Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs' weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

Molecular transport enhancement in pure metallic carbon nanotube porins.

Nanofluidic channels impose extreme confinement on water and ions, giving rise to unusual transport phenomena strongly dependent on the interactions at the channel-wall interface. Yet how the electronic properties of the nanofluidic channels influence transport efficiency remains largely unexplored. Here we measure transport through the inner pores of sub-1 nm metallic and semiconducting carbon nanotube porins. We find that water and proton transport are enhanced in metallic nanotubes over semiconducting nanotubes, whereas ion transport is largely insensitive to the nanotube bandgap value. Molecular simulations using polarizable force fields highlight the contributions of the anisotropic polarizability tensor of the carbon nanotubes to the ion-nanotube interactions and the water friction coefficient. We also describe the origin of the proton transport enhancement in metallic nanotubes using deep neural network molecular dynamics simulations. These results emphasize the complex role of the electronic properties of nanofluidic channels in modulating transport under extreme nanoscale confinement.

Sonochemical Synthesis and Ion Transport Properties of Surfactant-Stabilized Carbon Nanotube Porins.

Carbon nanotube porins (CNTPs), short segments of carbon nanotubes stabilized by a lipid coating, are a promising example of artificial membrane channels that mimic a number of key behaviors of biological ion channels. While the lipid-assisted synthesis of CNTPs may facilitate their subsequent incorporation into lipid bilayers, it limits the applicability of these pores in other self-assembled membrane materials and also precludes the use of large-scale purified CNT feedstocks. Here we demonstrate that CNTPs can be synthesized by sonochemical cutting of long CNT feedstocks in the presence of different surfactants, producing CNTS with transport properties identical with those obtained by the lipid-assisted procedure. Our results open up a wide variety of synthetic routes for CNTP production.

Fluids and Electrolytes under Confinement in Single-Digit Nanopores.

Confined fluids and electrolyte solutions in nanopores exhibit rich and surprising physics and chemistry that impact the mass transport and energy efficiency in many important natural systems and industrial applications. Existing theories often fail to predict the exotic effects observed in the narrowest of such pores, called single-digit nanopores (SDNs), which have diameters or conduit widths of less than 10 nm, and have only recently become accessible for experimental measurements. What SDNs reveal has been surprising, including a rapidly increasing number of examples such as extraordinarily fast water transport, distorted fluid-phase boundaries, strong ion-correlation and quantum effects, and dielectric anomalies that are not observed in larger pores. Exploiting these effects presents myriad opportunities in both basic and applied research that stand to impact a host of new technologies at the water-energy nexus, from new membranes for precise separations and water purification to new gas permeable materials for water electrolyzers and energy-storage devices. SDNs also present unique opportunities to achieve ultrasensitive and selective chemical sensing at the single-ion and single-molecule limit. In this review article, we summarize the progress on nanofluidics of SDNs, with a focus on the confinement effects that arise in these extremely narrow nanopores. The recent development of precision model systems, transformative experimental tools, and multiscale theories that have played enabling roles in advancing this frontier are reviewed. We also identify new knowledge gaps in our understanding of nanofluidic transport and provide an outlook for the future challenges and opportunities at this rapidly advancing frontier.

Co-Assembly of Carbon Nanotube Porins into Biomimetic Peptoid Membranes.

Robust and cost-effective membrane-based separations are essential to solving many global crises, such as the lack of clean water. Even though the current polymer-based membranes are widely used for separations, their performance and precision can be enhanced by using a biomimetic membrane architecture that consists of highly permeable and selective channels embedded in a universal membrane matrix. Researchers have shown that artificial water and ion channels, such as carbon nanotube porins (CNTPs), embedded in lipid membranes can deliver strong separation performance. However, their applications are limited by the relative fragility and low stability of the lipid matrix. In this work, we demonstrate that CNTPs can co-assemble into two dimension (2D) peptoid membrane nanosheets, opening up a way to produce highly programmable synthetic membranes with superior crystallinity and robustness. A combination of molecular dynamics (MD) simulations, Raman spectroscopy, X-ray diffraction (XRD), and atomic force microscopy (AFM) measurements to verify the co-assembly of CNTP and peptoids are used and show that it does not disrupt peptoid monomer packing within the membrane. These results provide a new option for designing affordable artificial membranes and highly robust nanoporous solids.

Nanofluidic computing makes a splash.

Ionic computing raises the possibility of devices that operate similarly to the human brain.

Breakdown of the Nernst-Einstein relation in carbon nanotube porins.

