JP2013090576A - Nucleic acid analyzing device and nucleic acid analyzer using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid analyzing device capable of highly accurately reading a base sequence of a nucleic acid with high discrimination ability in nucleic acid analysis by a tunnel current-type solid nanopore, and to provide a nucleic acid analyzer using the same.SOLUTION: The nucleic acid analyzing device for analyzing a nucleic acid in a sample by current measurement is characterized in that the discrimination ability to at least one among four bases constituting the nucleic acid by chemically modifying a nucleic acid base molecule or an organic compound or the like on an electrode surface of the tunnel current-type solid nanopore.

Description

  The present invention relates to a device and an analysis apparatus for performing sequence analysis of nucleic acids such as DNA and RNA using a thin film provided with nano-sized pores. In particular, the present invention relates to a nucleic acid analysis device using a nanopore having an electrode with a chemically modified molecule in the pore and an analysis apparatus using the same.

  A method using nanopores is attracting attention as an approach for realizing next-generation DNA sequencers. It is considered that the nanopore system has a great feature that analysis can be performed without labeling DNA, in other words, without using reagents such as enzymes and fluorescent dyes. Nanopores are roughly classified into two types according to the formation method. One is a so-called bio-nanopore in which a channel protein that forms nano-sized pores (hereinafter referred to as “nanopores”) is embedded in a bimolecular film, and the other is a so-called nanopore formed by subjecting a semiconductor material to microfabrication. Solid nanopore.

  In addition, two types of methods for analyzing DNA using such nanopores are currently proposed. The first method is a blocking current method. That is, when a liquid tank is provided on both sides of a thin film on which nanopores are formed, an electrolyte solution and an electrode are provided in each liquid tank, and a voltage is applied between the electrodes, an ionic current flows through the nanopore. The magnitude of the ion current is proportional to the cross-sectional area of the nanopore as a first order approximation. When DNA passes through the nanopore, the DNA blocks the nanopore and the effective cross-sectional area decreases, so that the ionic current decreases. This amount of decrease is called the blocking current. Based on the magnitude of the blocking current, the difference between the single strand and double strand of DNA can be identified. In one form of the biopore, it has been reported that the type of DNA base can be discriminated from the magnitude of the blocking current (Non-patent Document 1).

  However, the bimolecular film used for the biopore is a delicate thin film formed by gathering small organic molecules with a weak force, and it is considered that there is a problem in mechanical stability. In addition, since the process of incorporating the channel protein into the bilayer membrane depends on natural phenomena, there is a problem in the control and reproducibility of the number of channels. On the other hand, since solid nanopores use a semiconductor substrate or the like as a thin film, it is considered advantageous in terms of structural stability over biopores. In addition, since nanopores are formed mechanically, there is an advantage that industrial production is possible. In addition to a semiconductor substrate as a thin film material, there has also been reported an apparatus and method for analyzing biomolecules passing through solid nanopores provided on graphene (Patent Document 1). However, in solid nanopores, four types of base identification of DNA strands by blocking current have not been reported.

  The second method is a tunnel current method. That is, a method of measuring the tunnel current between the DNA passing through the nanopore and the electrode by applying a voltage between the electrodes facing the nanopore, and analyzing the DNA from the magnitude of the tunnel current Has been proposed (Patent Document 2). Although theoretical studies have reported that base identification can be made statistically based on the magnitude of the tunnel current (Non-Patent Document 2), there is no report that experimentally identified four types of bases in a DNA strand.

  As described above, the nanopore method is expected as the next-generation DNA sequencer, and from the viewpoint of industrial productivity, solid nanopores that can be formed by microfabrication of semiconductor materials are promising. In any of the methods, four types of base identification are not realized.

  As a related technique of the tunnel current method, there is a report that the discrimination ability for a plurality of kinds of DNA nucleosides is improved by modifying a 4-mercaptobenzoic acid on both the probe tip and the substrate surface using a scanning probe microscope (non-patent document). Reference 3). It has also been reported that the ability to discriminate complementary nucleobases is improved by modifying the nucleobase molecule at the probe tip of a scanning probe microscope (Non-patent Document 4). In the case of DNA, adenine (hereinafter referred to as A), thymine (hereinafter referred to as T), cytosine (hereinafter referred to as C), and guanine (hereinafter referred to as G) are complementary nucleobases.

International Publication No. WO2009 / 035647 US Patent US6627067

Clarke, J .; et al. Nat. Nanotech. (2009) Volume 4, pp. 265-270 Lagerqvist, J.A. et al. Nano Lett. (2006) Volume 6 Pages 779-782 Chang, S. et al. Nano Lett. (2010) Volume 10 Pages 1070-1075 Ohshiro, T. et al. , PNAS (2006) Volume 103 Pages 10-14

  Although it has been suggested that a method of analyzing a nucleic acid base by a tunnel current method using a solid nanopore can realize base discrimination (Non-patent Documents 2 and 3), a conventional method (Patent Document 2) ) Has a low base discrimination ability and it is difficult to read the base sequence of the nucleic acid with high accuracy.

  Accordingly, an object of the present invention is to provide a nucleic acid analysis device capable of reading a nucleic acid base sequence with high discrimination ability with high accuracy in a method for analyzing a nucleic acid base by a tunnel current method using solid nanopores, and a nucleic acid using the same. It is to provide an analysis device.

  At the same time, this analysis method allows analysis without using reagents such as enzymes and fluorescent dyes, enabling analysis at low cost, and further increases the readout base length that is limited to reagents, resulting in higher throughput than before. Is to provide a simple analysis technique.

  In order to solve the above problems, the main features of the nucleic acid analysis device of the present invention are as follows.

  (1) A nucleic acid analysis device that analyzes nucleic acid in a sample by current measurement, and is disposed on the solid substrate, the nanopore that passes through the solid substrate and through which the sample passes, and is disposed on the solid substrate. In order to improve the discrimination ability for at least one of the four types of bases constituting the nucleic acid on one end surface of the at least one electrode facing the nanopore. The nucleic acid is analyzed by applying a voltage between at least two electrodes and passing a current through the base constituting the nucleic acid in the sample while the sample is applied to the nanopore while the sample is applied to the nanopore. It is characterized by.

  The main features of the nucleic acid analyzer of the present invention are as follows.

  (2) A first solution that has the nucleic acid analysis device according to (1) and includes an introduction port for introducing a sample solution containing a nucleic acid to be analyzed and a discharge port for discharging the sample solution. A nucleic acid analyzing device disposed between a first solution tank and a second solution tank; a second solution tank having a tank, an inlet for introducing the solution, and a discharge port for discharging the solution; A nucleic acid driving means for moving the nucleic acid from the first solution tank to the second solution tank through the nanopore, and a first voltage source for applying a voltage between the first solution tank and the second solution tank; A first ammeter for detecting a current flowing between the first solution tank and the second solution tank via the nanopore, and a second voltage source for applying a voltage between electrodes provided in the nucleic acid analysis device; A second ammeter for detecting the current flowing between the electrodes provided in the nucleic acid analysis device, and at least Also includes a control means for controlling the nucleic acid driving means, and a data processing means for acquiring and processing data detected by at least the first and second ammeters, and entered the nanopore using the second voltage source. The present invention is characterized in that a nucleic acid in a sample solution is analyzed by passing a current through a base constituting the nucleic acid in the sample solution and measuring the current using a second ammeter.

  That is, the present invention is characterized in that the ability to discriminate a specific or a plurality of nucleobases is improved by chemically modifying a nucleobase molecule, an organic compound, or the like on the electrode surface of the tunnel current type solid nanopore.

  The present invention provides a nucleic acid analysis device having a high base discrimination ability and capable of reading a base sequence with high accuracy and a nucleic acid analysis apparatus using the same in a nucleobase analysis method by a tunnel current method using solid nanopores it can.

  In addition, since analysis can be performed without using reagents such as enzymes and fluorescent dyes, analysis can be performed at low cost, and further, the readout base length limited by the reagents becomes longer, so that it is possible to provide analysis technology with higher throughput than before. .

