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.

We demonstrate the automated and reproducible fabrication of sub-2-nm nanopores in 10-nm thick silicon nitride membranes, through controlled dielectric breakdown in solution. Our results reveal that under the appropriate conditions, nanopores can be fabricated with a size no larger than 2.0 ± 0.5-nm in diameter for a sample of N = 23 nanopores, with an average and standard deviation of 1.3 ± 0.6-nm. The dimensions of these nanopores are confirmed by using individual translocating DNA molecules as molecular rulers. We show that a 2.0-nm and a 2.1-nm diameter nanopore are capable of distinguishing single-stranded DNA versus double-stranded DNA, and that a 2.4-nm diameter nanopore can be used to investigate the overstretching transition in short dsDNA fragments. These results highlight the reliability and precision of the automated fabrication of nanopores via controlled dielectric breakdown, showing great promise for the manufacturing of future nanopore-based technologies.

Nanopore fabrication by controlled breakdown (CBD) overcomes many of the challenges of traditional nanofabrication techniques, by reliably forming solid-state nanopores sub-2 nm in size in a low-cost and scalable way for nucleic acid analysis applications. Herein, the breakdown kinetics of thin dielectric membranes immersed in a liquid environment are investigated in order to gain deeper insights into the mechanism of solid-state nanopore formation by high electric fields. For various fabrication conditions, we demonstrate that nanopore fabrication time is Weibull-distributed, in support of the hypothesis that the fabrication mechanism is a stochastic process governed by the probability of forming a connected path across the membrane (i.e. a weakest-link problem). Additionally, we explore the roles that various ions and solvents play in breakdown kinetics, revealing that asymmetric pH conditions across the membrane can significantly affect nanopore fabrication time for a given voltage polarity. These results, characterizing the stochasticity of the nanopore fabrication process and highlighting the parameters affecting it, should assist researchers interested in exploiting the potential of CBD for nanofluidic channel fabrication, while also offering guidance towards the conceivable manufacturing of solid-state nanopore-based technologies for DNA sequencing applications.

We present a methodology for preparing silicon nitride nanopores that provides in situ control of size with sub-nanometer precision while simultaneously reducing electrical noise by up to three orders of magnitude through the cyclic application of high electric fields in an aqueous environment. Over 90% of nanopores treated with this technique display desirable noise characteristics and readily exhibit translocation of double-stranded DNA molecules. Furthermore, previously used nanopores with degraded electrical properties can be rejuvenated and used for further single-molecule experiments.

We demonstrate the ability to slow DNA translocations through solid‐state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores—presently one of the largest hurdles facing nanopore‐based analysis—without affecting the signal quality or capture efficiency.