For over 100 years, the Nernst-Einstein relation has linked a charged particle's electrophoretic mobility and diffusion coefficient. Here we report experimental measurements of diffusion and electromigration of K+ ions in narrow 0.8-nm-diameter single-walled carbon nanotube porins (CNTPs) and demonstrate that the Nernst-Einstein relation in these channels breaks down by more than three orders of magnitude. Molecular dynamics simulations using polarizable force fields show that K+ ion diffusion in CNTPs in the presence of a single-file water chain is three orders of magnitude slower than bulk diffusion. Intriguingly, the simulations also reveal a disintegration of the water chain upon application of electric fields, resulting in the formation of distinct K+-water clusters, which then traverse the CNTP at high velocity. Finally, we show that although individual ion-water clusters still obey the Nernst-Einstein relation, the overall relation breaks down because of two distinct mechanisms for ion diffusion and electromigration.

Electrostatic gating of ion transport in carbon nanotube porins: A modeling study.

Carbon nanotube porins (CNTPs) are biomimetic membrane channels that demonstrate excellent biocompatibility and unique water and ion transport properties. Gating transport in CNTPs with external voltage could increase control over ion flow and selectivity. Herein, we used continuum modeling to probe the parameters that enable and further affect CNTP gating efficiency, including the size and composition of the supporting lipid membrane, slip flow in the carbon nanotube, and the intrinsic electronic properties of the nanotube. Our results show that the optimal gated CNTP device consists of a semiconducting CNTP inserted into a small membrane patch containing an internally conductive layer. Moreover, we demonstrate that the ionic transport modulated by gate voltages is controlled by the charge distribution along the CNTP under the external gate electric potential. The theoretical understanding developed in this study offers valuable guidance for the design of gated CNTP devices for nanofluidic studies, novel biomimetic membranes, and cellular interfaces in the future.

Electric Field Induced Biomimetic Transmembrane Electron Transport Using Carbon Nanotube Porins.

Cells modulate their homeostasis through the control of redox reactions via transmembrane electron transport systems. These are largely mediated via oxidoreductase enzymes. Their use in biology has been linked to a host of systems including reprogramming for energy requirements in cancer. Consequently, the ability to modulate membrane redox systems may give rise to opportunities to modulate underlying biology. The current work aims to develop a wireless bipolar electrochemical approach to form on-demand electron transfer across biological membranes. To achieve this goal, it is shown that by using membrane inserted carbon nanotube porins (CNTPs) that can act as bipolar nanoelectrodes, one can control electron flow with externally applied electric fields across membranes. Before this work, bipolar electrochemistry has been thought to require high applied voltages not compatible with biological systems. It is shown that bipolar electrochemical reaction via gold reduction at the nanotubes can be modulated at low cell-friendly voltages, providing an opportunity to use bipolar electrodes to control electron flux across membranes. The authors provide new mechanistic insight into this newly describe phenomena at the nanoscale. The results presented give rise to a new method using CNTPs to modulate cell behavior via wireless control of membrane electron transfer.

Early-Stage Aggregation and Crystalline Interactions of Peptoid Nanomembranes.

Fully synthetic peptoid membranes are known to mimic important features of biological membranes, with several advantages over other biomimetic membranes. A fundamental understanding of how the individual peptoid amphiphiles assemble in solution to form the bilayer membrane is key to unlocking their versatility for application in a broad range of processes. In this study, in situ X-ray scattering and molecular dynamics simulations are used to understand the early stages of assembly of three different peptoids that exhibit distinctly different crystallization kinetics. The in situ measurements reveal that the peptoids aggregate first into a nascent phase that is less crystalline than the assembled peptoid membrane. Anisotropic aromatic interactions are determined to be the dominant driving force in the early stages of membrane formation. These results provide key insights into how the peptoid assembly may be manipulated during the early stages of assembly and nucleation and growth.

Membrane fusion and drug delivery with carbon nanotube porins.

Drug delivery mitigates toxic side effects and poor pharmacokinetics of life-saving therapeutics and enhances treatment efficacy. However, direct cytoplasmic delivery of drugs and vaccines into cells has remained out of reach. We find that liposomes studded with 0.8-nm-wide carbon nanotube porins (CNTPs) function as efficient vehicles for direct cytoplasmic drug delivery by facilitating fusion of lipid membranes and complete mixing of the membrane material and vesicle interior content. Fusion kinetics data and coarse-grained molecular dynamics simulations reveal an unusual mechanism where CNTP dimers tether the vesicles, pull the membranes into proximity, and then fuse their outer and inner leaflets. Liposomes containing CNTPs in their membranes and loaded with an anticancer drug, doxorubicin, were effective in delivering the drug to cancer cells, killing up to 90% of them. Our results open an avenue for designing efficient drug delivery carriers compatible with a wide range of therapeutics.