It is the schematic of the structure of the tunnel current system solid nanopore which has two electrodes facing according to a 1st Example. It is the schematic of the structure (in the case of chemical modification molecule | numerator provision) of the tunnel current system solid nanopore which has two electrodes which concern on the 1st Example. It is the schematic of the structure of the tunnel current system solid nanopore which has 3-6 electrodes with a chemical modification molecule | numerator on the same plane concerning the 2nd Example. It is the tunnel current system solid nanopore which has two electrodes which concern on 3rd Example, and is the schematic of the structure which provided the chemical modification molecule | numerator only to one electrode. FIG. 6 is a schematic view of a structure according to a fourth embodiment, which is a tunnel current type solid nanopore having three electrodes on the same plane, to which chemically modified molecules are added except for one electrode. It is the schematic of the structure which is a tunnel current system solid nanopore which has four electrodes on the same plane concerning the 4th example, and gave the chemical modification molecule except for one electrode. It is the schematic of the structure which is a tunnel current system solid nanopore which has five electrodes on the same plane concerning the 4th example, and gave the chemical modification molecule except for one electrode. It is the schematic of the structure which is a tunnel current system solid nanopore which has six electrodes on the same plane concerning the 4th example, and gave the chemical modification molecule except for one electrode. FIG. 10 is a schematic diagram of a structure of a tunnel current type solid nanopore having eight electrodes with chemically modified molecules according to the fifth embodiment, in which the number of coplanar electrodes is two and the number of electrode layers is four. . It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator protruded from the opening concerning the 6th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is each process sectional drawing explaining an example of the manufacturing method of the tunnel current system solid nanopore of the structure which the chemical modification molecule | numerator does not protrude from an opening concerning the 7th Example. It is the schematic of one structure of the nucleic acid analysis apparatus using the nucleic acid analysis device by the tunnel current system solid nanopore which has two electrodes concerning an 8th Example. It is a graph of the time change of the tunnel current value measured when a DNA strand passes a nanopore concerning the 8th example. It is the schematic of one structure of the nucleic acid analyzer which used the nucleic acid analysis device by the tunnel current system solid nanopore which has four electrode pairs concerning a 9th Example. It is a graph of the time change of four tunnel electric current values measured when a DNA strand passes through the nanopore which has four electrode pairs concerning a 9th Example. It is the schematic explaining the example which comprised one electrode as a common electrode and comprised four electrode pairs in the nucleic acid analysis device by the tunnel current system solid nanopore which has five electrodes concerning a 10th Example.

First, embodiments will be described below.
The present invention relates to a device for analyzing a nucleobase using a tunneling current solid nanopore and an apparatus using the device. Therefore, the nucleic acid analysis device according to the present invention includes a solid substrate, at least one pore provided on the solid substrate, and at least two electrodes disposed on the solid substrate.

  The solid substrate can be formed of an electrical insulator material, for example, an inorganic material and an organic material (including a polymer material). Examples of the electrical insulator material include silicon, glass, quartz, polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polystyrene, and polypropylene. The size and thickness of the solid substrate are not particularly limited as long as nanopores can be provided. The solid substrate can be produced by a method known in the art, or can be obtained as a commercial product. For example, the solid substrate can be fabricated using techniques such as lithography and etching, laser ablation, injection molding, casting, molecular beam epitaxy, vapor deposition, chemical vapor deposition (CVD), electron beam or focused ion beam. The solid substrate may be coated to avoid adsorption of other molecules to the surface.

The solid substrate preferably has a thin film portion for providing nanopores. That is, a nanopore can be easily and efficiently formed on a solid substrate by providing a thin film portion having a material and thickness suitable for forming the nanopore on the solid substrate. Such a thin film portion may be the same material as the solid substrate or may be formed from another electrical insulator material. From the viewpoint of nanopore formation, the material of the thin film portion is preferably silicon, silicon oxide (SiO ₂ ), silicon nitride (SiN), silicon oxynitride (SiON), metal oxide, metal silicate, or the like. Further, the thin film portion may be a single layer or multiple layers. In the case of multiple layers, an electrode described later may be disposed between the layers of the thin film portion. The thickness of the thin film portion of the solid substrate is 5 nm to 200 nm, preferably 5 nm to 100 nm, more preferably 5 nm to 50 nm. The thin film portion can be formed on the solid substrate by techniques known in the art, for example, by low pressure chemical vapor deposition (LPCVD).

  The solid substrate is provided with at least one nanopore. In the present invention, the “nanopore” is a nanometer (nm) size hole, and is a hole penetrating the front and back of the thin film portion of the solid substrate, preferably the solid substrate.

Further, in the present invention, the “opening” is a through-hole opened in the solid substrate or the solid substrate thin film portion for forming the nanopore, and the portions exposed to the substrate surface side and the back surface side of the hole Shall point to. The shape of the opening has a contour shape formed by intersecting the side wall of the nanopore with the front surface or the back surface of the solid substrate or the solid substrate thin film portion.
During nucleic acid characterization, nucleobases, ions, etc. in the sample solution enter the nanopore from one opening and exit the nanopore from the same or opposite opening.

  The size of the nanopore (that is, the size of the opening. However, when an electrode having a chemically modified molecule is used as described later, the size of the effective nanopore may be different) depends on the type of analysis target. An appropriate size can be selected. The diameter of ssDNA (single-stranded DNA) is about 1.5 nm, and an appropriate range of the nanopore diameter for analyzing ssDNA is about 1.5 nm to 10 nm, preferably about 1.5 nm to 2.5 nm. The diameter of dsDNA (double-stranded DNA) is 2.6 nm, and an appropriate range of the diameter of the nanopore for analyzing dsDNA is about 3 nm to 10 nm, preferably about 3 nm to 5 nm. The depth of the nanopore can be changed by adjusting the thickness of the solid substrate or the thin film portion of the solid substrate.

  The depth of the nanopore is 2 times or more, preferably 3 times or more, more preferably 5 times or more the monomer unit constituting the nucleobase. For example, when the size is 3 or more nucleobases, the size is about 1 nm or more. Thereby, it is possible to enter the nanopore while controlling the shape and moving speed of the nucleobase, and it is possible to perform highly sensitive and highly accurate analysis.

  The shape of the nanopore is basically a circle, but it may be an ellipse or a polygon.

  At least one nanopore can be provided on the solid substrate, and when a plurality of nanopores are provided, it is preferable to arrange them regularly. Nanopores can be formed on a solid substrate by a known method in the art, for example, by irradiating an electron beam or an ion beam and using a lithography technique.

  At least two electrodes are disposed on the solid substrate. Examples of materials include gold, silver, platinum, palladium, rhodium, ruthenium, copper, iron, aluminum, titanium, nickel, tantalum, tungsten, cobalt, chromium, molybdenum, and niobium, and titanium nitride, tantalum nitride, and nitride. Metal nitrides such as tungsten, niobium nitride and chromium nitride, silicides such as tungsten silicide, titanium silicide, cobalt silicide and nickel silicide, carbon materials such as graphite, graphene and carbon nanotubes, semiconductors such as silicon and germanium, and mixtures of the above Etc.

  It is desirable that the electrode is provided facing the nanopore, has an acute-angle end, and the end faces the nanopore. The angle of the end is 10 to 80 degrees, preferably 10 to 45 degrees. Note that the apex portion of the electrode end portion is not necessarily a point in a strict sense, and is preferably a rounded shape having a radius of curvature of about 1.5 nm to 5 nm or less, more preferably about 1.5 nm to 2.5 nm. It may be. The shape and size of the electrodes other than the acute-angle end portions are not particularly limited, and can be appropriately selected according to the size of the solid substrate and nanopore used, the power source used, and the like.

  The thickness of the electrode at the end of the acute angle is 0.1 nm to 10 nm, preferably 0.1 nm to 5 nm, depending on the material used. More preferably, the thickness is 0.1 nm to 2 nm. The smaller the thickness of the electrode at the edge of the acute angle, the more the space through which the tunnel current is conducted can be limited, and the analysis with high resolution becomes possible. The thickness of the electrode other than the acute-angle end is not particularly limited, and can be appropriately selected according to the resistivity of the electrode material.

  It is preferable that the end part of the electrode is arranged facing the nanopore. More specifically, the electrode is disposed in a plane orthogonal to the nanopore central axis and the end of the thin film faces the nanopore. The end of the electrode may be disposed on the surface of the solid substrate or the thin film portion, or may be disposed inside the solid substrate or the thin film portion as long as it is provided facing the nanopore. For example, a solid substrate or a thin film portion may be an electrical insulator, an electrode may be disposed therein, and a cross section may be a laminated structure of an insulator-electrode-insulator, or an insulator-electrode-insulator- -It is good also as a structure which laminated | stacked the layer of-electrode-insulator-and an electrode.

