Dwell Time Prolongation and Identification of Single Nucleotides Passing through a Solid-State Nanopore by Using Ammonium Sulfate Aqueous Solution.

The ionic current blockades when poly(dT)60 or dNTPs passed through SiN nanopores in an aqueous solution containing (NH4)2SO4 were investigated. The dwell time of poly(dT)60 in the nanopores in an aqueous solution containing (NH4)2SO4 was significantly longer compared to that in an aqueous solution that did not contain (NH4)2SO4. This dwell time prolongation effect due to the aqueous solution containing (NH4)2SO4 was also confirmed when dCTP passed through the nanopores. In addition, when the nanopores were fabricated via dielectric breakdown in the aqueous solution containing (NH4)2SO4, the dwell time prolongation effect for dCTP still occurred even after the aqueous solution was displaced with the aqueous solution without (NH4)2SO4. Furthermore, we measured the ionic current blockades when the four types of dNTPs passed through the same nanopore, and the four types of dNTPs could be statistically identified according to their current blockade values.

Descriptive, injunctive, or the synergy of both? Experimenting normative information on behavioral changes under the COVID-19 pandemic.

Backgrounds: The effectiveness of citizens' behavioral changes to prevent the spread of SARS-CoV-2, such as avoiding large social events, relies on science communication from policymakers and collective action among peer citizens. Extant studies recognize the potential effects of information stimuli on citizens' behavioral changes, including what epidemiological experts request (injunctive information) and what surrounding people behave (descriptive information). Yet, they have insufficiently assessed the co-occurrence and possible interaction of multiple information stimuli. Methods: 1,819 Japanese citizens aged 18 or over were recruited for an experimental survey during March 1-3, 2021 and asked their views on a hypothetical wedding attendance in Japan while being exposed to randomly assigned normative information stimuli. Their willingness to attend a wedding asked before and after the intervention was measured. Infection risk perception was also asked as a mediating variable. Results: Findings suggest the constant supremacy of descriptive information and no synergistic effects in the interaction of multiple information stimuli. We also report that the effects of injunctive and descriptive information vary according to participants' risk perception, age, and trust in experts. Conclusion: Our experimental test enables a systematic assessment of multiple normative information and confirms the primacy of descriptive information as the main driver of behavioral change. Communication by medical experts has limitations but is still effective in specific categories of the population.

Sub-10-nm-thick SiN nanopore membranes fabricated using the SiO2sacrificial layer process.

In our previous studies, ultrathin SiN membranes down to 3 nm in thickness were fabricated using the poly-Si sacrificial layer process, and nanopores were formed in those membranes. The region of the SiN membrane fabricated using this process was small, and the poly-Si sacrificial layer remained throughout the other region. On the other hand, to reduce the noise of the current through the nanopore, it is preferable to reduce the capacitance of the nanopore chip by replacing the poly-Si layer with an insulator with low permittivity, such as SiO2. Thus, in this study, the fabrication of SiN membranes with thicknesses of 3-7 nm using the SiO2sacrificial layer process was examined. SiN membranes with thicknesses of less than 5 nm could not be formed when the thickness of the top SiN layer deposited onto the sacrificial layer was 100 nm. In contrast, SiN membranes down to 3.07 nm in thickness could be formed when the top SiN layer was 40 nm in thickness. This is thought to be due to the difference in membrane stress. Nanopores were then fabricated in the membranes via dielectric breakdown. The current noise of the nanopore membranes was approximately 3/5 that of membranes fabricated using the poly-Si sacrificial layer process. Last, ionic current blockades were measured when poly(dT)60passed through the nanopores, and the effective thickness of the nanopores was estimated based on those current-blockade values. The effective thickness was approximately 4.8 nm when the deposited thickness of the SiN membrane was 6.03 nm. On the other hand, the effective thickness and the deposited thickness were almost the same when the deposited thickness was 3.07 nm. This suggests it became difficult to form a shape in which the thickness of the nanopore edge was thinner than the deposited membrane thickness as the deposited thickness decreased.

