High-Voltage Biomolecular Sensing Using a Bacteriophage Portal Protein Covalently Immobilized within a Solid-State Nanopore.

The application of nanopores as label-free, single-molecule biosensors for electrical or optical probing of structural features in biomolecules has been widely explored. While biological nanopores (membrane proteins and bacteriophage portal proteins) and solid-state nanopores (thin films and two-dimensional materials) have been extensively employed, the third class of nanopores known as hybrid nanopores, where an artificial membrane substitutes the organic support membrane of proteins, has been only sparsely studied due to challenges in implementation. G20c portal protein contains a natural DNA pore that is used by viruses for filling their capsid with viral genomic DNA. We have previously developed a lipid-free hybrid nanopore by "corking" the G20c portal protein into a SiNx nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the G20c portal protein considerably improves the hybrid pore stability, lifetime, and voltage resilience. Moreover, we demonstrate electric-field-driven and motor protein-mediated transport of DNA molecules through this hybrid nanopore. Our integrated protein/solid-state device can serve as a robust and durable framework for sensing and sequencing at high voltages, potentially providing higher resolution, higher signal-to-noise ratio, and higher throughput compared to the more conventional membrane-embedded protein platforms.

Ionically Active MXene Nanopore Actuators.

Reversible electrochemical intercalation of cations into the interlayer space of 2D materials induces tunable physical and chemical properties in them. In MXenes, a large class of recently developed 2D carbides and nitrides, low intercalation energy, high storage capacitance, and reversible intercalation of various cations have led to their improved performance in sensing and energy storage applications. Herein, a coupled nanopore-actuator system where an ultrathin free-standing MXene film serves as a nanopore support membrane and ionically active actuator is reported. In this system, the contactless MXene membrane in the electric field affects the cation movement in the field through their (de)intercalation between individual MXene flakes. This results in reversible swelling and contraction of the membrane monitored by ionic conductance through the nanopore. This unique nanopore coupled to a mechanical actuation system could provide new insights into designing single-molecule biosensing platforms at the nanoscale.

A Long-Circulating Vector for Aptamers Based upon Polyphosphodiester-Backboned Molecular Brushes.

Aptamers face challenges for use outside the ideal conditions in which they are developed. These difficulties are most palpable in vivo due to nuclease activities, rapid clearance, and off-target binding. Herein, we demonstrate that a polyphosphodiester-backboned molecular brush can suppress enzymatic digestion, reduce non-specific cell uptake, enable long blood circulation, and rescue the bioactivity of a conjugated aptamer in vivo. The backbone along with the aptamer is assembled via solid-phase synthesis, followed by installation of poly(ethylene glycol) (PEG) side chains using a two-step process with near-quantitative efficiency. The synthesis allows for precise control over polymer size and architecture. Consisting entirely of building blocks that are generally recognized as safe for therapeutics, this novel molecular brush is expected to provide a highly translatable route for aptamer-based therapeutics.

Wafer-Scale Lateral Self-Assembly of Mosaic Ti3C2Tx MXene Monolayer Films.

Bottom-up assembly of two-dimensional (2D) materials into macroscale morphologies with emergent properties requires control of the material surroundings, so that energetically favorable conditions direct the assembly process. MXenes, a class of recently developed 2D materials, have found new applications in areas such as electrochemical energy storage, nanoscale electronics, sensors, and biosensors. In this paper, we present a lateral self-assembly method for wafer-scale deposition of a mosaic-type 2D MXene flake monolayer that spontaneously orders at the interface between two immiscible solvents. ReaxFF molecular dynamics simulations elucidate the interactions of a MXene flake with the solvents and its stability at the liquid/liquid interface, the prerequisite for MXene flakes self-assembly at the interface. Moreover, facile transfer of this monolayer onto a flat substrate (Si, glass) results in high-coverage monolayer films with uniform thickness and homogeneous optical properties. Multiscale characterization of the resulting films reveals the mosaic structure and sheds light on the electronic properties of the films, which exhibit good electrical conductivity over cm-scale areas.

Single-Molecule Sensing Using Nanopores in Two-Dimensional Transition Metal Carbide (MXene) Membranes.

Label-free nanopore technology for sequencing biopolymers such as DNA and RNA could potentially replace existing methods if improvements in cost, speed, and accuracy are achieved. Solid-state nanopores have been developed over the past two decades as physically and chemically versatile sensors that mimic biological channels, through which transport and sequencing of biomolecules have already been demonstrated. Of particular interest is the use of two-dimensional (2D) materials as nanopore substrates, since these can in theory provide the highest resolution readout (<1 nm of a biopolymer segment) and opportunities for electronic multiplexed readout through their interesting electronic properties. In this work, we report on nanopores comprising atomically thin flakes of 2D transition metal carbides called MXenes. We demonstrate a high-yield (60%), contamination-free, and alignment-free transfer method that involves their self-assembly at a liquid-liquid interface to large-scale (mm-sized) films composed of sheets, followed by nanopore fabrication using focused electron beams. Our work demonstrates the feasibility of MXenes, a class of hydrophilic 2D materials with over 20 compositions known to date, as nanopore membranes for DNA translocation and single-molecule sensing applications.

Assembling 2D MXenes into Highly Stable Pseudocapacitive Electrodes with High Power and Energy Densities.

Electrochemical capacitors (ECs) that store charge based on the pseudocapacitive mechanism combine high energy densities with high power densities and rate capabilities. 2D transition metal carbides (MXenes) have been recently introduced as high-rate pseudocapacitive materials with ultrahigh areal and volumetric capacitances. So far, 20 different MXene compositions have been synthesized and many more are theoretically predicted. However, since most MXenes are chemically unstable in their 2D forms, to date only one MXene composition, Ti3 C2 Tx , has shown stable pseudocapacitive charge storage. Here, a cation-driven assembly process is demonstrated to fabricate highly stable and flexible multilayered films of V2 CTx and Ti2 CTx MXenes from their chemically unstable delaminated single-layer flakes. The electrochemical performance of electrodes fabricated using assembled V2 CTx flakes surpasses Ti3 C2 Tx in various aqueous electrolytes. These electrodes show specific capacitances as high as 1315 F cm-3 and retain ≈77% of their initial capacitance after one million charge/discharge cycles, an unprecedented performance for pseudocapacitive materials. This work opens a new venue for future development of high-performance supercapacitor electrodes using a variety of 2D materials as building blocks.

Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors.

Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.