  Two or more electrodes are arranged for one nanopore, and the greater the number of electrodes, the better the discrimination ability against nucleobases. At least one of the two or more electrodes arranged is provided with a chemically modified molecule. When a plurality of electrodes to which chemically modified molecules are applied are used, all of them may be the same chemically modified molecule, or all may be different chemically modified molecules. The number of chemically modified molecule-attached electrodes and the number of types of chemically modified molecules are appropriately determined so as to obtain desired sensitivity.

  The electrode can be produced by a method known in the art, and can be disposed on the surface or inside of a solid substrate or a thin film portion. For example, when a titanium electrode is disposed inside a thin film portion, a titanium film having a desired thickness is deposited on the thin film by sputtering, and then the titanium film is patterned into a desired shape by lithography and dry etching. This can be achieved by depositing material.

  Chemically modified molecules are added to the electrode ends to enhance the discrimination ability for a specific or multiple nucleobases. The thickness of the chemically modified molecule is 2 nm or less, preferably 1 nm or less, more preferably 0.5 nm or less. For example, an electrode modified with T is used to improve discrimination ability for A, an electrode modified with G is used for C, an electrode modified with A is used for T, and An electrode modified with C is used, and an electrode modified with A is used for uracil. As the chemically modified molecule other than the nucleic acid molecule, for example, an organic compound such as 4-mercaptobenzoic acid may be used. Chemical modification molecules of nucleic acid molecules and organic compounds to the electrodes are performed using the electrical properties and chemical properties of the electrode material and the nucleic acid molecules and organic compounds. For example, by using a thiol derivative of a nucleobase, a nucleic acid molecule can be chemically modified on a gold electrode. The chemical modification of the organic compound is desirably performed by a process for forming a self-assembled monolayer. When applying different chemically modified molecules to multiple electrodes, the selectivity of the electrode to be chemically modified is changed by applying a voltage to the electrode, the difference in surface condition, etc., or unnecessary chemical modifying molecules are added. This is done by passing an electric current through the electrode to remove unnecessary chemically modified molecules.

  When chemically modified molecules are applied to the electrode ends of a conventional tunneling current type solid nanopore, the effective inter-electrode distance, effective nanopore diameter and effective nanopore area decrease, and DNA cannot pass through. There is a fear. Although details are also shown in Example 1, for example, a chemically modified molecule having a thickness of 1 nm at the tip of one electrode of a circular nanopore for single-stranded analysis having two opposite electrodes and a nanopore diameter and an interelectrode distance equal to 2 nm Is given, the distance between the tip of the chemically modified portion and the tip of the electrode that does not have a chemically modified molecule is 1 nm, so there is a high possibility that single-stranded DNA cannot pass through. Moreover, even if it can pass, if it passes the position shifted from between electrodes, a sensitivity will fall, and it is unpreferable.

  Therefore, the size and shape of nanopores before chemical modification, the distance between electrodes, the thin film portion and the electrode structure are adjusted so that the effective nanopore size after chemical modification is appropriate for the type of analysis target. To do. Specifically, for example, the diameter of the nanopore and the distance between the electrodes are increased by the thickness of the chemically modified molecule. In the case of a tunneling current type solid nanopore having a conventional structure, the edge of the electrode and the edge of the opening substantially coincide with each other, so that the chemically modified molecules at the edge of the electrode protrude from the edge of the opening by the thickness. When the mechanical and chemical strength of the chemically modified part is low, there is a possibility that deterioration of the function or peeling of the chemically modified molecules may occur due to the sample or sample solution entering the nanopore colliding with the chemically modified part. As a means for avoiding this, for example, the nanopore diameter is set to an appropriate size according to the type of analysis target, and only the electrode is attached to the chemically modified molecule so that the chemically modified molecule can be provided between the electrode tip and the edge of the opening. It is possible to recede by the thickness.

  The analyzer of the present invention preferably includes a mechanism for controlling the moving speed of the nucleic acid, that is, a sample moving mechanism. The sample moving mechanism, for example, allows the monomer in the nucleic acid to enter the nanopore of the analytical device one unit at a time in synchronization with the measurement of the current between the electrodes. As such a mechanism, for example, a sample driving device (arbitrary function generator, electrode, etc.) that drives a nucleic acid by electrophoresis can be used. By such a sample movement mechanism, the movement of the nucleic acid can be controlled so that the monomers in the nucleic acid sequentially enter and leave the nanopore, and the tunnel current corresponding to the monomer can be acquired over time. .

  As a method for controlling the moving speed of nucleic acid, there is a method for increasing the viscosity of a sample solution containing nucleic acid. For example, by controlling the temperature around the analysis device to lower the temperature of the sample solution and increase the viscosity of the sample solution, the Brownian motion of the nucleic acid can be suppressed. Alternatively, by adding a biopolymer other than the target to be measured to the sample solution, the viscosity of the sample solution can be increased, and at the same time the three-dimensional structure of the nucleic acid can be made linear. It becomes possible to do. At this time, as the biopolymer other than the measurement target, preferably a polymer having a size larger than the inner diameter of the nanopore, more preferably a three-dimensionally cross-linked polymer is used, so that the biopolymer other than the measurement target is converted into the nanopore. It is possible to eliminate a tunnel current change caused by a biopolymer that cannot enter and is not a measurement target.

  Another method for controlling the moving speed of the nucleic acid is a method of applying a pressure difference to each sample solution existing at the upper part and the lower part of the analytical device of the present invention. For example, the speed at which a nucleic acid passes through the nanopore can be reduced by applying a force to the nucleic acid opposite to the force with which the nucleic acid tries to pass through the nanopore by electrophoresis.

  By entering the nanopore of the analytical device while controlling the shape and / or moving speed of the nucleic acid, the main axis of the nucleic acid and the central axis of the nanopore are substantially coincident. By driving the nucleic acid by the sample moving mechanism, the nucleic acid passes through the nanopore, and the monomer constituting the nucleic acid sequentially passes between the electrodes formed on the nanopore sidewall. That is, the monomers arranged along the main axis direction of the nucleic acid sequentially pass between the electrodes, and the tunnel current between the electrodes changes at that time. Since the amount of change varies depending on the type of monomer, the monomer can be identified sequentially by measuring the tunnel current.

  In the present invention, nucleic acid characteristics can be analyzed using the above-described nucleic acid analysis device of the present invention and a nucleic acid analysis apparatus using the same. In the present invention, the term “nucleic acid” refers to a multimer (oligomer) or polymer (polymer) in which a plurality of small molecules (monomer, monomer) having a unit structure are linked, and a living body and a living body. It means something derived from something. Specifically, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), hybrid nucleic acid composed of DNA and RNA, and the like are included. In addition, the nucleic acid includes a polymer containing a sequence or a component that does not exist in nature. For example, a sequence such as poly (A), poly (T), poly (G), poly (C), or any sequence Also included are artificially synthesized polymer molecules having sequences. Nucleic acids also include nucleic acids prepared by nucleic acid amplification techniques known in the art (eg, polymerase chain reaction) and nucleic acids cloned into vectors. Methods for preparing samples containing these nucleic acids are well known in the art, and those skilled in the art can appropriately select a method for adjusting according to the type of nucleic acid.

  Hereinafter, the present invention will be described more specifically with reference to examples. In order to facilitate understanding, the analysis target is ssDNA. Examples 1 to 5 explain the configuration of the nucleic acid analysis device, Examples 6 to 7 explain the method of manufacturing the nucleic acid analysis device, and Examples 8 to 9 show the configuration of the nucleic acid analysis device using the nucleic acid analysis device. explain. However, the following examples do not limit the present invention.

<Example 1>
An example of the configuration of the nucleic acid analysis device for ssDNA according to the present invention will be described with reference to FIGS. 1A and 1B. Here, a top view and a cross-sectional view of the structure when chemically modifying molecules are applied to two electrodes in a tunnel current type solid nanopore having two electrodes facing each other (hereinafter referred to as a nanopore with two electrodes) are used. I will explain. The electrode 102 is sandwiched between insulating films 103. In the top view, the insulating film 103 is not shown in order to show the shape of the electrode 102, but the opening 101 of the insulating film 103 is shown. Further, although the electrode 102 is connected to the wiring, it is omitted in the drawing.

