Protein Motion and Configurations in a Form-Fitting Nanopore: Avidin in ClyA.

We probe the molecular dynamics and states of an avidin protein as it is captured and trapped in a voltage-biased cytolysin A nanopore using time-resolved single-molecule electrical conductance signals. The data for very large numbers of single-molecule events are analyzed and presented by a new method that provides clear visual insight into the molecular scale processes. Avidin in cytolysin A has surprisingly rich conductance spectra that reveal transient and more permanently trapped protein configurations in the pore and how they evolve into one another. We identify a long-lasting, stable, and low-noise configuration of avidin in the nanopore into which avidin can be reliably trapped and released. This may prove useful for single-molecule studies of other proteins that can be biotinylated and then transported by avidin to the pore via their coupling to avidin with biotin-avidin linking. We demonstrate the sensitivity of this system with detection of biotin attached to avidin captured by the pore.

Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore.

We report effective charges and diffusion constants of several different single-stranded DNA oligonucleotides trapped in an MspA nanopore. Nucleotide identity is found to have a substantial influence on effective charges and diffusion constants. These quantities are determined from escape time experiments for a DNA molecule attached to a NeutrAvidin molecule that, unlike the DNA, does not pass through the pore. Correlations are reported between oligonucleotide effective charges and current blockages, and between their diffusion constants and DNA-induced current blockage fluctuations. We also report an unanticipated source of current fluctuations that reflects a discrete blockage current level structure. We posit that this is associated with interactions between the NeutrAvidin molecule and the MspA nanopore.

Molecule-hugging graphene nanopores.

It has recently been recognized that solid-state nanopores in single-atomic-layer graphene membranes can be used to electronically detect and characterize single long charged polymer molecules. We have now fabricated nanopores in single-layer graphene that are closely matched to the diameter of a double-stranded DNA molecule. Ionic current signals during electrophoretically driven translocation of DNA through these nanopores were experimentally explored and theoretically modeled. Our experiments show that these nanopores have unusually high sensitivity (0.65 nA/Å) to extremely small changes in the translocating molecule's outer diameter. Such atomically short graphene nanopores can also resolve nanoscale-spaced molecular structures along the length of a polymer, but do so with greatest sensitivity only when the pore and molecule diameters are closely matched. Modeling confirms that our most closely matched pores have an inherent resolution of ≤ 0.6 nm along the length of the molecule.

Superheating and homogeneous single bubble nucleation in a solid-state nanopore.

We demonstrate extreme superheating and single bubble nucleation in an electrolyte solution within a nanopore in a thin silicon nitride membrane. The high temperatures are achieved by Joule heating from a highly focused ionic current induced to flow through the pore by modest voltage biases. Conductance, nucleation, and bubble evolution are monitored electronically and optically. Temperatures near the thermodynamic limit of superheat are achieved just before bubble nucleation with the system at atmospheric pressure. Bubble nucleation is homogeneous and highly reproducible. This nanopore approach more generally suggests broad application to the excitation, detection, and characterization of highly metastable states of matter.

Pressure-controlled motion of single polymers through solid-state nanopores.

Voltage-biased solid-state nanopores are well established in their ability to detect and characterize single polymers, such as DNA, in electrolytes. The addition of a pressure gradient across the nanopore yields a second molecular driving force that provides new freedom for studying molecules in nanopores. In this work, we show that opposing pressure and voltage bias enables nanopores to detect and resolve very short DNA molecules, as well as to detect near-neutral polymers.

Escape of DNA from a weakly biased thin nanopore: experimental evidence for a universal diffusive behavior.

We report experimental escape time distributions of double-stranded DNA molecules initially threaded halfway through a thin solid-state nanopore. We find a universal behavior of the escape time distributions consistent with a one-dimensional first passage formulation notwithstanding the geometry of the experiment and the potential role of complex molecule-liquid-pore interactions. Diffusion constants that depend on the molecule length and pore size are determined. Also discussed are the practical implications of long time diffusive molecule trapping in the nanopore.

Trapping DNA near a solid-state nanopore.

