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.

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.

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.

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.

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.

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.

Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes.

There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down 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 properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, 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.

Silicon Nanoribbon pH Sensors Protected by a Barrier Membrane with Carbon Nanotube Porins.

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.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Tuning crystallization pathways through sequence engineering of biomimetic polymers.

Two-step nucleation pathways in which disordered, amorphous, or dense liquid states precede the appearance of crystalline phases have been reported for a wide range of materials, but the dynamics of such pathways are poorly understood. Moreover, whether these pathways are general features of crystallizing systems or a consequence of system-specific structural details that select for direct versus two-step processes is unknown. Using atomic force microscopy to directly observe crystallization of sequence-defined polymers, we show that crystallization pathways are indeed sequence dependent. When a short hydrophobic region is added to a sequence that directly forms crystalline particles, crystallization instead follows a two-step pathway that begins with the creation of disordered clusters of 10-20 molecules and is characterized by highly non-linear crystallization kinetics in which clusters transform into ordered structures that then enter the growth phase. The results shed new light on non-classical crystallization mechanisms and have implications for the design of self-assembling polymer systems.

Impact of PEG additives and pore rim functionalization on water transport through sub-1 nm carbon nanotube porins.

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.

Structure of Carbon Nanotube Porins in Lipid Bilayers: An in Situ Small-Angle X-ray Scattering (SAXS) Study.

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.

Interpreting the widespread nonlinear force spectra of intermolecular bonds.

Single molecule force spectroscopy probes the strength, lifetime, and energetic details of intermolecular interactions in a simple experiment. A growing number of these studies have reported distinctly nonlinear trends in rupture force with loading rate that are typically explained in conventional models by invoking complex escape pathways. Recent analyses suggested that these trends should be expected even for simple barriers based on the basic assumptions of bond rupture dynamics and thus may represent the norm rather than the exception. Here we explore how these nonlinear trends reflect the two fundamental regimes of bond rupture: (i) a near-equilibrium regime, produced either by bond reforming in the case of a single bond or by asynchronized rupture of multiple individual bonds, and (ii) a kinetic regime produced by fast, non-equilibrium bond rupture. We analyze both single- and multi-bonded cases, describe the full evolution of the system as it transitions between near- and far-from-equilibrium loading regimes, and show that both interpretations produce essentially identical force spectra. Data from 10 different molecular systems show that this model provides a comprehensive description of force spectra for a diverse suite of bonds over experimentally relevant loading rates, removes the inconsistencies of previous interpretations of transition state distances, and gives ready access to both kinetic and thermodynamic information about the interaction. These results imply that single-molecule binding free energies for a vast number of bonds have already been measured.

Osmotically-driven transport in carbon nanotube porins.

We report the measurements of transport of ions and uncharged species through carbon nanotube (CNT) porins--short segments of CNTs inserted into a lipid bilayer membrane. Rejection characteristics of the CNT porins are governed by size exclusion for the uncharged species. In contrast, rejection of ionic species is governed by the electrostatic repulsion and Donnan membrane equilibrium. Permeability of monovalent cations follows the general trend in the hydrated ion size, except in the case of Cs(+) ions.

Practical single molecule force spectroscopy: how to determine fundamental thermodynamic parameters of intermolecular bonds with an atomic force microscope.

Single molecule force spectroscopy involves loading a chemical bond using an atomic force microscope and measuring the rupture forces required to break that bond. In 20 years since its inception this technique developed into a robust way to extract a nearly complete set of the information about the bond that includes the bond energy, the kinetic parameters of the bond, and the geometry of the transition state. In this article we review the basic physics of the measurements, the model that is used for data interpretation, and go over the ways to extract the bond information from the experimental data. We also discuss several practical aspects of the measurements that are helpful to the planning and analysis of single molecule force spectroscopy experiments.

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.

Lipid bilayer composition can influence the orientation of proteorhodopsin in artificial membranes.

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.