First, FIG. 1A shows a nanopore with a two-electrode having a conventional structure without chemically modifying molecules for comparison. The shape of the opening 101 was circular, and the nanopore diameter and the interelectrode distance were 2 nm in order to analyze ssDNA. FIG. 1B shows a structure in which chemically modified molecules 104 are applied to the surface of the electrode 102 according to the present invention. The thickness of the chemically modified molecule 104 is 1 nm or less. FIG. 1B (a) shows the chemical modification molecule 104 added to the two electrodes 102 of the two-electrode nanopore having the conventional structure shown in FIG. 1A.
Here, in FIG. 1B, the top view is marked with (a1) and the cross-sectional view is marked with (a2), but if there is no particular need to distinguish the top view or cross-sectional view, write. The same notation is adopted in the notation of the following drawings.

  In the case of FIG. 1B (a), the chemically modified molecule 104 at the tip of the electrode 102 protrudes from the edge of the opening 101 by the thickness, so that the effective area of the nanopore 105 is reduced and the ssDNA to be analyzed passes. It may not be possible.

  Therefore, as shown in FIG. 1B (b), the nanopore diameter and the distance between the electrodes are increased by the thickness of the chemically modified molecule 104 so that the distance between the chemically modified portions is 2 nm suitable for ssDNA analysis. . However, when the shape of the opening 101 is circular as shown in FIG. 1B (b), the size of the effective nanopore 105 in the vertical direction in the top view is larger than 2 nm, which is larger than the size suitable for the analysis of ssDNA. There is a possibility that the base sequence cannot be read with high accuracy.

Therefore, as shown in FIG. 1B (c), the effective nanopore 105 diameter after chemical modification is set to 2 nm suitable for ssDNA analysis. Specifically, the shape of the opening 101 is an ellipse, electrodes with chemically modified molecules facing in the major axis direction are arranged, and the distance between the chemically modified parts and the minor axis are 2 nm. With this structure, the base sequence can be read with high accuracy.
However, in the case of the structure shown in FIG. 1B (c), the chemically modified molecule 104 protrudes from the edge of the opening 101. Therefore, when the mechanical and chemical strength of the chemically modified portion is low, the nanopore 105 enters. When the coming sample or sample solution collides with the chemically modified portion, there is a possibility that the function is deteriorated or the chemically modified molecules 104 are peeled off. In that case, the opening 101 is a circle having a diameter of 2 nm suitable for the analysis of ssDNA, and only the electrode 102 of the chemically modified molecule 104 is attached so that the chemically modified molecule 104 can be applied between the tip of the electrode 102 and the edge of the opening 101. Retreat about the thickness. When retracted as shown in FIG. 1B (d), since the chemically modified molecules 104 do not protrude from the edge of the opening 101 even after chemical modification, it is possible to suppress the deterioration and peeling of the function of the chemically modified molecules 104 due to long-time use.

  The structure of FIG. 1B (d) is a structure that achieves both accuracy, reliability, and durability. However, since the structure is somewhat complicated, the manufacturing cost is relatively high. Choose a structure according to the required cost, accuracy, reliability and durability.

  In this embodiment, the chemically modified molecules 104 at the tip of each electrode may be the same or different. Moreover, the shape of the opening 101 may not be a circle or an ellipse, but may be a polygon similar to it.

<Example 2>
In Example 1, the tunnel current type solid nanopore having two electrodes on the same plane was shown as an example in which a chemically modified molecule was added. Here, a tunnel current having more than two electrodes on the same plane is shown. An embodiment in which chemically modified molecules are added to the system solid nanopore will be described with reference to FIG. FIG. 2 shows a top view of a solid nanopore having 3 to 6 electrodes 202 on the same plane. The electrode 202 is sandwiched between insulating films. In order to show the shape of the electrode 202, the insulating film is not shown, but the opening 201 is shown. Further, although the electrode 202 is connected to the wiring, it is omitted in the drawing.

  2 (a1) and (a2) are solid nanopores having three electrodes, FIGS. 2 (b1) and (b2) are solid nanopores having four electrodes, and FIGS. 2 (c1) and (c2) are solid nanopores having five electrodes. 2 (d1) and 2 (d2) are a top view and a cross-sectional view, respectively, of a solid nanopore having six electrodes.

  2 (a1), (b1), (c1), and (d1) have a structure in which the chemically modified molecule 204 protrudes from the opening 201, and FIGS. 2 (a2), (b2), (c2), ( In d2), the chemically modifying molecule 204 is located between the tip of the electrode 202 and the edge of the opening 201 as in FIG. 1B (d), and does not protrude from the opening 201. The former sets the effective nanopore diameter after chemical modification to 2 nm suitable for ssDNA, and the latter sets the nanopore diameter to 2 nm. The former has a relatively low manufacturing cost but may have low reliability and durability, and the latter has the opposite characteristics.

<Example 3>
In Examples 1 and 2, chemical modification molecules were applied to all of the two or more electrodes, but may be applied to only some of the electrodes without being applied to all of the electrodes. Here, in the nanopore with two electrodes, an example in which a chemically modified molecule is applied to only one of the two electrodes will be described with reference to FIG. FIG. 3 shows a top view and a cross-sectional view of a two-electrode nanopore having two electrodes on the same plane. The electrode 302 has a structure sandwiched between insulating films 303. Although the insulating film 303 is not shown in order to show the shape of the electrode 302, the opening 301 is shown. Further, although the electrode 302 is connected to a wiring, it is omitted in the drawing.

  In FIG. 3A, the shape of the opening 301 is circular, the tip of the electrode 302 and the edge of the opening 301 coincide, and a chemical modification molecule 304 is applied to one of the two electrodes 302, whereby This is a nanopore with two electrodes in which the distance between the electrode tips without modifying molecules is 2 nm suitable for the analysis of ssDNA. This structure corresponds to a structure in which one of the chemical modification portion in FIG. 1B (b) is removed and the diameter of the circular opening 101 is reduced, and the base sequence cannot be read with high accuracy as described in the first embodiment. Possibility and reliability / durability may be low.

  In FIG. 3B, the tip of the electrode 302 is coincident with the edge of the opening 301, and the chemical modification molecule 304 is applied to one electrode, so that the effective diameter of the nanopore 305 becomes 2 nm suitable for ssDNA analysis. Thus, a nanopore with two electrodes formed with nanopores. Specifically, the shape of the opening 301 is an ellipse, an electrode 302 facing in the major axis direction is arranged, the chemical modification molecule 304 is applied to only one of the two electrodes 302, and there is no chemical modification molecule tip and no chemical modification molecule The distance between the electrode tips and the minor axis are 2 nm. This structure corresponds to a structure in which one of the chemical modification portions in FIG. 1B (c) is removed and the major axis of the elliptical opening 101 is reduced, and the base sequence can be read with high accuracy as described in the first embodiment. On the other hand, reliability and durability may be low.

  In FIG. 3C, the diameter of the opening 301 is 2 nm suitable for the analysis of ssDNA, and the two electrodes 302 are both set back from the edge of the opening 301 by the thickness of the chemically modified molecule 304. It is a nanopore with two electrodes to which chemically modified molecules 304 are attached only to one side. In the case of manufacturing a structure in which the electrodes 302 are set back from the edge of the opening 301, it is difficult to manufacture structures in which the plurality of electrodes 302 are set back by different dimensions. Rather than the thickness of the chemically modified molecule 304. This structure corresponds to the structure in which one chemical modification portion in FIG. 1B (d) is removed, and as described in Example 1, the structure is compatible with accuracy, reliability, and durability. Since it is somewhat complicated, the manufacturing cost is relatively high.

<Example 4>
In Example 3, in the nanopore with two electrodes, an example in which a chemically modified molecule was given to only one of the two electrodes was shown, but here, a tunnel current method in which more than two electrodes are provided on the same plane In a solid nanopore, a chemical modification molecule is imparted to one or more electrodes, but as a specific example in which no chemical modification molecule is imparted to all electrodes, an example in which a chemical modification molecule is imparted except for one electrode is shown in the figure. It demonstrates using 4A-4D.

