To date, solid-state nanopores have been fabricated primarily through a focused-electronic beam via TEM. For mass production, however, a TEM beam is not suitable and an alternative fabrication method is required. Recently, a simple method for fabricating solid-state nanopores was reported by Kwok, H. et al. and used to fabricate a nanopore (down to 2 nm in size) in a membrane via dielectric breakdown. In the present study, to fabricate smaller nanopores stably—specifically with a diameter of 1 to 2 nm (which is an essential size for identifying each nucleotide)—via dielectric breakdown, a technique called “multilevel pulse-voltage injection” (MPVI) is proposed and evaluated. MPVI can generate nanopores with diameters of sub-1 nm in a 10-nm-thick Si3N4 membrane with a probability of 90%. The generated nanopores can be widened to the desired size (as high as 3 nm in diameter) with sub-nanometre precision, and the mean effective thickness of the fabricated nanopores was 3.7 nm.

4(5)-(2-mercaptoethyl)-1H-imidazole-2-carboxamide is a molecule that has multiple hydrogen bonding sites and a short flexible linker. When tethered to a pair of electrodes, it traps target molecules in a tunnel junction. Surprisingly large recognition-tunneling signals are generated for all naturally occurring DNA bases A, C, G,T, and 5-methyl-Cytosine. Tunnel current spikes are stochastic and broadly distributed, but characteristic enough so that individual bases can be identified as a tunneling probe is scanned over DNA oligomers. Each base yields a recognizable burst of signal, the duration of which is controlled entirely by the probe speed, down to speeds of 1 nm/s, implying a maximum off-rate of 3 s-1 for the recognition complex. The same measurements yield a lower bound on the on-rate of ~1 M-1s-1. Despite the stochastic nature of the signals, an optimized multi-parameter fit allows base-calling from a single signal peak with an accuracy that can exceed 80% when a single type of nucleotide is present in the junction, meaning that recognition-tunneling is capable of true single-molecule analysis. The accuracy increases to 95% when multiple spikes in a signal cluster are analyzed.

To slow the translocation of single-stranded DNA (ssDNA) through a solid-state nanopore, a nanopore was narrowed, and the effect of the narrowing on the DNA translocation speed was investigated. In order to accurately measure the speed, long (5.3 kb) ssDNA (namely, ss-poly(dA)) with uniform length (±0.4 kb) was synthesized. The diameters of nanopores fabricated by a transmission electron microscope were controlled by atomic-layer deposition. Reducing the nanopore diameter from 4.5 to 2.3 nm slowed down the translocation of ssDNA by more than 16 times (to 0.18 μs base(-1)) when 300 mV was applied across the nanopore. It is speculated that the interaction between the nanopore and the ssDNA dominates the translocation speed. Unexpectedly, the translocation speed of ssDNA through the 4.5 nm nanopore is more than two orders of magnitude higher than that of double-stranded DNA (dsDNA) through a nanopore of almost the same size. The cause of such a faster translocation of ssDNA can be explained by the weaker drag force inside the nanopore. Moreover, the measured translocation speeds of ssDNA and dsDNA agree well with those calculated by molecular-dynamics (MD) simulation. The MD simulation predicted that reducing the nanopore diameter to almost the same as that of ssDNA (i.e. 1.4 nm) decreases the translocation speed (to 1.4 μs base(-1)). Narrowing the nanopore is thus an effective approach for accomplishing nanopore DNA sequencing.

For nanopore sensing of various-sized molecules with high sensitivity, the size of the nanopore should be adjusted according to the size of each target molecule. For solid-state nanopores, a simple and inexpensive nanopore fabrication method utilizing dielectric breakdown of a membrane is widely used. This method is suitable for fabricating a small nanopore. However, it suffers two serious problems when attempting to fabricate a large nanopore: the generation of multiple nanopores and the non-opening failure of a nanopore. In this study, we found that nanopore fabrication by dielectric breakdown of a SiN membrane under high-pH conditions (pH ≥ 11.3) could overcome these two problems and enabled the formation of a single large nanopore up to 40 nm in diameter within one minute. Moreover, the ionic-current blockades derived from streptavidin-labelled and non-labelled DNA passing through the fabricated nanopore were clearly distinguished. The current blockades caused by streptavidin-labelled DNA could be identified even when its concentration is 1% of the total DNA.

For the nanopore sensing of various large molecules, such as probe-labelled DNA and antigen-antibody complexes, the nanopore size has to be customized for each target molecule. The recently developed nanopore fabrication method utilizing dielectric breakdown of a membrane is simple and quite inexpensive, but it is somewhat unsuitable for the stable fabrication of a single large nanopore due to the risk of generating multiple nanopores. To overcome this bottleneck, we propose a new technique called “two-step breakdown” (TSB). In the first step of TSB, a local conductive thin portion (not a nanopore) is formed in the membrane by dielectric breakdown. In the second step, the created thin portion is penetrated by voltage pulses whose polarity is opposite to the polarity of the voltage used in the first step. By applying TSB to a 20-nm-thick SiN membrane, a single nanopore with a diameter of 21–26 nm could be fabricated with a high yield of 83%.