WO2015068673A1 - Dna搬送制御デバイスおよびその製造方法、ならびにdnaシーケンシング装置 - Google Patents
Dna搬送制御デバイスおよびその製造方法、ならびにdnaシーケンシング装置 Download PDFInfo
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- WO2015068673A1 WO2015068673A1 PCT/JP2014/079162 JP2014079162W WO2015068673A1 WO 2015068673 A1 WO2015068673 A1 WO 2015068673A1 JP 2014079162 W JP2014079162 W JP 2014079162W WO 2015068673 A1 WO2015068673 A1 WO 2015068673A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6872—Methods for sequencing involving mass spectrometry
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/60—Detection means characterised by use of a special device
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
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- G01N2015/0038—Investigating nanoparticles
Definitions
- the present invention relates to a device for controlling the transport of a DNA strand and a method for producing the device.
- the present invention also relates to a DNA sequencing apparatus that reads the base sequence of a DNA strand.
- Nanopore DNA sequencer sequentially identifies base species by directly measuring changes in physical quantities based on individual base species contained in the DNA strand when the DNA strand passes through the nanopore. In addition to being capable of high-speed decoding, it is expected to lead to high throughput, low cost, and long base length decoding because it does not amplify template DNA with an enzyme and does not use a label such as a phosphor. .
- a physical quantity change based on the base species a method of measuring a tunnel current change across the DNA strand when DNA passes through the nanopore, a charge amount of the DNA strand, an ion current passing through the nanopore, and the like have been proposed.
- nanopores play a role in controlling the transport of single-molecule DNA strands.
- the first problem is to provide a method for stably mass-producing nanopores having a size through which only one DNA strand passes.
- the second problem is to delay the speed of the DNA strand passing through the nanopore to a speed sufficient for reading the DNA base sequence.
- the following two approaches have been proposed as a method for solving the above problems.
- Non-Patent Document 4 Polymethylmethacrylate is formed by self-organization of cylindrical and line / space-like fine domains by microphase separation of poly (styrene-b-methylmethacrylate) (PS-b-PMMA), which is a type of block copolymer. A pattern of about 10 nm to 100 nm can be obtained by removing the domain made of by etching. A method of processing a substrate using this fine domain as a mask has been studied.
- Non-Patent Document 5 when a block copolymer composed of a hydrophobic polymer chain and a hydrophilic polymer chain is used, and a fine cylinder composed of a hydrophilic polymer chain is formed by self-organization so as to penetrate the block copolymer thin film, It has been shown that dye molecules permeate through a fine cylinder (Non-Patent Document 5).
- the present invention utilizes the self-assembly of a block copolymer, has a minute size that allows only one molecule of DNA strands to permeate, and allows DNA sequences to be read to a speed at which base sequences can be read.
- An object is to find a method for easily obtaining nanopores capable of delaying the transport speed, and to provide a DNA transport control device excellent in reliability and durability using the method.
- the present inventors prepared a base material having pores, formed a self-organized thin film of a block copolymer on the base material, and formed by self-organization. It was conceived that a DNA passage control path (nanopore) composed of a cylindrical fine domain and a hole in a base material was formed to manufacture a DNA transport control device.
- the DNA transport control device of the present invention has nanopores of a size that allows only one molecule of DNA strands to pass through, and comprises a base material having pores and a block copolymer formed on the base material.
- the thin film is formed of a fine domain penetrating the thin film formed by self-organization of the block copolymer, and a matrix around the fine domain, and the nanopore is formed of the base material. It is composed of one of the apertures and a single fine domain.
- the DNA transport control device of the present invention has nanopores that can delay the transport speed of DNA strands to a speed at which a base sequence can be read, and its manufacturing method is also simple. According to the technique of the present invention, highly accurate nanopores can be stably produced, and thus it is very useful for the production of DNA sequencing devices.
- FIG. 1 is a schematic view showing the structure of a block copolymer thin film 20.