  4A to 4D show top views of solid nanopores having 3 to 6 electrodes 402 on the same plane. The electrode 402 is sandwiched between insulating films. In order to show the shape of the electrode 402, the insulating film is not shown, but the opening 401 is shown. Further, although the electrode 401 is connected to the wiring, it is omitted in the drawing. 4A is a solid nanopore having 3 electrodes, FIG. 4B is a solid nanopore having 4 electrodes, FIG. 4C is a solid nanopore having 5 electrodes, and FIG. 4D is a solid nanopore having 6 electrodes. 4A (a1), 4B (b1), 4C (c1), and 4D (d1) have a structure in which the opening 401 is circular and the chemically modified molecule 404 protrudes from the opening 401, as in FIG. 4A (a2), 4B (b2), 4C (c2), and 4D (d2), the opening 401 has an elliptical shape or similar shape as in FIG. 3B, and the chemically modified molecule 404 has an opening. 4A (a3), 4B (b3), 4C (c3), and 4D (d3) have a structure similar to that of FIG. 404 is between the tip of the electrode 402 and the edge of the opening 401, and does not protrude into the opening 401.

  4A (a1), 4B (b1), 4C (c1), and 4D in the case where the opening 401 is circular, the tip of the electrode 402 and the edge of the opening 401 coincide, and the chemically modified molecule 404 protrudes from the opening 401. As shown in (d1), the tip of the electrode without the chemically modified molecule is separated from the tip of the chemically modified portion of the other chemically modified molecule-attached electrode, and the position of the tip of the chemically modified portion and the tip of the electrode without the chemically modified molecule The size of the effective nanopore 405 determined by (1) becomes larger than the size suitable for ssDNA analysis, and the base sequence may not be read with high accuracy. Moreover, since the chemical modification molecule 404 protrudes from the opening 401, the reliability and durability may be low.

  On the other hand, as shown in FIGS. 4A (a2), 4B (b2), 4C (c2), and 4D (d2), the shape of the opening 401 is an ellipse or a similar shape, and the effective diameter of the nanopore 405 is If nanopores are formed to have a thickness of 2 nm suitable for ssDNA analysis, and the electrode 402 is disposed, the accuracy can be improved. However, since the chemically modified molecule 404 protrudes from the opening 401, the reliability and durability may be low. 4A (a2), 4B (b2), 4C (c2), and 4D (d2) show the case where there is one electrode without a chemically modified molecule. In this structure, the number of electrodes without a chemically modified molecule is as follows. Accordingly, it is necessary to adjust the shape of the opening 401 and the arrangement of the electrodes 402 so that the effective size of the nanopore 405 is suitable for the analysis target.

  On the other hand, as shown in FIGS. 4A (a3), 4B (b3), 4C (c3), and 4D (d3), the shape of the opening 401 is circular and the diameter is 2 nm suitable for the analysis of ssDNA. 3 is between the tip of the electrode 402 and the edge of the circular opening 401 and does not protrude into the opening 401, as in the structure of FIG. However, the manufacturing cost is relatively high. In the case of this structure, the shape of the opening 401 and the arrangement of the electrodes 402 do not depend on the number of electrodes without chemical modification molecules.

<Example 5>
In Examples 1 to 4, the structure in the case where a chemically modified molecule is applied to an electrode of a tunnel current type solid nanopore having two or more electrodes on the same plane and one electrode layer is described. . As the number of electrodes on the same plane increases, the manufacturing difficulty increases and the yield may decrease. Therefore, the number of electrodes on the same plane is set to 4 or less, and the number of stacked metal layers and insulating films is increased. The yield may be higher when the number of electrodes is increased. Here, the structure when chemically modifying molecules are applied to all electrodes of a tunneling current type solid nanopore having two electrodes on the same plane and four electrode layers will be described with reference to FIG. To do.

  FIG. 5 shows a top view and a cross-sectional view of an eight-electrode nanopore having two electrodes 502 on the same plane and four electrode 502 layers. Each electrode 502 has a structure separated by an insulating film 503. The insulating film 503 is not shown in order to show the shape of the electrode 502, but the opening 501 is shown. Further, although the electrode 502 is connected to the wiring, it is omitted in the drawing.

  In FIG. 5A, the shape of the opening 501 is circular, the edge of the electrode 502 and the edge of the opening 501 coincide with each other, and a chemical modification molecule 504 is applied to the electrode 502, whereby the distance between the chemical modification portion tips is changed. It is a nanopore with 8 electrodes of 2 nm suitable for analysis. Since the top view is the same as that of FIG. 1B (b), as in the case of the nanopore with electrode of FIG. 1B (b), even if the distance between the tips of the chemically modified portions is 2 nm, Since the size of the nanopore 505 is larger than 2 nm and larger than the size suitable for ssDNA analysis, there is a possibility that the base sequence cannot be read with high accuracy. In addition, since the chemically modified molecule 504 protrudes from the opening 501, the reliability and durability may be low.

  On the other hand, in FIG. 5B, similarly to FIG. 1B (c), the shape of the opening 501 is an ellipse, electrodes with chemical modification facing the major axis direction are arranged, and the distance and minor axis between the chemical modification parts are arranged. Is a nanopore with 8 electrodes having a thickness of 2 nm. Since the effective size of the nanopore 505 is suitable for ssDNA analysis, the base sequence can be read with high accuracy. However, since the chemically modified molecule 504 protrudes beyond the opening 501, the reliability and durability may be low.

  On the other hand, as shown in FIG. 5C, the shape of the opening 501 is circular, the diameter is 2 nm suitable for ssDNA analysis, and the chemically modified molecule 504 is between the tip of the electrode 502 and the edge of the circular opening 501. In the case of the structure that does not protrude from the opening 501, both accuracy and reliability / durability can be achieved, as in the structure of FIG. 1B (d), but the manufacturing cost is relatively high.

  In this embodiment, the number of the electrodes 502 on the same plane is two and the number of the layers of the electrodes 502 is four. However, the number and the number of the electrodes 502 can be arbitrarily selected. When the number of electrodes 502 on the same plane is 3 to 6, the top view is the same as FIG. In this embodiment, the case where the chemically modified molecules 504 are added to all the electrodes 502 is shown, but an electrode without the chemically modified molecules may be provided. Furthermore, the chemically modified molecules 504 at the tip of each electrode may be the same or different, and can be arbitrarily selected.

<Example 6>
An example of a method for producing a nucleic acid analysis device for ssDNA according to the present invention will be described with reference to FIGS. 6A to 6J. Here, an example of a method for producing a nanopore with two electrodes having two electrodes facing each other on the same plane, in which the tip of the electrode coincides with the edge of the opening and the chemically modified molecule protrudes from the opening will be described. 6A to 6J are manufacturing process flow charts showing the cross-sectional structure of the device. The manufacturing process is described below.

  A silicon nitride film 607 is deposited to a thickness of 20 nm by a CVD method on a (100) plane silicon single crystal substrate 606, and a titanium nitride film 608 to be a pad electrode is deposited to a thickness of 50 nm by sputtering to perform lithography and dry etching. Thus, the titanium nitride film 608 is patterned into a pad shape (100 μm square) (FIG. 6A).

  Next, a titanium nitride film 609 serving as an electrode for tunneling current measurement is deposited by CVD to a thickness of 5 nm (FIG. 6B) and patterned into the shape of the electrode (FIG. 6C). The electrode is a separated electrode with the position where the nanopore is formed as a gap 610. The width of the gap 610 is matched with the size of the opening to be formed. Here, it is 4 nm. In this embodiment, the electrodes are patterned as opposed to each other with the gap 610 interposed therebetween. However, the electrodes may be connected without the gap 610. However, in that case, it is necessary to cut the connected electrodes when forming the nanopore 616 (see FIG. 6I). After patterning the electrodes, a 20 nm silicon nitride film 611, a 60 nm silicon oxide film 612, and a 200 nm silicon nitride film 613 are deposited by CVD (FIG. 6D).

  Next, the silicon nitride film 611, the silicon oxide film 612, and the silicon nitride film 613 on the pad are removed by lithography and dry etching, and the titanium nitride 609 is exposed. Further, the 200 nm silicon nitride film 613 above the electrode gap is removed to expose the silicon oxide film 612 (FIG. 6E, the exposed area is 5 μm square). Thereafter, a silicon nitride film 614 having a thickness of 400 nm is deposited on the back surface by a CVD method, and a region in which a 1030 μm square silicon substrate 606 is exposed is formed by lithography and dry etching (FIG. 6F).

