Nanometer-diameter Pore Research Articles

Quantifying Molecular Transport through Lipid Electropores Induced by Nanosecond Pulsed Electric Fields

High amplitude, nanosecond-duration, pulsed electric fields (nsPEFs) induce the formation of nanometer-diameter pores in cell membranes. These transient lipid electropores facilitate the transport of normally impermeant ions and small molecules across the membrane. Quantitative measurements of molecular transport through lipid electropores are essential for the validation of electroporation models and for optimizing the nsPEF dose for application-specific responses. We calibrated the fluorescence intensity of the membrane integrity marker dye YO-PRO-1 in dense cell lysates using a fast confocal microscope in order to quantify the time-dependent molecular influx of YO-PRO-1 into living cells after nsPEF exposure. Using this method we were able to measure the number of YO-PRO-1 molecules that enter U-937 human histiocytic lymphoma cells following electropermeabilization by a single 6 ns, 20 MV/m pulse.

Engineering Biological Nanopore MspA for Sequencing DNA

The sequence of a long DNA molecule can be determined, in principle, by measuring the ionic current blockade the molecule produces as it permeates a nanometer-diameter pore in a thin insulating membrane. The difficulties in realizing this idea in practice are common to both biological and synthetic nanopores: the pore geometry does not permit isolation of a single nucleotide and the DNA molecule moves too fast through the nanopore for its sequence to be determined by the ionic current measurement. Here, we report our progress in engineering biological pore MspA for sequencing applications. It has been experimentally shown that DNA strands immobilized inside the MspA pore produce ionic current blockades that permit identification of a single nucleotide substitution in the DNA sequence [doi:10.1073/pnas.1001831107]. Through all-atom molecular dynamics simulations we investigate the molecular origin of such extreme sensitivity of the ionic current to the sequence and orientation of DNA strands. Furthermore, we demonstrate the feasibility of reducing the rate of DNA transport by introducing point mutations in the MspA structure. Spanning tens of microseconds, our simulations provide the most detailed account of the atomic-scale mechanics of DNA and ion transport through biological nanopores.

Influence of Size on the Rate of Mesoporous Electrodes for Lithium Batteries

High power rechargeable lithium batteries are a key target for transport and load leveling, in order to mitigate CO(2) emissions. It has already been demonstrated that mesoporous lithium intercalation compounds (composed of particles containing nanometer diameter pores separated by walls of similar size) can deliver high rate (power) and high stability on cycling. Here we investigate how the critical dimensions of pore size and wall thickness control the rate of intercalation (electrode reaction). By using mesoporous beta-MnO(2), the influence of these mesodimensions on lithium intercalation via single and two-phase intercalation processes has been studied in the same material enabling direct comparison. Pore size and wall thickness both influence the rate of single and two-phase intercalation mechanisms, but the latter is more sensitive than the former.

Stretching DNA using the electric field in a synthetic nanopore.

The mechanical properties of DNA over segments comparable to the size of a protein-binding site (3-10 nm) are examined using an electric-field-induced translocation of single molecules through a nanometer diameter pore. DNA, immersed in an electrolyte, is forced through synthetic pores ranging from 0.5 to 1.5 nm in radius in a 10 nm thick Si(3)N(4) membrane using an electric field. To account for the stretching and bending, we use molecular dynamics to simulate the translocation. We have found a threshold for translocation that depends on both the dimensions of the pore and the applied transmembrane bias. The voltage threshold coincides with the stretching transition that occurs in double-stranded DNA near 60 pN.

Zero- and one-dimensional 4He Bose fluids realized in nanometer pores

To realize helium quantum fluids in clusters and in one-dimensions, we have studied 4He and 3He adsorbed in nanometer diameter pores of zeolite and FSM-16. In each cage 13 Å in diameter of Na-Y zeolite covered with helium solid layer, a few helium atoms are suggested to be Bose and Fermi clusters for 4He and 3He atoms, respectively, of which symmetry difference results in completely different eigenstates and low temperature heat capacities. In the one-dimensional (1D) pores of 18 and 28 Å in FSM-16, 4He fluid nanotubes are formed on the pore walls preplated with solid-like helium layers. Absolute 1D Bose fluid is realized at sufficiently low temperatures and appropriate coverages, where the transverse motion in the fluid tube is in a ground state. We observed a temperature-linear term of the heat capacity probably due to the 1D phonon heat capacity of the 1D interacting Bose fluids.

Electrolytic transport through a synthetic nanometer-diameter pore

We have produced single, synthetic nanometer-diameter pores by using a tightly focused, high-energy electron beam to sputter atoms in 10-nm-thick silicon nitride membranes. Subsequently, we measured the ionic conductance as a function of time, bath concentration, and pore diameter to infer the conductivity and ionic mobility through the pores. The pore conductivity is found to be much larger than the bulk conductivity for dilute bath concentrations, where the Debye length is larger than the pore radius, whereas it is comparable with or less than the bulk for high bath concentrations. We interpret these observations by using multiscale simulations of the ion transport through the pores. Molecular dynamics is used to estimate the ion mobility, and ion transport in the pore is described by the coupled Poisson-Nernst-Planck and the Stokes equations that are solved self-consistently for the ion concentration and velocity and electrical potential. We find that the measurements are consistent with the presence of fixed negative charge in the pore wall and a reduction of the ion mobility because of the fixed charge and the ion proximity to the pore wall.

Sizing DNA Using a Nanometer-Diameter Pore

Each species from bacteria to human has a distinct genetic fingerprint. Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool. As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a nanometer-thick inorganic membrane with a tightly focused electron beam, as a transducer that detects single molecules of DNA and produces an electrical signature of the structure. When an electric field is applied across the membrane, a DNA molecule immersed in electrolyte is attracted to the pore, blocks the current through it, and eventually translocates across the membrane as verified unequivocally by gel electrophoresis. The relationship between DNA translocation and blocking current has been established through molecular dynamics simulations. By measuring the duration and magnitude of the blocking current transient, we can discriminate single-stranded from double-stranded DNA and resolve the length of the polymer.

Microfluidic Separation and Gateable Fraction Collection for Mass-Limited Samples

Integrating multiple analytical processes into microfluidic devices is an important research area required for a variety of microchip-based analyses. A microfluidic system is described that achieves preparative separations by intelligent fraction collection of attomole quantities of sample. The device consists of a main microfluidic channel used to perform electrophoresis, which is interconnected at 90 degrees to two vertically displaced channels via a nanocapillary array membrane. The membrane interconnect contains nanometer-diameter pores that provide fluidic communication between the channels. Sample injection and analyte collection are controlled by application of an electrical bias between the microfluidic channels across the nanocapillary array. After the separation, the automated transfer of the FITC-labeled Arg, Gln, and Gly bands occurs; a fluorescence detector located at the separation/collection channel interconnect is used to generate a triggering signal that initiates suitable voltages to allow near-quantitative transfer of analyte from the separation channel to the second fluidic layer. The ability to achieve such sample manipulations from mass-limited samples enables a variety of postseparation processing events.

Microscopic Kinetics of DNA Translocation through Synthetic Nanopores

We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 × 2.6 nm 2 cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.