- FIG. It is the figure which expanded the structural unit of the block copolymer thin film 20 typically.
- 3 is a schematic diagram showing a structure of a cylindrical fine domain 22.
- FIG. 2 is a schematic diagram showing a cross-sectional structure of a DNA transport control device 10.
- FIG. It is a figure explaining the method to control the arrangement
- FIG. 1 is a schematic diagram showing a cross-sectional structure of a DNA sequencing apparatus using the DNA transport control device of the present invention.
- Two solution cells 30 containing the electrolyte aqueous solution 33 communicate with each other through the passage 14 (nanopore) of the DNA transport control device 10.
- one solution cell 30 includes a DNA strand 31 as a sample whose sequence is to be read.
- the DNA strand passage path 14 of the DNA transport control device 10 is composed of a base hole 13 and a cylindrical fine domain 22.
- Each solution cell 30 is provided with an electrode 32, and the DNA strand 31 passes through the passage 14 in the DNA transport control device 10 by applying a voltage to both electrodes.
- the sensor When a sensor is required to read the base sequence when the DNA strand 31 passes through the passage 14 of the DNA transport control device 10, the sensor is installed before or after the DNA transport control device 10 or inside thereof. In FIG. 1, the sensor is omitted for simplification. There are no particular restrictions on the means for reading the DNA sequence and the configuration of the sensor therefor. Conventionally proposed are methods for measuring changes in tunneling current across a DNA strand, the amount of charge in the DNA strand, and the degree of blocking of ionic current passing through the passage when the DNA strand 31 passes through the passage route 14 of the DNA strand. Various methods are available.
- spectroscopic methods such as a Raman scattering and infrared absorption.
- a sensor capable of measuring a base sequence based on various methods may be used as appropriate.
- the DNA transport control device 10 of the present invention has a structure in which a block copolymer thin film 20 is formed on a base material 11 having a base hole 13 formed therein.
- having the block copolymer thin film 20 on the base material 11 only means that the block copolymer thin film 20 exists on the upper surface or the lower surface of the base material 11 or on both surfaces of the base material 11.
- the block copolymer thin film 20 exists only in the base hole 13, or the block copolymer thin film 20 is also formed in the base hole 13 in addition to the upper surface, the lower surface, or both surfaces of the base material 11. It is a concept that includes the case where it exists.
- the block copolymer thin film 20 has a cylindrical fine domain 22 penetrating through a thin film formed by self-organization by microphase separation.
- the shape of the fine domain 22 is desirably cylindrical as shown in the figure, but may not be cylindrical as long as it is formed so as to penetrate the thin film.
- the base material 11 is provided with a base hole 13 having a diameter D, and the diameter D is preferably 1 nm or more, and more preferably 30 nm or less.
- the diameter D is preferably 5 nm or more and 25 nm or less.
- it is especially preferable that the diameter D is 1 nm or more and 10 nm or less.
- the film thickness of the base material 11 is preferably 100 nm or less, particularly 50 nm or less so that fine holes can be formed. Further, when applied to an array reading method using a blocking current, 0.3 nm or more, otherwise In this case, the thickness is preferably 10 nm or more so as to have sufficient strength.
- the material of the base material 11 is not particularly limited as long as the base hole 13 can be opened.
- silicon nitride typically is used because it has corrosion resistance against the electrolyte solution 33 and is easy to open.
- the composition is particularly preferably Si 3 N 4 ), silicon oxide (SiO 2 ), hafnium oxide (HfO 2 ), or the like.
- the base material 11 may be used alone, it is preferable to provide a support substrate 12 in order to improve the hardness and handleability of the base material 11 as shown in FIG.
- a polymer chain or a coupling agent may be grafted on the surface of the base material 11 and chemically modified.
- the shape of the base hole 13 is preferably circular, but is not particularly required to be a perfect circle, and may be an ellipse, a polygon, or a shape in which they are distorted.
