WO2007040219A1

WO2007040219A1 - Nanoarray dna chip

Info

Publication number: WO2007040219A1
Application number: PCT/JP2006/319765
Authority: WO
Inventors: Seiichi Iwamatsu
Original assignee: Intellectual Property Bank Corp.
Priority date: 2005-10-04
Filing date: 2006-10-03
Publication date: 2007-04-12
Also published as: JPWO2007040219A1

Abstract

It is intended to provide a nanoarray DNA chip structure which is applicable mainly to a high-speed DNA base sequence analysis method with the use of surface-enhanced Raman scattering spectroscopy using a near-field optical microscope and other scanning DNA base sequence analysis methods. A nanoarray DNA chip using a means which comprises extending single molecular DNA chains in individual microchannels formed on the surface of a baseboard or a substrate, immobilizing the DNA chains therein and then arranging and aligning the same.

Description

Specification

Nanoarray DNA chip

Technical field

[0001] The present invention relates to a DNA chip structure necessary for analyzing base sequence information of a DNA strand.

Background art

Conventionally, a DNA chip has a DNA fixing end 104 formed of a functional group on the surface of a substrate 101 having a sliding glass force as shown in FIG. 7, and a DNA strand 103 is fixed on the DNA fixing end 104. It is customary to establish a microarray DNA chip by establishing a forest.

[0003] Patent Document 1: US6376166 "Apparatus and Method for The Analysis of Numeric Acids Hybridization on High Density NA Chips J Vladimir Poponin.

Disclosure of the invention

Problems to be solved by the invention

[0004] However, the conventional microarray DNA chip described above is disclosed in [Patent Document 1], such as a high-speed DNA base sequence analysis method by surface-enhanced Raman scattering spectroscopy using a near-field optical microscope and other scanning DNA bases. It cannot be applied to sequence analysis.

[0005] That is, in the high-speed DNA base sequence analysis method by surface-enhanced Raman scattering spectroscopy using a near-field optical microscope and other scanning DNA base sequence analysis methods, a single molecule macro DNA strand formed along the substrate surface is used. Or a DNA chip in which DNA strand fragments are arranged or arranged.

The present invention has been made in view of the above circumstances, and is mainly applied to a high-speed DNA base sequence analysis method by surface-enhanced Raman scattering spectroscopy using a near-field optical microscope and other scanning DNA base sequence analysis methods. The purpose is to provide a nanoarray DNA chip structure that can be used.

Means for solving the problem

[0007] In order to solve the above-mentioned problems and achieve the above-mentioned object, a DNA strand is extended and fixed in at least one narrow groove in a narrow groove formed on the substrate surface as a nanoarray DNA chip. Take the means to arrange and arrange.

[0008] In order to solve the above problems and achieve the above object, a DNA strand is extended into at least one narrow groove in a narrow groove formed on the surface of a thin film on a substrate as a nanoarray DNA chip. And then take a means of fixing and arranging and arranging.

[0009] In the nanoarray DNA chip according to claims 1 and 2, nanoparticles are fixed to the ends of the DNA strands, and means for bonding the nanoparticles to a pad portion formed on the substrate is taken.

[0010] The nanoparticle in the nanoarray DNA chip according to claim 3 takes a means consisting of any one of gold nanoparticles, glass nanoparticles, and synthetic resin nanoparticles.

[0011] The pad portion in the nanoarray DNA chip according to claim 3 takes a means consisting of either a gold thin film or a synthetic resin thin film.

[0012] The substrate of the nanoarray DNA chip described in claims 1 to 5 is a disk-shaped disk, and the fine grooves formed on the disk surface are concentric or spiral.

[0013] The substrate of the nanoarray DNA chip according to claims 1 to 5 is a cylindrical micro cylinder, and the fine grooves formed on the surface of the micro cylinder take a concentric or spiral shape.

[0014] The substrate of the nanoarray DNA chip according to claim 7 is a single substrate of a cylindrical microcylinder, and the fine grooves formed on the surface of the microcylinder are concentric or spiral.

[0015] The narrow groove according to claims 1 and 2 takes a measure as a V-shaped cross-sectional structure formed from the surface of a silicon single crystal substrate or a silicon single crystal film having a (111) crystal plane orientation.