Low-frequency noise induced by cation exchange fluctuation on the wall of silicon nitride nanopore.

Nanopore-based biosensors have attracted attention as highly sensitive microscopes for detecting single molecules in aqueous solutions. However, the ionic current noise through a nanopore degrades the measurement accuracy. In this study, the magnitude of the low-frequency noise in the ionic current through a silicon nitride nanopore was found to change depending on the metal ion species in the aqueous solution. The order of the low-frequency noise magnitudes of the alkali metal ionic current was consistent with the order of the adsorption affinities of the metal ions for the silanol surface of the nanopore (Li

Solid-state nanopores towards single-molecule DNA sequencing.

Nanopore DNA sequencing offers a new paradigm owing to its extensive potential for long-read, high-throughput detection of nucleotide modification and direct RNA sequencing. Given the remarkable advances in protein nanopore sequencing technology, there is still a strong enthusiasm in exploring alternative nanopore-sequencing techniques, particularly those based on a solid-state nanopore using a semiconductor material. Since solid-state nanopores provide superior material robustness and large-scale integrability with on-chip electronics, they have the potential to surpass the limitations of their biological counterparts. However, there are key technical challenges to be addressed: the creation of an ultrasmall nanopore, fabrication of an ultrathin membrane, control of the ultrafast DNA speed and detection of four nucleotides. Extensive research efforts have been devoted to resolving these issues over the past two decades. In this review, we briefly introduce recent updates regarding solid-state nanopore technologies towards DNA sequencing. It can be envisioned that emerging technologies will offer a brand new future in DNA-sequencing technology.

Stable fabrication of a large nanopore by controlled dielectric breakdown in a high-pH solution for the detection of various-sized molecules.

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.

Silicon nitride nanopore created by dielectric breakdown with a divalent cation: deceleration of translocation speed and identification of single nucleotides.

Nanopore DNA sequencing with a solid-state nanopore requires deceleration of the ultrafast translocation speed of single-stranded DNA (ssDNA). We report an unexpected phenomenon: controlled dielectric breakdown (CBD) with a divalent metal cation, especially Ca2+, provides a silicon nitride nanopore with the ability to decelerate ssDNA speed to 100 µs per base even after solution replacement. This speed is two orders of magnitude slower than that for CBD with a conventional monovalent metal cation. Temperature dependence experiments revealed that the enthalpic barrier for a nanopore created via CBD with Ca2+ is 25-30kBT, comparable to that of a biological nanopore. The slowing effect originates from the strong interaction between ssDNA and divalent cations, which were coated on the sidewall of the nanopore during the CBD process. In addition, we found that the nanopore created via CBD with Ca2+ can decelerate the speed of even single-nucleotide monomers, dNMPs, to 0.1-10 ms per base. The four single nucleotides could be statistically identified according to their blockade currents. Our approach is simple and practical because it simultaneously allows nanopore fabrication, ssDNA deceleration and the identification of nucleotide monomers.

Challenges of Single-Molecule DNA Sequencing with Solid-State Nanopores.

A powerful DNA sequencing tool with high accuracy, long read length and high-throughput would be required more and more for decoding the complicated genetic code. Solid-state nanopore has attracted many researchers for its promising future as a next-generation DNA sequencing platform due to the processability, the robustness and the large-scale integratability. While the diverse materials have been widely explored for a solid-state nanopore, silicon nitride (Si3N4) is especially preferable from the viewpoint of mass production based on semiconductor fabrication process. Here, as a nanopore sensing mechanism, we focused on the ionic blockade current method which is the most developed technique. We also highlight the main challenges of Si3N4 nanopore-based DNA sequencer that should be addressed: the fabrication of ultra-small nanopore and ultra-thin membrane, the modulation of DNA translocation speed and the detection of base-specific signals. In this chapter, we discuss the recent progress relating to solid-state nanopore DNA sequencing, which helps to provide a comprehensive information about the current technical situation.