- the diameter D of the base hole 13 means the diameter of the perfect circle drawn so that it may have the area in the figure.
- the base material 11 can be manufactured according to a known method as disclosed in, for example, Japanese Patent Laid-Open No. 8-248198.
- a silicon nitride or silicon oxide film to be the base material 11 is formed on a silicon wafer to be the support substrate 12, and then the silicon wafer on the back surface is removed by an anisotropic etching technique using a TMAH solution or an aqueous KOH solution to open the window. It can be manufactured by opening it.
- FIB processing using a particle beam such as gallium ion or helium ion
- EB processing using a focused electron beam
- photolithography can be employed.
- Direct processing techniques such as FIB processing and EB processing are suitable when a single base hole 13 is opened.
- FIB processing and EB processing are suitable when a device capable of parallel reading is manufactured by arraying the base holes 13, processing by photolithography is suitable in terms of the time required for processing.
- FIG. 2 is a schematic view showing the structure of the block copolymer thin film 20.
- the block copolymer thin film 20 has a structure in which cylindrical fine domains 22 are formed in a matrix 21 (continuous phase) by microphase separation.
- the block copolymer thin film is formed on the surface of the base material 11 having the base hole 13.
- a part of the cylindrical fine domain 22 is omitted in order to clearly show the positional relationship between the base hole 13 and the cylindrical fine domain 22.
- the block copolymer thin film 20 is formed only on the surface of the base material 11, but as will be described later, the block copolymer thin film 20 is formed on both surfaces of the base material 11 and inside the base hole 13. May be formed.
- the cylindrical fine domains 22 are arranged and distributed in the matrix 21 and are oriented in the direction penetrating the block copolymer thin film 20. As shown in FIG. 2B, the cylindrical fine domains 22 form a regularly arranged pattern so as to have a hexagonal close-packed structure on the horizontal plane of the block copolymer thin film 20.
- the diameter D of the base hole 13 is shown larger than the diameter d of the cylindrical fine domain 22, which has a configuration corresponding to the first embodiment of the present invention described later.
- the diameter D of the base hole 13 is smaller than the diameter d of the cylindrical fine domain 22 in the second embodiment. Since it can be said that the diameter d and the center-to-center distance L of the cylindrical fine domain 22 are substantially determined by the block copolymer to be used, if the diameter d and the center-to-center distance L are measured in advance for each block copolymer, the values thereof are obtained. Based on this, the size of the opening of the base hole 13 can be determined, or an appropriate block copolymer can be selected based on the size of the opening of the base hole 13.
- FIG. 3 is a schematic enlarged view of the structural unit of the block copolymer thin film 20.
- the block copolymer thin film 20 is formed of the block copolymer 40 alone or as a main component thereof.
- the molecule of the block copolymer 40 is a diblock copolymer, a chemical structure in which a hydrophobic polymer chain 41 and a hydrophilic polymer chain 42 are bonded to each end as shown in FIG. have.
- the side surface of the cylinder as shown in FIG. This is preferable because the block copolymer 40 can be easily arranged. Then, with the binding portion of the hydrophobic polymer chain 41 and the hydrophilic polymer chain 42 as a boundary, the matrix 21 having the hydrophobic polymer chain 41 as a main component and the cylinder having the hydrophilic polymer chain 42 as a main component
- the fine domains 22 are formed by self-organization by microphase separation.
- the block copolymer 40 may be synthesized by an appropriate method, but in order to improve the regularity of the microphase-separated structure, a synthesis method that reduces the molecular weight distribution as much as possible, for example, a living polymerization method or an atom transfer radical It is preferable to use a polymerization (ATRP) method.
- the block copolymer 40 may be an AB type diblock copolymer in which a hydrophobic polymer chain 41 and a hydrophilic polymer chain 42 are bonded to each other end as shown in FIG. Alternatively, it may be an ABA type triblock copolymer. Further, it may be an ABC type block copolymer having a third polymer chain and comprising three or more polymer chains. Further, in addition to the block copolymer in which the polymer chains are connected in series, a star-type block copolymer in which each polymer chain is bonded at one point may be used.