Water-ion permselectivity of narrow-diameter carbon nanotubes.

Carbon nanotube (CNT) pores, which mimic the structure of the aquaporin channels, support extremely high water transport rates that make them strong candidates for building artificial water channels and high-performance membranes. Here, we measure water and ion permeation through 0.8-nm-diameter CNT porins (CNTPs)-short CNT segments embedded in lipid membranes-under optimized experimental conditions. Measured activation energy of water transport through the CNTPs agrees with the barrier values typical for single-file water transport. Well-tempered metadynamics simulations of water transport in CNTPs also report similar activation energy values and provide molecular-scale details of the mechanism for water entry into these channels. CNTPs strongly reject chloride ions and show water-salt permselectivity values comparable to those of commercial desalination membranes.

Author Correction: High permeability sub-nanometre sieve composite MoS2 membranes.

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

Carbon nanotube porin diffusion in mixed composition supported lipid bilayers.

Carbon nanotube porins (CNTPs), short pieces of carbon nanotubes capable of self-inserting into a lipid bilayer, represent a simplified model of biological membrane channels. We have used high-speed atomic force microscopy (HS-AFM) and all-atom molecular dynamics (MD) simulations to study the behavior of CNTPs in a mixed lipid membrane consisting of DOPC lipid with a variable percentage of DMPC lipid added to it. HS-AFM data reveal that the CNTPs undergo diffusive motion in the bilayer plane. Motion trajectories extracted from the HS-AFM movies indicate that CNTPs exhibit diffusion coefficient values broadly similar to values reported for membrane proteins in supported lipid bilayers. The data also indicate that increasing the percentage of DMPC leads to a marked slowing of CNTP diffusion. MD simulations reveal a CNTP-lipid assembly that diffuses in the membrane and show trends that are consistent with the experimental observations.

High permeability sub-nanometre sieve composite MoS2 membranes.

Two-dimensional membranes have gained enormous interest due to their potential to deliver precision filtration of species with performance that can challenge current desalination membrane platforms. Molybdenum disulfide (MoS2) laminar membranes have recently demonstrated superior stability in aqueous environment to their extensively-studied analogs graphene-based membranes; however, challenges such as low ion rejection for high salinity water, low water flux, and low stability over time delay their potential adoption as a viable technology. Here, we report composite laminate multilayer MoS2 membranes with stacked heterodimensional one- to two-layer-thick porous nanosheets and nanodisks. These membranes have a multimodal porous network structure with tunable surface charge, pore size, and interlayer spacing. In forward osmosis, our membranes reject more than 99% of salts at high salinities and, in reverse osmosis, small-molecule organic dyes and salts are efficiently filtered. Finally, our membranes stably operate for over a month, implying their potential for use in commercial water purification applications.

Towards single-species selectivity of membranes with subnanometre pores.

Synthetic membranes with pores at the subnanometre scale are at the core of processes for separating solutes from water, such as water purification and desalination. While these membrane processes have achieved substantial industrial success, the capability of state-of-the-art membranes to selectively separate a single solute from a mixture of solutes is limited. Such high-precision separation would enable fit-for-purpose treatment, improving the sustainability of current water-treatment processes and opening doors for new applications of membrane technologies. Herein, we introduce the challenges of state-of-the-art membranes with subnanometre pores to achieve high selectivity between solutes. We then analyse experimental and theoretical literature to discuss the molecular-level mechanisms that contribute to energy barriers for solute transport through subnanometre pores. We conclude by providing principles and guidelines for designing next-generation single-species selective membranes that are inspired by ion-selective biological channels.

Strong Differential Monovalent Anion Selectivity in Narrow Diameter Carbon Nanotube Porins.

Inner pores of carbon nanotubes combine extremely fast water transport and ion selectivity that could potentially be useful for high-performance water desalination and separation applications. We used dye-quenching halide assays and stopped-flow spectrometry to determine intrinsic permeability of three small monovalent halide anions (chloride, bromide, iodide) and one pseudohalide anion (thiocyanate) through narrow 0.8 nm diameter carbon nanotube porins (CNTPs). These measurements revealed unexpectedly strong differential ion selectivity with permeabilities of different ions varying by up to 2 orders of magnitude. Removal of the negative charge from the nanotube entrance increased anion permeability by only a relatively small factor, indicating that electrostatic repulsion was not a major determinant of CNTP selectivity. First principle molecular dynamics simulations revealed that the origin of this strong differential ion selectivity is partial dehydration of anions upon entry into the narrow CNTP channels.

Decoupling copolymer, lipid and carbon nanotube interactions in hybrid, biomimetic vesicles.

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.