Nanopore Fabrication by Controlled Dielectric Breakdown

Nanofabrication techniques for achieving dimensional control at the nanometer scale are generally equipment-intensive and time-consuming. The use of energetic beams of electrons or ions has placed the fabrication of nanopores in thin solid-state membranes within reach of some academic laboratories, yet these tools are not accessible to many researchers and are poorly suited for mass-production. Here we describe a fast and simple approach for fabricating a single nanopore down to 2-nm in size with sub-nm precision, directly in solution, by controlling dielectric breakdown at the nanoscale. The method relies on applying a voltage across an insulating membrane to generate a high electric field, while monitoring the induced leakage current. We show that nanopores fabricated by this method produce clear electrical signals from translocating DNA molecules. Considering the tremendous reduction in complexity and cost, we envision this fabrication strategy would not only benefit researchers from the physical and life sciences interested in gaining reliable access to solid-state nanopores, but may provide a path towards manufacturing of nanopore-based biotechnologies.Comment: 19 pages, 4 figures. Supplementary information contains 22 pages, 11 figures and 2 tables - A version of this manuscript was first submitted for publication on April 23rd, 2013. It is currently under review at another journa

Descreening of Field Effect in Electrically Gated Nanopores

This modeling work investigates the electrical modulation characteristics of field-effect gated nanopores. Highly nonlinear current modulations are observed in nanopores with non-overlapping electric double layers, including those with pore diameters 100 times the Debye screening length. We attribute this extended field-effect gating to a descreening effect, i.e. the counter-ions do not fully relax to screen the gating potential due to the presence of strong ionic transport

The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic

Microcantilever actuation generated by redox-induced surface stress

Electrochemically-induced changes in surface stress at the solid-liquid interface are measured using a differential cantilever-based sensor. The simultaneous, in situ measurements of the current (charge) and interfacial stress changes are performed by employing an AFM cantilever as both the working electrode (in a conventional three-probe electrochemical cell configuration) and as the mechanical transducer (bending of the cantilever). The custom-built instrument achieves a surface stress sensitivity of 1x10-4 N/m and a dynamic range of 5x105. Combining electrochemistry with cantilever-based sensing provides the extra surface characterization capability essential for the interpretation of the origin of the surface stress.The objective of the present study is to gain a better understanding of the mechanisms responsible for the nanomechanical motion of cantilever sensors during adsorption and absorption processes. The study of these simple model systems will lead to a general understanding of the cantilever-based sensor's response and provide insights into the physical origin of the measured surface stress.The surface stress generated by the electrochemically-controlled absorption of ions into a thin polypyrrole film is investigated. A compressive change in surface stress of about -2 N/m is measured when the polymer is electrochemically switched between its oxidized and neutral (swollen) state. The volume change of the polymer phase with respect to the gold-coated cantilever is shown to be responsible for the mechanical motion observed.The potential-induced surface stress and surface energy change on an Au(111)-textured cantilever, in a 0.1 M HClO4 electrolyte, are simultaneously measured. These measurements revealed that for solid electrodes these two thermodynamic parameters are significantly different. In the double layer region, a surface stress change of -0.55 +/-0.06 N/m is measured during ClO4- adsorption whereas the surface energy variation is smaller by one order of magnitude. The origin of the surface stress change at the metal-electrolyte interface is understood by the variation in electron density at the surface which alters the inter-atomic bonds strength between surface atoms, while the specificity of adsorption of ions is found to be mostly responsible for the fine structure of the surface stress profile

Analysis of nanopore data: classification strategies for an unbiased curation of single-molecule events from DNA nanostructures

Nanopores are versatile single-molecule sensors that are being used to sense increasingly complex mixtures of structured molecules, with applications in molecular data storage and disease biomarker detection. However, increased molecular complexity presents additional challenges to the analysis of nanopore data including more translocation events being rejected for not matching an expected signal structure and a greater risk of selection bias entering this event curation process. To highlight these challenges, here we present the analysis of a model molecular system consisting of a nanostructured DNA molecule attached to a linear DNA carrier. We make use of recent advances in the event segmentation capabilities of Nanolyzer, a graphical analysis tool provided for nanopore event fitting, and describe approaches to event substructure analysis. In the process, we identify and discuss important sources of selection bias in that emerge in the analysis of this molecular system and consider the complicating effects of molecular conformation and variable experimental conditions (e.g. pore diameter). We then present additional refinements to existing analysis techniques, allowing for improved separation of multiplexed samples, fewer translocation events rejected as false negatives, and a wider range of experimental conditions for which accurate molecular information can be extracted. Increasing the coverage of analyzed events within nanopore data is not only important for characterizing complex molecular samples with high fidelity, but is also becoming essential to the generation of accurate, unbiased training data as machine learning approaches to data analysis and event identification continue to increase in prevalence