- hydrophilic polymer chain 42 constituting the block copolymer 40 examples include polyethylene oxide (PEO), polylactic acid (PLA), polyacrylamide (for example, N, N-dimethylacrylamide), and polyhydroxyalkyl methacrylate (for example, polyhydroxyethyl).
- PEO polyethylene oxide
- PLA polylactic acid
- PLA polyacrylamide
- polyhydroxyalkyl methacrylate for example, polyhydroxyethyl
- Methacrylates PHEMA, etc.
- ionic polymers eg polymers of unsaturated carboxylic acids such as polyacrylic acid or polyacrylmethacrylic acid, nucleic acids, or salts thereof
- polyethylene oxide polylactic acid or The thing which consists of polyhydroxyethyl methacrylate is mentioned.
- hydrophobic polymer chain 41 constituting the block copolymer 40 examples include polystyrene (PS), polyalkyl methacrylate (for example, polymethyl methacrylate (PMMA)), polyvinyl pyridine, polyalkyl siloxane (for example, polydimethyl siloxane), and polyalkyl.
- PS polystyrene
- PMMA polymethyl methacrylate
- PVC polyvinyl pyridine
- polyalkyl siloxane for example, polydimethyl siloxane
- polyalkyl The thing containing dienes (for example, polybutadiene) etc. is mentioned.
- the hydrophobic polymer chain 41 preferably has a side chain (liquid crystalline side chain) which is a rod-like molecule having a mesogenic group for expressing liquid crystallinity.
- Examples of the mesogenic group include a group having a skeleton based on azobenzene, stilbene, benzylideneaniline, biphenyl, naphthalene, or cyclohexane.
- examples of the spacer group bonded to the mesogenic group include an alkyl group, an alkoxy group, and an alkoxyalkyl group.
- the spacer group is preferably linear and preferably has 4 or more carbon atoms.
- the spacer group present between the main chain of the hydrophobic polymer chain and the mesogenic group preferably has 5 or more carbon atoms, particularly 8 or more, and particularly 10 or more.
- hydrophobic polymer chain 41 having such a side chain examples include those in which the alkyl moiety in the polyalkyl methacrylate is partially or entirely substituted with such a liquid crystalline side chain.
- Polyethylene oxide is particularly preferable as the hydrophilic polymer chain 42 combined with the hydrophobic polymer chain 41 having a liquid crystalline side chain.
- the matrix 21 composed of the hydrophobic polymer chain 41 having a liquid crystalline portion exhibits a liquid crystal phase.
- the liquid crystalline side chains are homeotropically oriented with respect to the upper surface (that is, the free surface) of the block copolymer thin film 20, and as a result, the cylindrical fine domains 22 are formed in the block copolymer thin film 20. It becomes easy to orient in the direction which stands upright with respect to the surface and penetrates the thin film.
- the orientation of the cylindrical fine domains 22 often varies depending on the film thickness of the block copolymer thin film 20, the process temperature during self-assembly, the surface condition of the base material, etc., and its control is difficult There is.
- a liquid crystalline block copolymer By using a liquid crystalline block copolymer, it is possible to easily obtain an orientation in a direction in which the cylindrical fine domain 22 stands upright with respect to the block copolymer thin film 20 and penetrates the thin film.
- the self-organized structure of the block copolymer is defined by the composition ratio of each block, that is, the ratio of the volume occupied by each polymer chain constituting the block copolymer.
- the composition ratio increases from 0.5 to 1.0
- the self-organized structure that is, the shape of the domain, changes from a lamellar (plate-like) shape to a cylindrical shape and further to a spherical shape.
- L / d between the center distance L and the diameter d of the cylinder shape while maintaining the cylinder shape, it is necessary to increase the composition ratio.
