Fabrication and characterization of nanopores with insulated transverse nanoelectrodes for DNA sensing in salt solution.

We report on the fabrication, simulation, and characterization of insulated nanoelectrodes aligned with nanopores in low-capacitance silicon nitride membrane chips. We are exploring these devices for the transverse sensing of DNA molecules as they are electrophoretically driven through the nanopore in a linear fashion. While we are currently working with relatively large nanopores (6-12 nm in diameter) to demonstrate the transverse detection of DNA, our ultimate goal is to reduce the size sufficiently to resolve individual nucleotide bases, thus sequencing DNA as it passes through the pore. We present simulations and experiments that study the impact of insulating these electrodes, which is important to localize the sensing region. We test whether the presence of nanoelectrodes or insulation affects the stability of the ionic current flowing through the nanopore, or the characteristics of DNA translocation. Finally, we summarize the common device failures and challenges encountered during fabrication and experiments, explore the causes of these failures, and make suggestions on how to overcome them in the future.

The role of pore geometry in single nanoparticle detection.

We observe single nanoparticle translocation events via resistive pulse sensing using silicon nitride pores described by a range of lengths and diameters. Pores are prepared by focused ion beam milling in 50 nm-, 100 nm-, and 500 nm-thick silicon nitride membranes with diameters fabricated to accommodate spherical silica nanoparticles with sizes chosen to mimic that of virus particles. In this manner, we are able to characterize the role of pore geometry in three key components of the detection scheme, namely, event magnitude, event duration, and event frequency. We find that the electric field created by the applied voltage and the pore's geometry is a critical factor. We develop approximations to describe this field, which are verified with computer simulations, and interactions between particles and this field. In so doing, we formulate what we believe to be the first approximation for the magnitude of ionic current blockage that explicitly addresses the invariance of access resistance of solid-state pores during particle translocation. These approximations also provide a suitable foundation for estimating the zeta potential of the particles and/or pore surface when studied in conjunction with event durations. We also verify that translocation achieved by electro-osmostic transport is an effective means of slowing translocation velocities of highly charged particles without compromising particle capture rate as compared to more traditional approaches based on electrophoretic transport.

Polystyrene particles reveal pore substructure as they translocate.

In this article, we report resistive-pulse sensing experiments with cylindrical track-etched PET pores, which reveal that the diameters of these pores fluctuate along their length. The resistive pulses generated by polymer spheres passing through these pores have a repeatable pattern of large variations corresponding to these diameter changes. We show that this pattern of variations enables the unambiguous resolution of multiple particles simultaneously in the pore, that it can detect transient sticking of particles within the pore, and that it can confirm whether any individual particle completely translocates the pore. We demonstrate that nonionic surfactant has a significant impact on particle velocity, with the velocity decreasing by an order of magnitude for a similar increase in surfactant concentration. We also show that these pores can differentiate by particle size and charge, and we explore the influence of electrophoresis, electroosmosis, and pore size on particle motion. These results have practical importance for increasing the speed of resistive-pulse sensing, optimizing the detection of specific analytes, and identifying particle shapes.

A hydrophobic entrance enhances ion current rectification and induces dewetting in asymmetric nanopores.

Hydrophobic interactions and local dewetting of hydrophobic cavities have been identified as a key mechanism for ionic gating in biological voltage-gated channels in a cell membrane. Hydrophobic interactions are responsible for rectification of the channels, i.e. the ability to transport ions more efficiently in one direction compared to the other. We designed single polymer nanopores with a hydrophobic gate on one side in the form of a single layer of C10 or C18 thiols. This nanoporous system behaves like an ionic diode whose direction of rectification is regulated by the pH of the electrolyte. In addition, reversible dewetting of the hydrophobic region of the pore was observed as voltage-dependent ion current fluctuations in time between conducting and non-conducting states. The observations are in accordance with earlier molecular dynamics simulations, which predicted the possibility of spontaneous and reversible dewetting of hydrophobic pores.

Ag nanotubes and Ag/AgCl electrodes in nanoporous membranes.