Elucidating the Dynamics of Polymer Transport through Nanopores using Asymmetric Salt Concentrations

While notable progress has been made in recent years both experimentally and theoretically in understanding the highly complex dynamics of polymer capture and transport through nanopores, there remains significant disagreement between experimental observation and theoretical prediction that needs to be resolved. Asymmetric salt concentrations, where the concentrations of ions on each side of the membrane are different, can be used to enhance capture rates and prolong translocation times of electrophoretically driven polymers translocating through a nanopore from the low salt concentration reservoir, which are both attractive features for single-molecule analysis. However, since asymmetric salt concentrations affect the electrophoretic pull inside and outside the pore differently, it also offers a useful control parameter to elucidate the otherwise inseparable physics of the capture and translocation process. In this work, we attempt to paint a complete picture of the dynamics of polymer capture and translocation in both symmetric and asymmetric salt concentration conditions by reporting the dependence of multiple translocation metrics on voltage, polymer length, and salt concentration gradient. Using asymmetric salt concentration conditions, we experimentally observe the predictions of tension propagation theory, and infer the significant impact of the electric field outside the pore in capturing polymers and in altering polymer conformations prior to translocation

Screening for Group A Streptococcal disease via Solid-State Nanopore Detection of PCR Amplicons

Single molecule detection methods are becoming increasingly important for diagnostic applications. Practical Early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a PCR-based assay targeting group A streptococcus (strep A) which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices both for adapting PCR-based assays to solid-state nanopore readout as well as analytical approaches by which to decide on sample status

Efficient simulation of arbitrary multi-component first-order binding kinetics for improved assay design and molecular assembly

Traditional ELISA, long the workhorse for specific target protein detection using microplate wells, is nearing its fundamen-tal limit of sensitivity. New opportunities in healthcare call for in vitro diagnostic tests with ultra-high sensitivity. Magnetic bead-based ELISA formats have been developed that can reach unprecedented sensitivities order of magnitude better than are allowed for by the rate constants for a single ligand-receptor interaction. However, these ultra-high sensitivity assays are highly vulnerable to a host of confounding factors, including nonspecific binding from background molecules and loss of low-abundance target to tube walls and during wash steps. Moreover, optimization of workflow is often time-consuming and expensive. In this work, we present a simulation tool that allows users to graphically define arbitrary binding assays, includ-ing fully reversible first-order binding kinetics, timed addition of extra components, and timed wash steps. The tool is freely available as a user-friendly webapp. The framework is lightweight and fast, allowing for inexpensive simulation and visuali-zation of arbitrarily complex assay schemes, including but not limited to digital immunoassays, DNA hybridization, and enzyme kinetics, for validation and optimization of assay designs without requiring any programming knowledge from the user. We demonstrate some of these capabilities and provide practical guidance on assay simulation design

Schematic of the fabrication setup.

<p>A computer-controlled custom current amplifier is used to apply voltages up to ±20 V and measure the current with sub-nA sensitivity from one of the two Ag/AgCl electrodes positioned on either sides of the membrane. It is noteworthy to realize that this experimental setup is identical (with the exception of the custom current amplifier replacing the commonly used Axopatch 200B) to the instrumentation used to study DNA or proteins translocation through nanopores.</p

Time-to-pore creation as a function of experimental conditions.

<p>a) Semi-log plot of fabrication time of individual nanopores created in 30-nm-thick SiN_x membranes in 1 M KCl buffered as indicated, versus the applied voltage and the calculated applied electric field. The number of separate nanopores each data point is averaged over is indicated in parentheses. The vast majority of nanopores plotted are sub-5-nm in size (i.e. <7 nS). b) Semi-log plot of fabrication time versus pH for the data plotted in a). c) Semi-log plot of fabrication time of individual nanopores created in 10-nm-thick SiN_x membranes in 1 M Cl-based electrolyte buffered at pH 10 for different cationic species versus the applied voltage and the calculated applied electric field. All 66 nanopores plotted are sub-5-nm in size (i.e. <20 nS).</p