- the maximum value of L / d is considered to be about 2.5.
- the composition ratio is increased, the domain shape changes from a cylindrical shape to a spherical shape.
- this restriction can be greatly relaxed in a liquid crystalline block copolymer in which a liquid crystalline side chain is introduced into a hydrophobic polymer chain, and a cylindrical domain having an L / d exceeding 10 may be formed. It becomes possible. This is because, in the case of a hydrophobic polymer chain having a liquid crystalline side chain, a conformation in which the polymer chain is linearly stretched by the arrangement of the liquid crystalline portion is formed.
- cylindrical fine domains 22 composed of hydrophilic polymer chains 42 are arranged so as to penetrate the block copolymer thin film 20.
- the diameter d of the cylindrical fine domain 22 can be controlled in the range of 1 nm at the minimum and 100 nm at the maximum according to the molecular weight of the block copolymer used.
- the block copolymer thin film 20 has a structure in which cylindrical fine domains 22 that are hydrophilic, that is, can contain an aqueous solution, are arranged in a matrix 21 that is hydrophobic, that is, not soluble in water.
- the present inventors diligently studied and led to the discovery of the effect of delaying the transport speed of the DNA strand, which is the first effect of the present invention. That is, the DNA strand passes through the cylindrical fine domain 22 in a state immersed in an aqueous solution, and the passage speed is high in water in a micropore not filled with a hydrophilic polymer chain or in a bulk state. The present inventors have found that it is extremely slow compared with the transport speed of the DNA strand in the molecular gel, and completed the present invention.
- the cylindrical fine domains 22 are surrounded by a hydrophobic matrix 21, and the ends of the hydrophilic polymer chains 42 filling the inside thereof are connected to the cylindrical fine domains 22 and hydrophobic. It has a structure fixed to the interface of the matrix 21, that is, the cylinder-shaped side surface of the cylindrical fine domain. It is considered that the density of the hydrophilic polymer chain 42 in the cylindrical fine domain 22 is almost equal to the solid state in the dry state.
- a thin film having such a structure is immersed in an aqueous solution, low molecules such as water and electrolyte contained in the aqueous solution diffuse into the hydrophilic cylindrical domain 22.
- the hydrophilic polymer chain 42 in the cylindrical fine domain 22 has a high density without being significantly swollen. Does not drop. That is, the inside of the cylindrical fine domain 22 is predicted to be a fine space filled with an ultra-high density gel. Whether or not high molecular weight DNA strands can enter and pass through such a fine space has been completely unknown, but the present inventors have conducted various studies and finally made a potential difference before and after the membrane. It was found that the DNA strand passes through the inside of the cylindrical fine domain 22 by being provided.
- the transport speed of the DNA chain is the diameter d of the cylindrical fine domain 22, the height of the cylindrical fine domain 22, that is, the thickness of the block copolymer thin film 20, the density of the hydrophilic polymer chain 42 in the cylindrical fine domain 22, etc. It is possible to control by.
- the diameter d of the cylindrical fine domain 22 needs to be controlled in consideration of the relationship with the base hole 13 as will be described later.
- the thickness of the block copolymer thin film 20 is arbitrary, but in order to stably obtain a structure in which the cylindrical fine domain 22 penetrates the film with good reproducibility, it is 10 nm or more, particularly 20 nm or more, and 500 nm or less. In particular, it is desirable that the range is 100 nm or less.
- a cylindrical shape is most preferable from the viewpoint of controlling the DNA transport speed, but a lamellar microdomain or a co-continuous domain oriented so as to penetrate the thin film is also suitable. Regardless of the difference, it has been found that the effect of delaying the transport speed of DNA strands can be obtained, and it does not exclude application of these structures.
- the block copolymer thin film 20 arranged so that the cylindrical fine domains 22 penetrate can be manufactured by the following method.