  Next, the silicon substrate 606 exposed from the back surface is anisotropically etched with an aqueous potassium hydroxide solution to form a thin film portion 615 having no silicon substrate 606 of about 30 μm square (FIG. 6G). The silicon oxide film 612 of the thin film portion 615 is removed by wet etching with hydrofluoric acid (FIG. 6H), and nanopores 616 are formed by irradiating an electron beam with a transmission electron microscope (FIG. 6I). The shape of the opening is an ellipse having a major axis of 4 nm and a minor axis of 2 nm, and there is an electrode in the major axis direction. After the formation of the nanopore 616, chemically modified molecules 604 are applied to the two electrode surfaces (FIG. 6J). The thickness of the chemically modified molecule 604 is about 1 nm, and the distance between the two chemically modified portions is about 2 nm. The top view is the same as FIG. 1B (c). Here, 4-mercaptobenzoic acid was used as the chemically modified molecule 604.

In the present embodiment, the method for manufacturing a nanopore with two electrodes having two electrodes facing each other on the same plane has been described. However, a nanopore with an electrode having one electrode layer and more than two electrodes is manufactured. The case can be realized by changing pad patterning (FIG. 6A) and electrode patterning (FIG. 6C). When a plurality of electrode layers are used as shown in FIG. 5B, silicon nitride films and titanium nitrides are alternately deposited after FIG. 6B, and the lowermost titanium nitride is collectively collected as in FIG. 6C. Pattern. Furthermore, it is only necessary to prepare pads as many as the number of electrodes, and to connect each electrode and pad by wiring.
In this embodiment, the chemically modified molecules 604 are applied to all the electrodes. However, all the chemically modified molecules 604 may be different or the same, or electrodes having no chemically modified molecules may be mixed.

<Example 7>
In Example 6, a method for producing nanopores with electrodes having a structure in which chemically modified molecules protrude from the openings has been described. Here, an example of a method for producing a two-electrode nanopore having a structure that has two electrodes facing each other on the same plane, the chemically modifying molecule is between the electrode tip and the edge of the opening, and does not protrude into the opening. To do. 7A to 7K are manufacturing process flow charts showing the cross-sectional structure of the device. The manufacturing process is described below.

  A silicon nitride film 707 is deposited by a CVD method to a thickness of 50 nm on a silicon single crystal substrate 706 having a crystal plane orientation of (100) plane, an amorphous silicon film 717 is deposited by a CVD method to a thickness of 10 nm, and a pad electrode and A titanium nitride film 708 is deposited by sputtering to a thickness of 50 nm, and the titanium nitride film 708 is patterned into a pad shape (100 μm square) by lithography and dry etching (FIG. 7A).

  Next, a gold / titanium film 718 is deposited by sputtering (FIG. 7B) and patterned into the shape of an electrode (FIG. 7C). In this example, 5 nm of gold was used as the electrode material, and 1 nm of titanium was used as the adhesive layer between the gold and the underlying amorphous silicon film 717. Gold was chosen as a conductive material that is difficult to oxidize. The electrode is a separated electrode with a gap 710 at the position where the nanopore is formed. The width of the gap 710 is matched with the size of the nanopore formed in FIG. Here, it is 4 nm. In this embodiment, the electrodes are patterned as opposed to each other with the gap 710 interposed therebetween, but may be connected electrodes without a gap. In that case, it is necessary to cut the connected electrodes when the nanopore 716 is formed. After patterning the electrodes, a 10 nm amorphous silicon film 719, a 60 nm silicon oxide film 712, and a 200 nm silicon nitride film 713 are deposited by CVD (FIG. 7D).

  Next, the amorphous silicon film 719, the silicon oxide film 712, and the silicon nitride film 713 above the pad are removed by lithography and dry etching, and the gold / titanium film 718 is exposed. Further, the 200 nm silicon nitride film 713 above the electrode gap is removed to expose the silicon oxide film 712 (FIG. 7E, the exposed area is 5 μm square). Thereafter, a 400 nm silicon nitride film 714 is deposited on the back surface by CVD, and a region where a 1030 μm square silicon substrate 706 is exposed is formed by lithography and dry etching (FIG. 7F).

  Next, the silicon substrate 706 exposed from the back surface is anisotropically etched with an aqueous potassium hydroxide solution to form a thin film portion 715 having no silicon substrate 706 of about 30 μm square (FIG. 7G).

  The silicon oxide film 712 on the surface side of the thin film portion 715 is removed by wet etching using hydrofluoric acid, and the silicon nitride film 707 on the back surface side of the thin film portion 715 is removed by wet etching using hot phosphoric acid (FIG. 6H). Then, nanopores 716 are formed by irradiating an electron beam with a transmission electron microscope (FIG. 7I). The shape of the opening is a circle with a diameter of 4 nm. The distance between the two electrodes facing each other is also 4 nm. After the formation of the nanopore 716, exposure to oxygen plasma oxidizes the amorphous silicon films 717 and 719 sandwiching the upper and lower electrodes. Since the amorphous silicon film becomes a silicon oxide film, the volume is increased and the nanopore is reduced. The power and time of the oxygen plasma are adjusted so that the diameter of the nanopore after oxidation becomes 2 nm. As a result of the oxygen plasma exposure, the circular opening with a diameter of 4 nm formed by the amorphous silicon films above and below the electrode becomes a circular opening with a diameter of 2 nm due to the silicon oxide film. On the other hand, the gold of the electrode material is difficult to oxidize. No increase in volume due to, and the distance between the two electrodes does not change (FIG. 7J). Thereafter, chemically modified molecules 704 are applied to the two electrode surfaces (FIG. 7K). The thickness of the chemically modified molecule 704 is about 1 nm, and the distance between the two chemically modified portions is about 2 nm. The top view is the same as FIG. 1B (d). Here, 4-mercaptobenzoic acid was used as the chemically modified molecule 704.

  In the present embodiment, the method for manufacturing a nanopore with two electrodes having two electrodes facing each other on the same plane has been described. However, a nanopore with an electrode having one electrode layer and more than two electrodes is manufactured. The case can be realized by changing the patterning of the pad (FIG. 7A) and the patterning of the electrode (FIG. 7C). Further, when a plurality of electrode layers are used as shown in FIG. 5C, an amorphous silicon film and a gold / titanium film are alternately deposited after FIG. 7B, and the lowest gold / titanium is formed as in FIG. 7C. Pattern up to the film. Furthermore, it is only necessary to prepare pads as many as the number of electrodes, and to connect each electrode and pad by wiring.

  In this embodiment, chemically modified molecules 704 are applied to all electrodes, but all chemically modified molecules 704 may be different or the same. Further, electrodes without chemically modifying molecules may be mixed.

<Example 8>
One configuration of the nucleic acid analysis apparatus using the nucleic acid analysis device according to the present invention will be described with reference to FIG. The nucleic acid analyzer includes a first solution tank 821, a second solution tank 822, a nucleic acid analysis device 823 using nanopores with electrodes to which chemically modified molecules are divided to divide both solution tanks, and a first solution tank 821 through a nanopore 816. Nucleic acid driving means 824, 825, and control and data processing means 826 that move between the first solution tank 822 and the second solution tank 822. In this embodiment, the nucleic acid driving means 824 and 825 are arranged in both solution tanks. However, in addition to or instead of this, the nucleic acid driving means 824 and 825 may be incorporated in the nucleic acid analysis device 823. Both solution tanks are provided with inlets 827 and 828 for introducing the solution and outlets 829 and 830 for discharging the solution, respectively. Furthermore, electrodes 831 and 832 are provided in both solution tanks, and the electrodes 831 and 832 are connected to a voltage source 833 and an ammeter 834 whose polarity and output are variable. These electrodes 831 and 832 may be incorporated in the nucleic acid driving means 824 and 825, respectively.

  In this embodiment, an example in which a nanopore with two electrodes in which a chemically modified molecule 804 is added to all electrodes 802 is used for the nucleic acid analysis device 823 is shown. A voltage source 835 having variable polarity and output and an ammeter 836 are connected between the two electrodes 802. The voltage sources 833 and 835, the ammeters 834 and 836, and the nucleic acid driving means 824 and 825 are connected to the control and data processing means 826.

