Rena Akahori

Fabricating nanopores with diameters of sub-1 nm to 3 nm using multilevel pulse-voltage injection

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.

Slowing single-stranded DNA translocation through a solid-state nanopore by decreasing the nanopore diameter

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.

A novel side-gated ultrathin-channel nanopore FET (SGNAFET) sensor for direct DNA sequencing

A novel side-gated ultrathin-channel nanopore FET (SGNAFET), for fast and label-free DNA sequencing with high resolution and sensitivity, is proposed. The goal of the SGNAFET is to identify the four types of nucleotides in DNA by changes in the channel current of the SGNAFET. Aiming to reach that goal, a SGNAFET with channel thickness (tch.) of 2 or 4 nm was successfully operated and could detect DNA translocations through its nanopore on the basis of changes in its channel current.

Discrimination of three types of homopolymers in single-stranded DNA with solid-state nanopores through external control of the DNA motion

To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane. We demonstrated that a single-stranded DNA could be inserted into and removed from a nanopore in our experimental system. The number of different ionic-current levels observed while DNA remained in the nanopore corresponded to the number of different types of homopolymers in the DNA.

Side-gated ultrathin-channel nanopore FET sensors

A side-gated, ultrathin-channel nanopore FET (SGNAFET) is proposed for fast and label-free DNA sequencing. The concept of the SGNAFET comprises the detection of changes in the channel current during DNA translocation through a nanopore and identifying the four types of nucleotides as a result of these changes. To achieve this goal, both p- and n-type SGNAFETs with a channel thicknesses of 2 or 4 nm were fabricated, and the stable transistor operation of both SGNAFETs in air, water, and a KCl buffer solution were confirmed. In addition, synchronized current changes were observed between the ionic current through the nanopore and the SGNAFETs drain current during DNA translocation through the nanopore.

Slowing the translocation of single-stranded DNA by using nano-cylindrical passage self-assembled by amphiphilic block copolymers

We report a novel approach to slow the translocation of single-stranded DNA (ssDNA) by employing polyethylene oxide (PEO) filled nano-cylindrical domains as transportation channels. DNA strands were demonstrated to electrophoretically translocate through PEO filled cylindrical domains with diameters of 2 and 9 nm, which were self-assembled by amphiphilic liquid crystalline block copolymers. The average translocation rate of ssDNA strands was effectively reduced to an order of 10 μs per nucleotide, which was 1-2 orders slower than that attained by utilizing conventional solid-state nanopore devices.

Two-step breakdown of a SiN membrane for nanopore fabrication: Formation of thin portion and penetration

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%.

Fabrication and analysis of SiN nanopores for direct DNA sequencing

This paper summarizes the latest developments regarding solid-state nanopores achieved in our laboratory. For direct DNA sequencing with solid-state nanopores, a simple method has been developed to precisely fabricate nanopores with diameters of 1 to 3 nm. By measuring DNA translocation events through the fabricated nanopores, it was confirmed that single-stranded DNA (ssDNA) can pass through nanopores with diameters as small as 1.2 nm. In addition, it was discovered that the translocation speed of ssDNA is higher than that for double-stranded DNA (dsDNA) by three or four orders of magnitude. This difference in speed was explained by a difference in the forces acting on the DNA. From molecular dynamics (MD) simulations, it was found that the force of solution-DNA interactions on dsDNA is approximately 10 times larger than that on ssDNA during DNA translocation through a nanopore.