- a block copolymer 40 having a predetermined chemical structure and composition is synthesized.
- the living polymerization method or the atom transfer radical polymerization (ATRP) method to the polymerization reaction in terms of controlling the molecular weight, composition, and molecular weight distribution.
- ATRP atom transfer radical polymerization
- the shape, size, distance between domains, and the like vary. Therefore, it is desirable to adjust the polymerization reaction so that the predetermined structure is self-organized.
- the obtained block copolymer 40 is dissolved in a solvent to form a film on the surface of the base material 11 having the base holes 13.
- the solvent is not particularly limited as long as the block copolymer is uniformly dissolved, and a commonly used organic solvent such as toluene or chloroform can be used. Since the block copolymer 40 is amphiphilic, there may be no solvent that can be uniformly dissolved depending on the chemical composition of the polymer chains to be combined. In that case, a mixed solvent obtained by mixing a plurality of solvents may be applied.
- a method such as spin coating or dip coating may be applied as appropriate to the formation of the block copolymer thin film 20.
- the block copolymer thin film 20 is disposed on both surfaces of the base material 11.
- the film forming conditions such as the concentration of the block copolymer solution, the type of solvent, the number of revolutions in the case of spin coating, the number of rotations in the case of dip coating, and the like so that the block copolymer thin film 20 has a predetermined thickness. Good.
- the degree of filling of the block copolymer into the base hole 13 provided in the base material 11 can also be controlled by the film forming conditions.
- the block copolymer molecules 40 in the block copolymer thin film 20 formed by the above method are in a random state with rapid evaporation of the solvent. That is, the microphase separation process of the block copolymer is frozen in the middle, and the target cylindrical fine domains often do not exist in the thin film. Accordingly, the block copolymer thin film 20 formed on the base material 11 is annealed to advance the microphase separation process, and the cylindrical fine domains are regularly formed in a uniform state in the block copolymer thin film. It is good to form a structure arranged in a.
- the annealing process is a process for forming a structure in which the free energy of the thin film is minimized by holding the block copolymer 40 in a state of motion in the block copolymer thin film 20.
- the annealing treatment is a heat treatment (thermal annealing) above the glass transition temperature of the polymer chain constituting the block copolymer 40, or a treatment that swells the film by exposing the block copolymer thin film 20 to solvent vapor (solvent annealing). ) Or the like.
- the cylindrical fine domains 22 need to be self-assembled so as to penetrate the block copolymer thin film 20, but depending on the type of the block copolymer, the orientation of the cylindrical fine domains 22 may be annealed. May be controlled by Therefore, it is desirable to carefully select an annealing method according to the type of block copolymer. For example, when a block copolymer PS-b-PEO having polystyrene (PS) as the hydrophobic polymer chain 41 and polyethylene oxide (PEO) as the hydrophilic polymer chain 42 is used, a cylindrical shape composed of PEO by solvent annealing. Fine domains can be self-assembled to penetrate the PS-b-PEO thin film.
- PS polystyrene
- PEO polyethylene oxide
- the liquid crystalline portion exhibits liquid crystallinity by orienting a randomly dispersed isotropic phase above the liquid crystal transition temperature in a certain direction below the liquid crystal transition temperature. Therefore, when a liquid crystalline block copolymer is used, a uniform microphase separation structure can be obtained by first heating to a temperature higher than the liquid crystal transition temperature and then cooling to a temperature lower than the liquid crystal transition temperature.
- a liquid crystal transition temperature is used. It is preferable that the annealing treatment is performed after being heated to 100 ° C. or higher and then cooled to 90 ° C. which is lower than the liquid crystal transition temperature and higher than the glass transition temperature.
- the fine domains formed by the self-organization of the block copolymer 40 have a periodic structure in which a large number of domains are arranged at a constant period.
- This periodic structure is derived from the principle of microphase separation in which block copolymer molecules undergo phase separation on the molecular order.