  When analyzing the nucleic acid 837, the nucleic acid 837 to be analyzed is mixed with the buffer solution, and only the buffer solution is introduced into the first solution tank 821 from the inlet 827 and into the second solution tank 822 through the inlet 828. The nucleic acid 837 is moved from the first solution tank 821 to the second solution tank 822 by the nucleic acid driving means 824 and 825. The current flowing between the two electrodes 831 and 832 using the voltage source 833 simultaneously with the driving of the nucleic acid, that is, the ionic current flowing through the nanopore 816 is measured using the ammeter 834. When the nucleic acid 837 is introduced into the nanopore 816, the ion current value decreases. When the ion current value decreases, the speed at which the nucleic acid 837 passes through the nanopore 816 is adjusted by the nucleic acid driving means 824 and 825 so that the base species can be identified by the tunnel current flowing between the electrodes 802. The base type is identified by a difference in a tunnel current value flowing under a constant voltage or a difference in current-voltage characteristics of each base.

  Here, a method for performing base identification of a DNA strand based on a difference in a tunnel current value flowing under a constant voltage will be described. There are four types of bases in the DNA chain, ATGC, and a unique current value is observed for each type of these bases and sent to the control and data processing means 826. An example is shown in FIG. A tunnel current when a polymer in which only one type of each base is connected passes through the nanopore 816 is measured in advance, and a current value corresponding to each base type is obtained and stored in the memory of the control and data processing means. The base sequence of the DNA strand to be analyzed can be known by comparing the current value obtained at the time of measuring the tunnel current of the DNA strand to be analyzed with the current value corresponding to each base type measured in advance.

  After all the bases constituting the nucleic acid 837 pass between the electrodes and the base identification is completed, the same nucleic acid 837 may be driven in the reverse direction to perform base identification again and perform verification. If the base species cannot be identified by a single measurement because the speed at which the nucleic acid 837 passes through the nanopore 816 is high, the identification may be made statistically by repeatedly measuring the same base. When the analysis of the nucleic acid 837 is completed, the nucleic acid 837 is discharged from the discharge port 830.

  In this embodiment, the tunnel current is measured using a nucleic acid analysis device having one nanopore. However, a large number of nanopores are arranged on the same substrate, and simultaneous measurement of the tunnel current of a large number of nucleic acids to be analyzed is performed. As a result, throughput can be improved.

<Example 9>
In Example 8, one configuration of a nucleic acid analyzer using a two-electrode nanopore provided with a chemically modified molecule was described. For a single nanopore, it is possible to analyze the base sequence with higher discriminating ability and higher accuracy by identifying the type of base with tunnel current flowing between various electrodes using more than two electrodes. Can do. Here, FIG. 10 shows a configuration of a nucleic acid analyzer using electrode-equipped nanopores having more than two electrodes for one nanopore among nucleic acid analyzers using electrode-attached nanopores to which chemically modified molecules are attached. Will be described. Here, as shown in FIG. 5C, an example using a nanopore with eight electrodes in which the number of electrodes on the same plane is two and the number of electrode layers is four will be described. Eight electrodes are composed of two electrode pairs of four layers, the uppermost electrode pair is modified with T, the discrimination ability for A is improved, and the second electrode pair from the uppermost layer is modified with C, The discriminating ability for G is raised, the third electrode pair from the uppermost layer is modified with A, the discriminating ability for T is raised, the lowermost electrode pair is modified with G, and the discriminating ability for C is raised.

  In the same manner as in Example 8, the apparatus includes a first solution tank 1021, a second solution tank 1022, a nucleic acid analyzing device 1023 using a nanopore with an electrode provided with a chemically modified molecule 1004 that divides both solution tanks, and a nucleic acid 1037 through a nanopore 1016. Is constituted by nucleic acid driving means 1024 and 1025 for moving the first solution tank 1021 and the second solution tank 1022, and control and data processing means 1026. In this embodiment, the nucleic acid driving means 1024 and 1025 are arranged in both solution tanks. However, in addition to or instead of this, the nucleic acid driving means 1024 and 1025 may be built in the nucleic acid analysis device 1023. Both solution tanks are provided with inlets 1027 and 1028 for introducing the solution and outlets 1029 and 1030 for discharging the solution, respectively. Furthermore, both solution tanks are provided with electrodes 1031 and 1032, and the electrodes 1031 and 1032 are connected to a voltage source 1033 having variable polarity and output and an ammeter 1034. These electrodes 1031 and 1032 may be incorporated in the nucleic acid driving means 1024 and 1025, respectively. A voltage source 1035, 1038, 1040, 1042 of variable polarity and output and an ammeter 1036, 1039, 1041, 1043 are connected to each of the four electrode pairs. Voltage sources 1033, 1035, 1038, 1040, 1042, ammeters 1034, 1036, 1039, 1041, 1043, and nucleic acid driving means 1024, 1025 are connected to control and data processing means 1026. In this embodiment, four voltage sources and ammeters are connected to four electrode pairs. However, one set of voltage source and ammeter may be switched to four electrode pairs by using a switch. . In that case, the switch is also connected to the control and data processing means.

  When analyzing the nucleic acid 1037, the nucleic acid 1037 to be analyzed is mixed with the buffer solution, and only the buffer solution is introduced into the first solution tank 1021 through the inlet 1027 and into the second solution tank 1022 through the inlet 1028. The nucleic acid 1037 is moved from the first solution tank 1021 to the second solution tank 1022 by the nucleic acid driving means 1024 and 1025. Simultaneously with the driving of the nucleic acid, the voltage source 1033 is used to measure the current flowing between the two electrodes 1031 and 1032, that is, the ionic current flowing through the nanopore 1016, using the ammeter 1034. When the nucleic acid 1037 is introduced into the nanopore 1016, the ionic current value decreases. When the current value decreases, the nucleic acid driving means 1024 and 1025 increase the speed at which the nucleic acid 1037 passes through the nanopore 1016 so that the base species can be identified by the tunnel current flowing between the four electrode pairs of the nucleic acid analysis device 1023. Adjust. The base type is identified by a difference in a tunnel current value flowing under a constant voltage or a difference in current-voltage characteristics of each base.

  Here, a method for performing base identification of a DNA strand based on a difference in a tunnel current value flowing under a constant voltage will be described. In advance, a tunnel current flowing between four electrode pairs of the nucleic acid analysis device 1023 when a polymer in which only one type of each base is connected passes through the nanopore 1016 is measured, and current values corresponding to the four types of bases are obtained. It is stored in the memory of the control and data processing means 1026. Among the four electrode pairs, the uppermost electrode pair has a high selectivity sensitivity to A, and when A is between the electrodes, a sufficiently large current flows as compared to bases other than A, and therefore, it can be distinguished from other bases.

  On the other hand, the discriminating ability of three types of bases other than A may be low. Similarly, the second electrode pair from the top layer can distinguish G and bases other than G, the third electrode pair from the top layer can distinguish T and bases other than T, and the bottom electrode pair is C. And bases other than C can be distinguished. When a DNA strand to be analyzed passes between four electrode pairs within a certain time, a tunnel current between the four electrode pairs changes within a certain time. Since each tunnel current change provides highly sensitive data for each different base, the base sequence of the DNA strand to be analyzed can be known by complementing each data.

  Specifically, when the base sequence of the DNA strand to be analyzed is AGTCTAGGC, the time dependence of the tunnel current obtained when the DNA strand passes between four electrode pairs will be described with reference to FIG. From the time dependence of the tunnel current flowing between the uppermost electrode pair, it can be seen that the base sequence of the DNA strand to be analyzed is at least “A ?????? A ???”. Here, “?” Is any base of A, T, G, and C. Next, from the dependence of the tunnel current between the second electrode pair from the top layer, it can be seen that at least “? G? G ?? G?”. Similarly, by obtaining data identifying bases with high selection sensitivity from the time dependence of the tunnel current between each electrode pair, and complementing each other, it is possible to know the base sequence of the DNA strand to be analyzed. .

  In the same manner as in Example 8, after all the bases constituting the nucleic acid 1037 pass between the four electrode pairs and the base identification is completed, the same nucleic acid 1037 is driven in the reverse direction to perform base identification again. Verification may be performed. If the base species cannot be identified by a single measurement because the speed at which the nucleic acid 1037 passes through the nanopore 1016 is high, the identification may be made statistically by repeatedly measuring the same base. When the analysis of the nucleic acid 1037 is completed, the nucleic acid 1037 is discharged from the discharge port 1030.