The invention's effect

[0016] According to the present invention, the arrangement and sequence of DNA strands (including RNA) are improved, and DNA base sequence analysis methods mainly by surface-enhanced Raman scattering spectroscopy using a near-field optical microscope, and other scanning DNAs. The DNA base sequence analysis by the base sequence analysis method can be performed at high speed and with high accuracy, and the DNA base sequence analysis by the multi-probe can be performed at high speed and with high accuracy.

Brief Description of Drawings FIG. 1 is a schematic diagram of a main part of a nanoarray DNA chip according to the present invention.

FIG. 2 is a schematic view of the main part of a nanoarray DNA chip according to the present invention.

FIG. 3 is a schematic diagram of the main part of a nanoarray DNA chip according to the present invention.

FIG. 4 is a schematic view of the main part of a disk-shaped nanoarray DNA chip according to the present invention.

FIG. 5 is a schematic view of the main part of a microcylinder nanoarray DNA chip according to the present invention.

FIG. 6 is a schematic cross-sectional view of the main part of a microarray DNA chip in the prior art.

FIG. 7 is a cross-sectional view of a V-shaped fine groove formed on a surface of a silicon single crystal substrate and a single crystal film according to the present invention.

Explanation of symbols

[0018] 101 substrate

102 narrow groove

103 DNA strand

104 DNA anchor

201 thin film

301 node

302 nanoparticles

401, 501 DNA channel in narrow groove

601 V-shaped narrow groove

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the nanoarray DNA chip according to the embodiment of the present invention will be described in detail with reference to the drawings.

[0020] FIG. 1 is a schematic diagram of a main part showing an example of a nanoarray DNA chip according to the present invention.

) Is a schematic plan view, (b) is a schematic cross-sectional view in the X direction including the DNA strand, and (c) is a schematic cross-sectional view in the Y direction including the DNA strand.

[0021] The nanoarray DNA chip has a structure in which the ends of the DNA strand 103 extended in the narrow groove 102 in which the surface force of the substrate 101 is also formed are fixed at the DNA fixing end 104, and are arranged and arranged in a line. Become. [0022] The substrate 101 of the nanoarray DNA chip may be a slide glass, an engineering plastic plate typified by an acrylic resin board, a silicone resin board, or a polycarbonate resin board, and preferably a quartz plate having a low thermal expansion coefficient. It consists of a silicon substrate.

[0023] The narrow grooves 102 formed from the surface of the substrate 101 of the nanoarray DNA chip are linear or spiral or have a depth of about 1 nm to 2 nm in diameter of the DNA strand and a width of about 2 nm in diameter of the DNA strand. Concentric circular shape, DNA strand fragments and single molecule macro DNA strands (average length: 5cm), X-ray exposure (resolution: 0.5nm) and ion beam exposure After forming a resist film on the substrate surface by the method (resolution: 0.5 nm) or imprint method (resolution: 2 nm), the resist film is etched by ion etching, plasma etching, or chemical etching. It is formed by removing.

[0024] In order to place and arrange a single-stranded or double-stranded DNA strand 103 extending in a narrow groove 102 formed on the surface of a nanoarray DNA chip substrate 101, an atomic force microscope probe is used as a microtweezer. And a method in which nanoparticles are fixed to the ends of DNA strands with functional groups and moved with optical tweezers.

[0025] In order to place and arrange a single-stranded or double-stranded DNA strand 103 extending in a narrow groove 102 formed on the surface of a nanoarray DNA chip substrate 101, an atomic force microscope probe is used as a microtweezer. And a method in which nanoparticles are fixed to the ends of DNA strands with functional groups and moved with optical tweezers.

[0026] To place and arrange the single-stranded or double-stranded DNA strand 103 extending in the narrow groove 102 formed on the surface of the nanoarray DNA chip substrate 101 and to fix it, the entire DNA strand 103 Can be adsorbed, or the chemically modified end of DNA strand 103 (DNA fixed end 104) can be fixed with a functional group.

In the fixing operation, when the entire DNA strand 103 is adsorbed, the inner surface of the narrow groove 102 is made hydrophobic to adsorb the entire DNA strand 103 into the narrow groove 102, or the inside of the narrow groove 102 is hydrophilic. In this case, the DNA strand 103 can be fixed in the narrow groove 102 by adsorbing a divalent cation in the narrow groove 102.