- the DNA transport control device 10 is required to transport the DNA strands 31 individually. Therefore, in order to control the transport of the DNA strand 31, the passage path 14 (nanopore) is independently arranged at a predetermined position alone, or at a predetermined position at a predetermined interval for parallel processing. Must be placed.
- the passage path 14 composed of the base hole 13 and the cylindrical fine domain 22 arranged alone or at a predetermined interval at a predetermined position is formed. To do.
- the device 10 in the DNA transport according to the present invention is a block having the above-described characteristics in the base material 11 in which the base hole 13 is opened.
- a copolymer thin film 20 is formed.
- the diameter D of the base hole 13 is larger than the diameter d of the cylindrical fine domain 22.
- the base hole 13 may be processed by a top-down method typified by semiconductor fine processing, and an opening can be disposed at a target position.
- the diameter D of the obtained base hole is limited in terms of processing technology, and as described above, it is difficult to obtain a hole with a single nano-order diameter with good reproducibility.
- the diameter D of the base hole 13, the diameter d of the cylindrical fine domain 22, and the center interval L of the cylindrical fine domain 22 have the relationship of the following formula 1, the cylindrical fine is formed on or inside the base hole 13.
- the domains 22 are arranged in a self-aligned manner, and as a result, a passing path 14 having a smaller diameter than the base hole 13 can be obtained.
- Formula 1 d ⁇ D ⁇ L
- the cylindrical fine domain 22 is formed so as to penetrate the block copolymer thin film 20.
- one cylindrical fine domain 22 is arranged on the base hole 13 in a self-aligning manner.
- the center of the base hole 13 and the cylindrical fine domain 22 do not necessarily coincide with each other, but the DNA chain can pass through the passage 14 constituted by the cylindrical fine domain 22 and the base hole 13. There is no problem in the operation as a transport device.
- FIGS. 5B to 5D can be exemplified as another configuration in which the relationship of Expression 1 is established.
- FIG. 5B shows a state where a part of the block copolymer thin film 20 is also formed inside the base hole 13
- FIG. 5C shows a part of the block copolymer thin film 20 where the base hole 13 is formed.
- FIG. 5D schematically shows a state where the film is formed on both surfaces, and a state where the block copolymer thin film 20 is formed inside the base hole 13.
- a passage 14 having a diameter smaller than the diameter D of the base hole 13 can be easily obtained. Processing of the passage 14 is easier and costs involved in manufacturing a DNA transport control device. Can be expected. Furthermore, since the size of the passage path 14 is defined by the diameter d of the cylindrical fine domain 22, the dimensional reproducibility is extremely good.
- the diameter d of the cylindrical fine domain 22 is preferably 20 nm or less, particularly preferably 10 nm or less, so that a plurality of DNA strands do not enter the cylindrical fine domain at one time. It is preferably 1 nm or more, particularly 1.4 nm or more so that it is difficult for the chain to enter.
- the ratio D / d between the diameter D of the base hole 13 and the diameter d of the cylindrical fine domain 22 is preferably as large as possible. According to Equation 1, for that purpose, it is necessary to increase the ratio L / d between the center-to-center distance L of the cylindrical fine domains 22 and the diameter d of the cylindrical fine domains 22. As described above, the magnitude of the L / d value is governed by the microphase separation phenomenon of the block copolymer, but it can be made larger than usual by applying the liquid crystalline block copolymer.
- the base hole 13 has a function of limiting the DNA strand transported in the passage route 14 to one molecule, and the cylindrical fine domain 22 delays the transport speed of the DNA strand. Contributes only to function.
- the pore diameter limitation is relaxed compared to solid state nanopores that do not have cylindrical microdomains 22.
- the pore diameter is set to 1 to 2 nm, which is equivalent to the size of one molecule of DNA strand.
- the effect of delaying the DNA strand can be expressed by the cylindrical fine domain 22, so that the base hole 13 has a size that can limit the number of DNA strands passing at one time, specifically, For this, it is sufficient if it can be 10 nm or less, particularly 3 nm or less.