We demonstrate that voltage-biased solid-state nanopores can transiently localize DNA in an electrolyte solution. A double-stranded DNA (dsDNA) molecule is trapped when the electric field near the nanopore attracts and immobilizes a non-end segment of the molecule across the nanopore orifice without inducing a folded molecule translocation. In this demonstration of the phenomenon, the ionic current through the nanopore decreases when the dsDNA molecule is trapped by the nanopore. By contrast, a translocating dsDNA molecule under the same conditions causes an ionic current increase. We also present finite-element modeling results that predict this behavior for the conditions of the experiment.

Origins and consequences of velocity fluctuations during DNA passage through a nanopore.

We describe experiments and modeling results that reveal and explain the distribution of times that identical double-stranded DNA (dsDNA) molecules take to pass through a voltage-biased solid-state nanopore. We show that the observed spread in this distribution is caused by viscous-drag-induced velocity fluctuations that are correlated with the initial conformation of nanopore-captured molecules. This contribution exceeds that due to diffusional Brownian motion during the passage. Nevertheless, and somewhat counterintuitively, the diffusional Brownian motion determines the fundamental limitations of rapid DNA strand sequencing with a nanopore. We model both diffusional and conformational fluctuations in a Langevin description. It accounts well for passage time variations for DNA molecules of different lengths, and predicts conditions required for low-error-rate nanopore-strand DNA sequencing with nanopores.

Ice lithography for nanodevices.

We report the successful application of a new approach, ice lithography (IL), to fabricate nanoscale devices. The entire IL process takes place inside a modified scanning electron microscope (SEM), where a vapor-deposited film of water ice serves as a resist for e-beam lithography, greatly simplifying and streamlining device fabrication. We show that labile nanostructures such as carbon nanotubes can be safely imaged in an SEM when coated in ice. The ice film is patterned at high e-beam intensity and serves as a mask for lift-off without the device degradation and contamination associated with e-beam imaging and polymer resist residues. We demonstrate the IL preparation of carbon nanotube field effect transistors with high-quality trans-conductance properties.

Solid-state nanopore for detecting individual biopolymers.

Solid-state nanopores have been fabricated and used to characterize single DNA and protein molecules. Here we describe the details on how these nanopores were fabricated and characterized, the nanopore sensing system setup, and protocols of using these nanopores to characterize DNA and protein molecules.

Base dependent DNA-carbon nanotube interactions: activation enthalpies and assembly-disassembly control.

We quantify the base dependent interactions between single stranded DNA and single walled carbon nanotubes (SWNTs) in solution. DNA/SWNT hybrids hold the promise of applications ranging from nanoscale electronics and assembly of nanotube based materials, to drug delivery and DNA sequencing. These applications require control over the hybrid assembly and disassembly. Our analytical assay reveals the order of nucleobase binding strengths with SWNTs as G>C>A>T. Furthermore, time dependent fixed temperature experiments that probe the kinetics of the dissociation process provide values for the equilibrium constants and dissociation enthalpies that underlie the microscopic interactions. Quantifying the base dependency of hybrid stability shows how insight into the energetics of the component interactions facilitates control over hybrid assembly and disassembly.

Nanoscale dynamics of Joule heating and bubble nucleation in a solid-state nanopore.

We present a mathematical model for Joule heating of an electrolytic solution in a nanopore. The model couples the electrical and thermal dynamics responsible for rapid and extreme superheating of the electrolyte within the nanopore. The model is implemented numerically with a finite element calculation, yielding a time and spatially resolved temperature distribution in the nanopore region. Temperatures near the thermodynamic limit of superheat are predicted to be attained just before the explosive nucleation of a vapor bubble is observed experimentally. Knowledge of this temperature distribution enables the evaluation of related phenomena including bubble nucleation kinetics, relaxation oscillation, and bubble dynamics.

Ion selectivity of graphene nanopores.

As population growth continues to outpace development of water infrastructure in many countries, desalination (the removal of salts from seawater) at high energy efficiency will likely become a vital source of fresh water. Due to its atomic thinness combined with its mechanical strength, porous graphene may be particularly well-suited for electrodialysis desalination, in which ions are removed under an electric field via ion-selective pores. Here, we show that single graphene nanopores preferentially permit the passage of K(+) cations over Cl(-) anions with selectivity ratios of over 100 and conduct monovalent cations up to 5 times more rapidly than divalent cations. Surprisingly, the observed K(+)/Cl(-) selectivity persists in pores even as large as about 20 nm in diameter, suggesting that high throughput, highly selective graphene electrodialysis membranes can be fabricated without the need for subnanometer control over pore size.

