, in contrast to previous reports with solvent containing bilayers. These results establish CNTPs as a promising biomimetic platform for developing cell interfaces, creating stochastic sensors, and water desalination.

Carbon nanotubes (CNTs) have narrow hydrophobic inner pores, highly reminiscent of protein channels, that enable fast transport of water and ions. Their rigid structure and chemical robustness allow for diverse ex vivo applications. We have recently reported biomimetic membrane channels cased on carbon nanotubes—carbon nanotube porins (CNTPs). We have shown that CNTPs self-insert into lipid membranes and form stable pores. In this study, we used electrophysiological single-channel measurements to quantify ion conductance and selectivity of 0.8 nm diameter CNTPs. We report the conductance of single CNTP channels, as well as the scaling of the conductance with the ion concentration. We also used the reversal potential measurements to characterize the selectivity of the ion transport and found that CNTPs are highly selective to cations over anions. We will discuss the mechanism of this selectivity, and demonstrate ways to control it. These findings are important and relevant for developing CNTPs into a potential candidate nanopore for water treatment applications. Our studies on intrinsic characterization of CNTP transport properties will also enable researchers to consider this new material for a variety of biophysical applications.

Light-activated bioelectronic silicon nanowire transistor devices are made by fusing proteoliposomes containing a bacteriorhodopsin (bR) proton pump onto the nanowire surface. Under green-light illumination, bR pumps protons toward the nanowire, and the pH gradient developed by the pump changes the transistor output. Furthermore, co-assembly of small biomolecules that alter the bilayer permeability to other ions can upregulate and downregulate the response of field-effect transistor devices.

Bacteriorhodopsin (bR) is a well-characterized photoactivated proton pump capable of creating a proton gradient using the energy of absorbed light. We are developing a bioelectronic platform that couples bacteriorhodopsin activity with the electronic response of a nanowire field-effect transistor. To accomplish this task we use hierarchical assembly of lipids and membrane proteins into a 1-D lipid bilayer structure covering the channel region of the transistor device. This presentation discusses the device preparation, characterization, and overall performance. In addition, we present results showing how other ion channels and ionophores, co-assembled with the protein embedded bilayer, can be used to change various performance characteristics of the device, independent of one-another. The ability to use components of the biological "toolkit" to fine-tune bioelectronic devices could have important implications for design of next generation biointerfaces.

Short carbon nanotubes spontaneously insert into lipid bilayers and live cell membranes to form channels with useful and tunable transport properties that make them a promising biomimetic nanopore platform for developing cell interfaces, studying nanofluidic transport in biological channels, and creating stochastic sensors. Synthetic analogues of biological membrane channels that match the latter's high efficiency and exquisite selectivity for transporting ions and molecules could find many applications. Although it is possible to produce nanopores of a size comparable to that of protein channels, replicating their affinity and transport properties remains challenging. Jia Geng et al. now show that short (10-nm-long) single-wall carbon nanotubes spontaneously insert into lipid bilayers and live cell membranes to form channels with useful and tuneable transport properties. These carbon-nanotube channel-forming molecules or porins offer a promising biomimetic nanopore platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors. There is much interest in developing synthetic analogues of biological membrane channels1 with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up2 and top-down3 methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties4,5,6,7,8 and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels1. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane9,10. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls11,12, and short tubes have been forced into membranes to create sensors13, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70–100 picosiemens under physiological conditions. Despite their structural simplicity, these ‘CNT porins’ transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

Acidity of the local microenvironments is an important parameter for detection of pathological conditions and understanding of cellular mechanisms. Silicon nanoribbon field effect transistor (FET) has been proved to be a high-throughput and sensitive platform for pH sensing. However, the efficiency of these sensors is limited by the biocompatibility of silicon material and its tendency for bio-fouling. Recent studies have demonstrated sub-2nm diameter carbon nanotube (CNT) porins embedded in lipid bilayers are fast and efficient proton conductors. In this research, we focus on a bioelectronic approach toward designing pH-sensing devices. We will describe an organic/inorganic hybrid device that combines nanoribbon FET with self-assembled CNT/lipid bilayer for pH sensing in physiological conditions. Finally, we will also discuss its potential application in nanometer spatial resolution intracellular biosensing.

Author(s): Tunuguntla, Ramya H; Bangar, Mangesh A; Kim, Kyunghoon; Stroeve, Pieter; Grigoropoulos, Costas; Ajo-Franklin, Caroline M; Noy, Aleksandr

Ion and water transport through biological membranes by osmosis is essential in many biological processes and the complex nature of biological pores and channels involved in these processes is not fully understood. We are developing simplified robust models of ion channels based on smooth, narrow and hydrophobic pores of carbon nanotubes. Incorporation of these pores into liposomes gives us a convenient system to study osmotically-induced transport using dynamic light scattering. We will present our measurements of the transport of charged and uncharged chemical species through the carbon nanotube pores and also discuss the role of the charges at the nanotube pore ends in regulating the ion transport selectivity.

Cells differ in their individual permeabilities depending on what lipids and proteins are present in the membrane and by regulating the number of channel proteins present in their system. The maintenance and regulation of ion gradients, specifically the electrochemical proton gradient, ΔμH+, across biological membranes has been established as a necessary intermediate in biological energy transduction that is crucial for proper cellular function. In recent years, artificial membrane channels have attracted much attention due to the key role of proton channels in performing a number of specific functions in different cells. Carbon nanotube (CNT) structures are similar to biological channels (e.g. aquaporins) with their smooth, narrow (ca. 1.5 nm), hydrophobic inner pores. The hydrophobic walls of the CNT facilitate the formation of 1D hydrogen bonded water chains and results in weak interactions with water molecules that enable nearly frictionless water transport. CNTs can provide a functional mimic of biological channels, and to that end we have created shortened CNT porins and investigated osmotically-driven transport of protons, ions and uncharged species through these pores. This presentation will discuss measurements of transport efficiency and transport selectivity in these biomimetic membrane channels and compare them with properties of biological ion channels.

Living systems control transport of ions or small molecules across biological membranes using ion channels that form highly efficient and selective pores in lipid bilayers. Although bottom-up synthesis and top-down fabrication could produce pores of comparable size, an unresolved challenge remains to build nanopore scaffolds that fully replicate transport properties of membrane channels. We will show that pores in lipid membranes formed by ultra-short carbon nanotubes (CNTs) have transport properties that come remarkably close to that goal. These carbon nanotube channels can transport water, protons, small ions, and DNA and their ion-rejection properties can be controlled by the charge at the pore mouth. Interestingly, these pores also display the stochastic "gating" behavior common for biological ion channels. We attribute this effect to a spontaneous reversible ionic "penetration-exclusion" transition, suggesting that it represents a general feature of nanofluidic transport in sub-2-nm pores. Overall, transmembrane CNT ion channels represent a robust and versatile biomimetic scaffold for studying fundamentals of transport in biological channels, artificial cell design, and stochastic sensing.