[0028] FIG. 2 is a schematic diagram of a main part showing another example of the nanoarray DNA chip according to the present invention,

(a) is a schematic plan view, (b) is a schematic cross-sectional view in the X direction including a DNA strand, and (c) is a DNA strand. It is a schematic cross-sectional view in the Y direction.

[0029] In the nanoarray DNA chip, narrow grooves 102 are formed in a thin film 201 formed on the surface of a substrate 101, and the ends of DNA strands 103 extended into the narrow grooves 102 are fixed by DNA fixing ends 104. The structure is arranged and arranged linearly.

The substrate 101 of the nanoarray DNA chip is preferably made of a quartz plate or a silicon substrate, in addition to a glass slide, an engineering plastic plate such as an acrylic resin plate, a silicone resin plate or a polycarbonate plate.

[0031] Nano-array DNA chip substrate 101 The material for the thin film 201 on the surface 101 is an engineering plastic material such as acrylic resin, silicone resin, polyimide resin or fluorine resin, rubber-based resin. Silicon oxide (quartz), silicon and other ceramic materials are metal materials and carbon materials. In particular, when forming the thin film 201 shown in the figure by the exposure technique, a photosensitive acrylic resin, silicone resin, polyimide resin or fluorine resin, rubber-based resin is used.

[0032] The thickness of the nanoarray DNA chip The narrow groove 102 formed in the thin film 201 having a thickness of about 1 to 2 nm has a width of about 2 nm in the diameter of the DNA strand and has a predetermined shape such as a linear shape, a spiral shape or a concentric shape X-ray exposure method (resolution: <0.5nm), ion beam exposure method (resolution: 0.5nm) and imprint method (solution) Image power: 2 nm).

[0033] To place and arrange the single-stranded or double-stranded DNA strand 103 in the narrow groove 102 formed in the thin film 201 on the surface of the nanoarray DNA chip substrate 101, an atomic force microscope probe is used. Use micro tweezers or a method in which nanoparticles are fixed to the ends of DNA strands with functional groups and moved with optical tweezers.

[0034] Nanoarray DNA chip substrate 101 To place and arrange single-stranded or double-stranded DNA strands 103 in narrow grooves 102 formed in thin film 201 on surface 101, and to fix and insert DNA strand 103, The entire strand 103 can be adsorbed, or the chemically modified end of the DNA strand 103 (DNA fixed end 104) can be fixed with a functional group.

[0035] When the entire DNA strand 103 is adsorbed in the fixing operation, the inner surface of the narrow groove 102 is made hydrophobic to adsorb the entire DNA strand 103 into the narrow groove 102, or the inside of the narrow groove 102 is hydrophilic. of In this case, the DNA strand 103 can be fixed in the narrow groove 102 by adsorbing a divalent cation in the narrow groove 102.

[0036] FIG. 3 is a schematic diagram of the main part showing another example of the nanoarray DNA chip according to the present invention, ( _a ) is a schematic plan view, and (b) is a schematic cross-sectional view in the X direction including a DNA strand. It is.

That is, nanoparticles 302 are immobilized on the DNA fixed ends 104 of the ends of the DNA strands 103 arranged and arranged in the narrow grooves 102 on the surface of the substrate 101, and the nanoparticles 302 are formed on the substrate 101. Bonded to the pad portion 301.

[0038] The nanoparticle (B) 302 has a nano-sized particle force including gold nanoparticles, glass nanoparticles, and synthetic resin nanoparticles having a diameter of about lOnm.

[0039] The pad portion 301 is made of a thin film including a gold thin film or a synthetic resin thin film having a shape of a thickness of lnm or more and an area of about lOnm square or more.

[0040] The bonding process is performed by pressing the probe tip of the cantilever against the nanoparticle on the pad portion 301 and pressing, thermocompression bonding, or ultrasonic bonding.

[0041] The compatibility between the pad part 301 material and the nanoparticle 302 material is the most reliable and reliable bonding with the combination of gold nanoparticle and gold thin film pad. The combination of glass nanoparticles and gold thin film pads is extremely difficult to bond. The combination of synthetic resin nanoparticles and synthetic resin pads can be bonded fairly reliably. The combination of glass nanoparticles and synthetic resin pad can be bonded with a certain degree of certainty.