De novo sequencing and variant calling with nanopores using PoreSeq.

The accuracy of sequencing single DNA molecules with nanopores is continually improving, but de novo genome sequencing and assembly using only nanopore data remain challenging. Here we describe PoreSeq, an algorithm that identifies and corrects errors in nanopore sequencing data and improves the accuracy of de novo genome assembly with increasing coverage depth. The approach relies on modeling the possible sources of uncertainty that occur as DNA transits through the nanopore and finds the sequence that best explains multiple reads of the same region. PoreSeq increases nanopore sequencing read accuracy of M13 bacteriophage DNA from 85% to 99% at 100× coverage. We also use the algorithm to assemble Escherichia coli with 30× coverage and the λ genome at a range of coverages from 3× to 50×. Additionally, we classify sequence variants at an order of magnitude lower coverage than is possible with existing methods.

Electrical pulse fabrication of graphene nanopores in electrolyte solution.

Nanopores in graphene membranes can potentially offer unprecedented spatial resolution for single molecule sensing, but their fabrication has thus far been difficult, poorly scalable, and prone to contamination. We demonstrate an in-situ fabrication method that nucleates and controllably enlarges nanopores in electrolyte solution by applying ultra-short, high-voltage pulses across the graphene membrane. This method can be used to rapidly produce graphene nanopores with subnanometer size accuracy in an apparatus free of nanoscale beams or tips.

Pressure-voltage trap for DNA near a solid-state nanopore.

We report the formation of a tunable single DNA molecule trap near a solid-state nanopore in an electrolyte solution under conditions where an electric force and a pressure-induced viscous flow force on the molecule are nearly balanced. Trapped molecules can enter the pore multiple times before escaping the trap by passing through the pore or by diffusing away. Statistical analysis of many individually trapped molecules yields a detailed picture of the fluctuation phenomena involved, which are successfully modeled by a one-dimensional first passage approach.

Nanometer-thin solid-state nanopores by cold ion beam sculpting.

Recent work on protein nanopores indicates that single molecule characterization (including DNA sequencing) is possible when the length of the nanopore constriction is about a nanometer. Solid-state nanopores offer advantages in stability and tunability, but a scalable method for creating nanometer-thin solid-state pores has yet to be demonstrated. Here we demonstrate that solid-state nanopores with nanometer-thin constrictions can be produced by "cold ion beam sculpting," an original method that is broadly applicable to many materials, is easily scalable, and requires only modest instrumentation.

Thermal activation and saturation of ion beam sculpting.

We report a material-dependent critical temperature for ion beam sculpting of nanopores in amorphous materials under keV ion irradiation. At temperatures below the critical temperature, irradiated pores open at a rate that soon saturates with decreasing temperature. At temperatures above the critical temperature, the pore closing rate rises rapidly and eventually saturates with increasing temperature. The observed behavior is well described by a model based on adatom diffusion, but is difficult to reconcile with an ion-stimulated viscous flow model.

Nanopore fabrication in amorphous Si: Viscous flow model and comparison to experiment.

Nanopores fabricated in free-standing amorphous silicon thin films were observed to close under 3 keV argon ion irradiation. The closing rate, measured in situ, exhibited a memory effect: at the same instantaneous radius, pores that started larger close more slowly. An ion-stimulated viscous flow model is developed and solved in both a simple analytical approximation for the small-deformation limit and in a finite element solution for large deformations. The finite-element solution exhibits surprising changes in cross-section morphology, which may be extremely valuable for single biomolecule detection, and are untested experimentally. The finite-element solution reproduces the shape of the measured nanopore radius versus fluence behavior and the sign and magnitude of the measured memory effect. We discuss aspects of the experimental data not reproduced by the model, and successes and failures of the competing adatom diffusion model.

Probing surface charge fluctuations with solid-state nanopores.

We identify a contribution to the ionic current noise spectrum in solid-state nanopores that exceeds all other noise sources in the frequency band 0.1-10 kHz. Experimental studies of the dependence of this excess noise on pH and electrolyte concentration indicate that the noise arises from surface charge fluctuations. A quantitative model based on surface functional group protonization predicts the observed behaviors and allows us to locally measure protonization reaction rates. This noise can be minimized by operating the nanopore at a deliberately chosen pH.