  In this embodiment, the tunnel current is measured using a nucleic acid analysis device having one nanopore. However, a large number of nanopores are arranged on the same substrate, and simultaneous measurement of the tunnel current of a large number of nucleic acids to be analyzed is performed. As a result, throughput can be improved.

<Example 10>
An example of the electrode pair configuration of the nucleic acid analysis device according to the present invention will be described.
When measuring the tunnel current of the nucleic acid to be analyzed between the electrode pair, the two electrodes constituting the electrode pair may not be the same chemically modified molecule, but may be different chemically modified molecules, or one electrode may be not chemically modified. good. In a nucleic acid analysis device having three or more electrodes, one of the electrodes can be connected to the ground as a common electrode, and (n−1) pairs of electrodes can be configured by n electrodes (n ≧ 3) . Here, an example in which one of the electrodes 1202 is a common electrode and four electrode pairs are configured in a nucleic acid analysis device having five electrodes 1202 will be described with reference to FIG.

  FIG. 12 has five electrodes 1202 on the same plane, and the electrode layer is composed of one layer. One of the five electrodes 1202 is not chemically modified, and different chemically modified molecules 1204 are applied to the remaining four electrodes. Here, the five electrodes 1202 are described separately from the electrodes a, b, c, d, and e. The electrode a is not chemically modified, the electrode b is modified with T, the discriminating ability for A is increased, the electrode c is modified with C, the discriminating ability for G is increased, and the electrode d is modified with A. In addition, the discrimination ability for T is improved, and the electrode e is modified with G to increase the discrimination ability for C. The electrode a is a common electrode, and four electrode pairs of electrodes a and b, a and c, a and d, and a and e are configured. A is identified by the electrode pair ab, G is identified by the electrode pair ac, T is identified by the electrode pair ad, and C is identified by the electrode pair ae.

  In this embodiment, the common electrode is not chemically modified, but may be chemically modified. Further, in this embodiment, all the electrodes are on the same plane, but two or more electrode layers may be present on different planes. Further, the number of electrodes may be increased to increase the accuracy.

101, 201, 301, 401, 501, 1201 ... opening,
102, 202, 302, 402, 502, 802, 831, 832, 1002, 1202 ... electrodes,
103,303,403,503,803,1003 ... insulating film,
104,204,304,404,504,604,704,804,1004,1204 ... chemically modified molecule,
105, 205, 305, 405, 505 ... effective nanopores,
606, 706 ... silicon substrate,
607, 611, 613, 614, 713, 714 ... silicon nitride film,
608, 609, 708 ... titanium nitride film,
610, 710 ... gap,
612, 712, 720 ... silicon oxide film,
615, 715 ... Thin film part,
616, 716, 816, 1016 ... nanopore,
717, 719 ... amorphous silicon film,
718 ... gold / titanium film,
821, 1021 ... first solution tank,
822, 1022 ... second solution tank,
823, 1023 ... nucleic acid analysis device,
824, 825, 1024, 1025 ... nucleic acid driving means,
826, 1026 ... control and data processing means,
827, 828, 1027, 1028 ... inlet,
829, 830, 1029, 1030 ... discharge port,
833, 835, 1033, 1035, 1038, 1040, 1042 ... voltage source,
834, 836, 1034, 1036, 1039, 1041, 1043 ... ammeter,
837, 1037 ... nucleic acid.

Claims (15)

A nucleic acid analysis device for analyzing nucleic acid in a sample by current measurement,
A solid substrate;
A nanopore provided through the solid substrate and through which the sample passes;
At least two electrodes disposed on the solid substrate, each having one end face facing the nanopore;
With
At least one end surface of the electrode facing the nanopore is provided with a chemically modified molecule for improving the discrimination ability for at least one of the four types of bases constituting the nucleic acid,
Analyzing the nucleic acid by applying a voltage between the at least two electrodes and causing a current to flow through a base constituting the nucleic acid in the entered sample while the sample enters the nanopore. Nucleic acid analysis device. One end surface of the electrode to which the chemically modifying molecule is applied is in the inner direction of the nanopore rather than the opening end of the opening having a contour shape formed by crossing the side wall of the nanopore and the surface or back surface of the solid substrate. With a protruding arrangement structure,
2. The nucleic acid according to claim 1, wherein an effective nanopore size determined by the arrangement structure is a size that allows the nucleic acid to be analyzed to pass through and a size that allows current to flow through the base. Analytical device. One end face of the electrode to which the chemically modified molecule is applied has an opening end of an opening having a contour shape formed by intersecting a side wall of the nanopore and the front or back surface of the solid substrate, or the individual from the open end. Having an arrangement structure arranged on the substrate side,
2. The nucleic acid analysis device according to claim 1, wherein the size of the opening is a size that allows the nucleic acid to be analyzed to pass through and a size that allows current to flow through the base.   2. The solid substrate according to claim 1, wherein the solid substrate is made of any one of silicon, glass, quartz, polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polystyrene, and polypropylene. Nucleic acid analysis device. The solid substrate has a thin film portion having a thickness of 5 to 200 nm;
The nucleic acid analysis device according to claim 1, wherein the nanopore is provided in the thin film portion.   The thin film portion is any of silicon, glass, quartz, polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polystyrene, polypropylene, silicon oxide, silicon nitride, silicon oxynitride, metal oxide, and metal silicate. The nucleic acid analysis device according to claim 5, wherein the nucleic acid analysis device is made of any one material.   The electrode is a metal material such as gold, silver, platinum, palladium, rhodium, ruthenium, copper, iron, aluminum, titanium, nickel, tantalum, tungsten, cobalt, chromium, molybdenum, niobium, or titanium nitride, nitride Metal nitride material that is one of tantalum, tungsten nitride, niobium nitride, chromium nitride, or silicide material that is any of tungsten silicide, titanium silicide, cobalt silicide, nickel silicide, graphite, graphene, or carbon nanotube 2. The nucleic acid analyzing device according to claim 1, wherein the nucleic acid analyzing device is made of any one of a carbon material and a semiconductor material which is either silicon or germanium.   The nucleic acid analysis device according to claim 1, wherein the electrode has a thickness in a range of 0.1 to 10 nm.   The nucleic acid analysis according to claim 1, wherein the chemically modified molecule is composed of any one of a nucleic acid molecule contained in the sample to be analyzed or an organic compound having molecular recognition ability for the nucleic acid molecule. device.   The nucleic acid analysis device according to claim 1, wherein the chemically modified molecule has a thickness of 2 nm or less.   The diameter of the opening is circular when the shape of the opening is circular, the short diameter of the opening is elliptical, and the diameter of the inscribed circle of the polygon is 10 nm or less. The nucleic acid analysis device according to 1.   The nucleic acid includes at least one of single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, a hybrid nucleic acid composed of DNA and RNA, and an artificially synthesized polymer molecule. The nucleic acid analysis device according to claim 1.   The nucleic acid analysis device according to claim 1, wherein the depth of the nanopore is twice or more as large as a monomer unit constituting the nucleic acid. The nucleic acid analysis device according to any one of claims 1 to 13,
A first solution tank comprising an inlet for introducing a sample solution containing a nucleic acid to be analyzed, and an outlet for discharging the sample solution;
A second solution tank comprising an inlet for introducing the solution and an outlet for discharging the solution;
Nucleic acid driving for moving the nucleic acid from the first solution tank to the second solution tank through the nanopore having the nucleic acid analysis device disposed between the first solution tank and the second solution tank. Means,
A first voltage source for applying a voltage between the first solution tank and the second solution tank;
A first ammeter that detects a current flowing between the first solution tank and the second solution tank via the nanopore;
A second voltage source for applying a voltage between electrodes provided in the nucleic acid analysis device;
A second ammeter for detecting a current flowing between electrodes provided in the nucleic acid analysis device;
Control means for controlling at least the nucleic acid driving means;
Data processing means for obtaining and processing at least data detected by the first and second ammeters,
Using the second voltage source, a current is passed through the base constituting the nucleic acid in the sample solution that has entered the nanopore, and the current is measured using the second ammeter, whereby the nucleic acid in the sample solution is measured. A nucleic acid analyzer characterized in that   15. The nucleic acid analyzer according to claim 14, wherein the nucleic acid driving device is built in the nucleic acid analysis device.

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