FIG. 4 is a schematic diagram of the main part of a disk-shaped nanoarray DNA chip showing an application example according to the present invention, where (a) is a schematic plan view and (b) is a schematic cross-sectional view.

That is, a narrow groove formed concentrically or spirally on the surface of the disk-shaped substrate 101.

The DNA strand 401 in the narrow groove in which the DNA strand 103 is arranged and arranged in 102 is fixed at the DNA fixing end 104.

[0044] The DNA strand 103 may be a force suitable for a single molecule macro DNA strand. If the length of the DNA strand is different, the DNA fixing end 104 is connected to a single DNA strand. It can be fixed to both ends.

[0045] FIG. 5 is a schematic diagram of a main part of a microcylinder-shaped nanoarray DNA chip showing another application example according to the present invention, ( _a ) is a schematic external view, and (b) is a schematic cross-sectional view. is there. [0046] That is, in a micro-cylinder substrate (including a base) 101 of a cylindrical or columnar shape, the inside of a narrow groove in which DNA strands 103 are arranged and arranged in narrow grooves 102 formed concentrically or spirally on the surface of DN. A strand 501 is immobilized at the DNA anchoring end 104.

[0047] The DNA strand 103 may be a force suitable for a single-molecule macro DNA strand. If the length of the DNA strand is different, the DNA fixing end 104 is connected to a single DNA strand. It can be fixed to both ends.

FIG. 7 shows another embodiment of the present invention, in which a V-shaped fine groove 601 is formed from the surface of a substrate 101 made of silicon single crystal, and a DNA strand 103 is arranged in the V-shaped fine groove 601. (A) Cross-sectional view of V-shaped fine groove -1 and 601 are formed from the surface of the silicon single crystal thin film 201 formed on the surface of the substrate 101, and the DNA strand 103 is arranged in the V-shaped fine groove 601. (b) Cross section of V-shaped narrow groove-2.

[0049] The V-shaped fine groove 601 is formed of a (111) crystal plane orientation silicon single crystal substrate or a silicon single crystal thin film 201 having a surface force formed on the substrate surface by various exposure methods, followed by a KOH aqueous solution or the like. V-shaped narrow groove 601 having a V-shaped cross-sectional structure is formed by anisotropic chemical etching using, and after removing the resist film, DNA strand 103 in the bottom direction in the V-shaped narrow groove 601 is dropped and arranged.

[0050] The narrow groove structure is not limited to the V-shaped cross-sectional structure, and may be other cross-sectional structures such as a U-shape and a semicircular shape. The V-shaped cross-sectional structure may be applied to other nanoarray DNA chips. Needless to say.

Claims

The scope of the claims

[1] A nanoarray DNA chip characterized in that a DNA strand is stretched and fixed and arranged and arranged in at least one narrow groove formed on a substrate surface.

[2] A nanoarray DNA chip characterized in that a DNA strand is stretched and fixed in a narrow groove formed on the surface of a thin film on a substrate surface and arranged and arranged in at least one narrow groove.

[3] A nanoarray DNA chip characterized in that nanoparticles are fixed to the ends of the DNA strands according to claims 1 and 2, and the nanoparticles are bonded to a pad formed on the substrate. P.

[4] A nanoarray DNA chip, wherein the nanoparticles according to claim 3 are composed of any one of gold nanoparticles, glass nanoparticles, and synthetic resin nanoparticles.

[5] The nanoarray DNA chip according to claim 3, wherein the pad portion is made of either a gold thin film or a synthetic resin thin film.

[6] A nanoarray DNA chip according to any one of claims 1 to 5, wherein the substrate is a disk-shaped disk, and the fine grooves formed on the disk surface are concentric or spiral.

[7] The nanoarray according to any one of claims 1 to 5, wherein the substrate is a cylindrical microcylinder, and the narrow grooves formed on the surface of the microcylinder are concentric or spiral. DNA chip.

[8] The nanoarray DNA according to claim 7, wherein the substrate is a cylindrical microcylinder substrate, and the fine grooves formed on the surface of the microcylinder are concentric or spiral. Chip.

[9] The narrow groove according to claims 1 and 2 is a V-shaped cross-sectional structure formed on a surface of a silicon single crystal substrate or a silicon single crystal film having a (111) crystal plane orientation. DNA chip.