Miniaturization of the entire experimental setup is a key requirement for widespread application of nanodevices. For nanopore biosensing, integrating electrodes onto the nanopore membrane and controlling the pore length is important for reducing the complexity and improving the sensitivity of the system. Here we present a method to achieve these goals, which relies on electroless plating to produce Ag nanotubes in track-etched polymer nanopore templates. By plating from one side only, we create a conductive nanotube that does not span the full length of the pore, and thus can act as a nanoelectrode located inside the nanopore. To give optimal electrochemical behavior for sensing, we coat the Ag nanotube with a layer of AgCl. We characterize the behavior of this nanoelectrode by measuring its current-voltage response and find that, in most cases, the response is asymmetric. The plated nanopores have initial diameters between 100 and 300 nm, thus a range suitable for detection of viruses.

DNA translocation through graphene nanopores.

We report on DNA translocations through nanopores created in graphene membranes. Devices consist of 1-5 nm thick graphene membranes with electron-beam sculpted nanopores from 5 to 10 nm in diameter. Due to the thin nature of the graphene membranes, we observe larger blocked currents than for traditional solid-state nanopores. However, ionic current noise levels are several orders of magnitude larger than those for silicon nitride nanopores. These fluctuations are reduced with the atomic-layer deposition of 5 nm of titanium dioxide over the device. Unlike traditional solid-state nanopore materials that are insulating, graphene is an excellent electrical conductor. Use of graphene as a membrane material opens the door to a new class of nanopore devices in which electronic sensing and control are performed directly at the pore.

Versatile ultrathin nanoporous silicon nitride membranes.

Single- and multiple-nanopore membranes are both highly interesting for biosensing and separation processes, as well as their ability to mimic biological membranes. The density of pores, their shape, and their surface chemistry are the key factors that determine membrane transport and separation capabilities. Here, we report silicon nitride (SiN) membranes with fully controlled porosity, pore geometry, and pore surface chemistry. An ultrathin freestanding SiN platform is described with conical or double-conical nanopores of diameters as small as several nanometers, prepared by the track-etching technique. This technique allows the membrane porosity to be tuned from one to billions of pores per square centimeter. We demonstrate the separation capabilities of these membranes by discrimination of dye and protein molecules based on their charge and size. This separation process is based on an electrostatic mechanism and operates in physiological electrolyte conditions. As we have also shown, the separation capabilities can be tuned by chemically modifying the pore walls. Compared with typical membranes with cylindrical pores, the conical and double-conical pores reported here allow for higher fluxes, a critical advantage in separation applications. In addition, the conical pore shape results in a shorter effective length, which gives advantages for single biomolecule detection applications such as nanopore-based DNA analysis.

Solid-state nanopore technologies for nanopore-based DNA analysis.

Nanopore-based DNA analysis is a new single-molecule technique that involves monitoring the flow of ions through a narrow pore, and detecting changes in this flow as DNA molecules also pass through the pore. It has the potential to carry out a range of laboratory and medical DNA analyses, orders of magnitude faster than current methods. Initial experiments used a protein channel for its pre-defined, precise structure, but since then several approaches for the fabrication of solid-state pores have been developed. These aim to match the capabilities of biochannels, while also providing increased durability, control over pore geometry and compatibility with semiconductor and microfluidics fabrication techniques. This review summarizes each solid-state nanopore fabrication technique reported to date, and compares their advantages and disadvantages. Methods and applications for nanopore surface modification are also presented, followed by a discussion of approaches used to measure pore size, geometry and surface properties. The review concludes with an outlook on the future of solid-state nanopores.

Nanopore-based single-molecule DNA analysis.

Nanopore-based DNA analysis is a single-molecule technique with revolutionary potential. It promises to carry out a range of analyses, orders of magnitude faster than current methods, including length measurement, specific sequence detection, single-molecule dynamics and even de novo sequencing. The concept involves using an applied voltage to drive DNA molecules through a narrow pore that separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the pore, they block the flow of ions and, thus, their structure and length can be determined based on the degree and duration of the resulting current reductions. In this review, I explain the nanopore-based DNA analysis concept and briefly explore its historical foundations, before discussing and summarizing all experimental results reported to date. I conclude with a summary of the obstacles that must be overcome for it to realize its promised potential.