Identification of four single-stranded DNA homopolymers with a solid-state nanopore in alkaline CsCl solution.

DNA sequencing via solid-state nanopores is a promising technique with the potential to surpass the performance of conventional sequencers. However, the identification of all four nucleotide homopolymers with a typical SiN nanopore is yet to be clearly demonstrated because a guanine homopolymer rapidly forms a G-quadruplex in a typical KCl aqueous solution. To address this issue, we introduced an alkaline CsCl aqueous solution, which denatures the G-quadruplex into a single-stranded structure by disrupting the hydrogen-bonding network between the guanines and preventing the binding of the K+ ion to G-quartets. Using this alkaline CsCl solution, we provided a proof-of-principle that single-stranded DNA homopolymers of all four nucleotides could be statistically identified according to their blockade currents with the same single nanopore. We also confirmed that a triblock DNA copolymer of three nucleotides exhibited a trimodal Gaussian distribution whose peaks correspond to those of the DNA homopolymers. Our findings contribute to the development of practical DNA sequencing with a solid-state nanopore.

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%.

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.

Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction.

The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 µs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.

Multichannel detection of ionic currents through two nanopores fabricated on integrated Si3N4 membranes.

Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip.

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 SGNAFET's drain current during DNA translocation through the nanopore.

Prevention of Dielectric Breakdown of Nanopore Membranes by Charge Neutralization.

To achieve DNA sequencing using a solid-state nanopore, it is necessary to reduce the electric noise current. The noise current can be decreased by reducing the capacitance (C) of the nanopore device. However, we found that an electric-charge difference (ΔQ) between the electrolyte in one chamber and the electrolyte in another chamber occurred. For low capacitance devices, this electric-charge imbalance can lead to unexpectedly high voltage (ΔV = ΔQ/C) which disrupted the membrane when the two electrolytes were independently poured into the chambers. We elucidated the mechanism for the generation of initial defects and established new procedures for preventing the generation of defects by connecting an electric bypass between the chambers.

Deceleration of single-stranded DNA passing through a nanopore using a nanometre-sized bead structure.

DNA sequencing with a solid-state nanopore requires a reduction of the translocation speeds of single-stranded DNA (ssDNA) over 10 µs/base. In this study, we report that a nanometre-sized bead structure constructed around a nanopore can reduce the moving speed of ssDNA to 270 µs/base by adjusting the diameter of the bead and its surface chemical group. This decelerating effect originates from the strong interaction between ssDNA and the chemical group on the surface of the bead. This nanostructure was simply prepared by dip coating in which a substrate with a nanopore was immersed in a silica bead solution and then dried in an oven. As compared with conventional approaches, our novel method is less laborious, simpler to perform and more effective in reducing ssDNA translocation speed.

Fabrication of 3-nm-thick Si3N4 membranes for solid-state nanopores using the poly-Si sacrificial layer process.

To improve the spatial resolution of solid-state nanopores, thinning the membrane is a very important issue. The most commonly used membrane material for solid-state nanopores is silicon nitride (Si3N4). However, until now, stable wafer-scale fabrication of Si3N4 membranes with a thickness of less than 5 nm has not been reported, although a further reduction in thickness is desired to improve spatial resolution. In the present study, to fabricate thinner Si3N4 membranes with a thickness of less than 5 nm in a wafer, a new fabrication process that employs a polycrystalline-Si (poly-Si) sacrificial layer was developed. This process enables the stable fabrication of Si3N4 membranes with thicknesses of 3 nm. Nanopores were fabricated in the membrane using a transmission electron microscope (TEM) beam. Based on the relationship between the ionic current through the nanopores and their diameter, the effective thickness of the nanopores was estimated to range from 0.6 to 2.2 nm. Moreover, DNA translocation through the nanopores was observed.

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.

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.