Robert Y. Henley

High-Bandwidth Protein Analysis Using Solid-State Nanopores

High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D0) by a factor of ~50, and are voltage-independent in the regime tested. We reason that deviations of D from D0 are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.

Slow DNA Transport through Nanopores in Hafnium Oxide Membranes

We present a study of double- and single-stranded DNA transport through nanopores fabricated in ultrathin (2-7 nm thick) freestanding hafnium oxide (HfO2) membranes. The high chemical stability of ultrathin HfO2 enables long-lived experiments with <2 nm diameter pores that last several hours, in which we observe >50 000 DNA translocations with no detectable pore expansion. Mean DNA velocities are slower than velocities through comparable silicon nitride pores, providing evidence that HfO2 nanopores have favorable physicochemical interactions with nucleic acids that can be leveraged to slow down DNA in a nanopore.

Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays

In recent years, nanopores have emerged as exceptionally promising single-molecule sensors due to their ability to detect biomolecules at subfemtomole levels in a label-free manner. Development of a high-throughput nanopore-based biosensor requires multiplexing of nanopore measurements. Electrical detection, however, poses a challenge, as each nanopore circuit must be electrically independent, which requires complex nanofluidics and embedded electrodes. Here, we present an optical method for simultaneous measurements of the ionic current across an array of solid-state nanopores, requiring no additional fabrication steps. Proof-of-principle experiments are conducted that show simultaneous optical detection and characterization of ssDNA and dsDNA using an array of pores. Through a comparison with electrical measurements, we show that optical measurements are capable of accessing equivalent transmembrane current information.

Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins

Go with the flow Enhanced water transport occurs in a number of narrow pore channels, such as biological aquaporins. Tunuguntla et al. thoroughly characterized molecular transport through narrow carbon nanotubes (CNTs) (see the Perspective by Siwy and Fornasiero). In contrast to previous studies, the authors focused on water and ion transport through relatively short (∼10-nm) fragments of CNTs embedded in lipid bilayer membranes. Strong confinement generated highly accelerated water flow compared with that observed in biological water transporters. A key factor in the transport rate was the tunable rearrangement of intermolecular hydrogen bonding. Furthermore, by changing the charges at the mouth of the nanotube, the authors were able to alter the ion selectivity. Science, this issue p. 792; see also p. 753 Small-diameter carbon nanotubes show promising transport properties and selectivity for water purification applications. Fast water transport through carbon nanotube pores has raised the possibility to use them in the next generation of water treatment technologies. We report that water permeability in 0.8-nanometer-diameter carbon nanotube porins (CNTPs), which confine water down to a single-file chain, exceeds that of biological water transporters and of wider CNT pores by an order of magnitude. Intermolecular hydrogen-bond rearrangement, required for entry into the nanotube, dominates the energy barrier and can be manipulated to enhance water transport rates. CNTPs block anion transport, even at salinities that exceed seawater levels, and their ion selectivity can be tuned to configure them into switchable ionic diodes. These properties make CNTPs a promising material for developing membrane separation technologies.

Direct and Scalable Deposition of Atomically Thin Low-Noise MoS2 Membranes on Apertures.

Molybdenum disulfide (MoS2) flakes can grow beyond the edge of an underlying substrate into a planar freestanding crystal. When the substrate edge is in the form of an aperture, reagent-limited nucleation followed by edge growth facilitate direct and selective growth of freestanding MoS2 membranes. We have found conditions under which MoS2 grows preferentially across micrometer-scale prefabricated solid-state apertures in silicon nitride membranes, resulting in sealed membranes that are one to a few atomic layers thick. We have investigated the structure and purity of our membranes by a combination of atomic-resolution transmission electron microscopy, elemental analysis, Raman spectroscopy, photoluminescence spectroscopy, and low-noise ion-current recordings through nanopores fabricated in such membranes. Finally, we demonstrate the utility of fabricated ultrathin nanopores in such membranes for single-stranded DNA translocation detection.

Nanopore-Based Conformational Analysis of a Viral RNA Drug Target

Nanopores are single-molecule sensors that show exceptional promise as a biomolecular analysis tool by enabling label-free detection of small amounts of sample. In this paper, we demonstrate that nanopores are capable of detecting the conformation of an antiviral RNA drug target. The hepatitis C virus uses an internal ribosome entry site (IRES) motif in order to initiate translation by docking to ribosomes in its host cell. The IRES is therefore a viable and important drug target. Drug-induced changes to the conformation of the HCV IRES motif, from a bent to a straight conformation, have been shown to inhibit HCV replication. However, there is presently no straightforward method to analyze the effect of candidate small-molecule drugs on the RNA conformation. In this paper, we show that RNA translocation dynamics through a 3 nm diameter nanopore is conformation-sensitive by demonstrating a difference in transport times between bent and straight conformations of a short viral RNA motif. Detection is possible because bent RNA is stalled in the 3 nm pore, resulting in longer molecular dwell times than straight RNA. Control experiments show that binding of a weaker drug does not produce a conformational change, as consistent with independent fluorescence measurements. Nanopore measurements of RNA conformation can thus be useful for probing the structure of various RNA motifs, as well as structural changes to the RNA upon small-molecule binding.

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

Compared to conventional methods, single molecule, real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC per cent bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-ng quantities is impractical due to inefficient delivery of DNA molecules into the confines of zero-mode waveguides, zeptolitre optical cavities in which DNA sequencing proceeds. Here we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000 bp long DNA template within seconds from a sub-ng input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

Electrophoretic Deformation of Individual Transfer RNA Molecules Reveals Their Identity

It has been hypothesized that the ribosome gains additional fidelity during protein translation by probing structural differences in tRNA species. We measure the translocation kinetics of different tRNA species through ∼3 nm diameter synthetic nanopores. Each tRNA species varies in the time scale with which it is deformed from equilibrium, as in the translocation step of protein translation. Using machine-learning algorithms, we can differentiate among five tRNA species, analyze the ratios of tRNA binary mixtures, and distinguish tRNA isoacceptors.

Response to Comment on “Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins”

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.

Label-Free Single-Molecule Thermoscopy Using a Laser-Heated Nanopore

When light is used to excite electronic transitions in a material, nonradiative energy during relaxation is often released in the form of heat. In this work, we show that photoexcitation of a silicon nitride nanopore using a focused visible laser results in efficient localized photothermal heating, which reduces the nearby electrolyte viscosity and increases the ionic conductance. In addition, a strong localized thermal gradient in the pore vicinity is produced, evidenced by finite-element simulations and experimental observation of both ion and DNA thermophoresis. After correcting for thermophoresis, the nanopore current can be used as a nanoscale thermometer, enabling rapid force thermoscopy. We utilize this to probe thermal melting transitions in synthetic and native biomolecules that are heated at the nanopore. Our results on single molecules are validated by correspondence to bulk measurements, which paves the way to various biophysical experiments that require rapid temperature and force control on individual molecules.