- a feature of this embodiment is that the base sequence of DNA can be read by a blocking current method.
- a blocking current method As shown in FIG. 1, when a voltage is applied while the DNA transport control device is immersed in an aqueous solution of a low molecular electrolyte typified by potassium chloride, an ionic current derived from the electrolyte passing through the nanopore flows. . If a DNA strand is contained in the aqueous solution, an event occurs in which the DNA strand passes through the nanopore. When the ion current at this time is measured, the current value changes corresponding to the type of base passing through the nanopore.
- a method of reading the base sequence from the amount of current change is a blocking current method.
- the blocking current method is an excellent method in that it is not necessary to separately prepare a sensor for reading the arrangement.
- the thickness of the nanopore needs to be equal to the length of one base. That is, the nanopores need not only have a small diameter but also a thickness of several nanometers or less. When the diameter of the nanopore has a distribution in the thickness direction, the thickness of the portion with the smallest diameter needs to be several nanometers or less.
- Such ultrathin nanopores can be manufactured by opening a very thin base material made of a material such as graphene as the base material. Note that a material other than graphene can be used in the same manner as long as the thickness of the base material can be several nm. In addition, even a thicker base material can be used in the same manner as long as the nanopore end portion can be processed to be equally thin by processing.
- the thickness of the opening of the base material is about several nanometers, even if the diameter can be an ideal diameter equivalent to the diameter of one molecule of DNA strand, the speed at which the change in current value can be measured. It is difficult to delay the transport speed of the DNA strand.
- the present invention by making the passage route a hybrid composed of the base hole 13 of the base material 11 and the cylindrical fine domain 22 in the block copolymer thin film 20, the effect of delaying the transport speed of the DNA strand can be obtained.
- the DNA transport control device is particularly useful for a DNA sequencing apparatus using a blocking current method.
- FIGS. 5 (e) to 5 (g) show a configuration in which the relationship of Formula 2 of the present embodiment is established.
- FIG. 5 (e) shows a state in which the block copolymer thin film 20 is formed on the surface of the base material 11 having the base hole 13
- FIG. 5 (f) shows the block copolymer thin film 20 on the surface of the base material 11.
- FIG. 5G schematically shows the state in which the block copolymer thin film 20 is formed on both surfaces of the base material 11.
- the diameter d of the cylindrical fine domain 22 is excessively large, the hydrophilic polymer chain in the cylindrical fine domain is likely to swell and the effect of delaying the transport of DNA is not sufficiently exhibited.
- the diameter d is preferably 30 nm or less, and the diameter d is preferably 5 nm or more, and particularly preferably 10 nm or more when aligning the base hole 13 and the cylindrical fine domain 22 described later.
- FIG. 6 is a diagram for explaining a method of controlling the arrangement of the cylindrical fine domains 22 by graphoepitaxy using a partition member.
- graphoepitaxy means that the arrangement of fine domains obtained by self-organization of the block copolymer is controlled by a three-dimensional guide such as the wall surface of the partition member.
- FIG. 6A and 6B show an example in which the partition member 15 is arranged so as to form a hexagonal hollow space on the surface of the base material 11.
- the cylindrical fine domains 22 are self-assembled in a hexagonal arrangement, when the wall surface of the partition member 15 is present, the arrangement corresponds to the shape of the wall surface of the partition member 15, that is, the arrangement formed by the cylindrical fine domains 22.
- the lattice is arranged with the densest orientation along the wall surface. This is due to the effect that the free energy is minimized when the cylinder is closely packed in a limited space. Therefore, when a hexagonal space exists as shown in FIG. 6A, the cylindrical fine domains 22 are arranged along each wall surface of the partition member 15.
- the cylindrical fine domains 22 can be arranged at the center of the space. It can. That is, if the base hole 13 is arranged in the center of the hexagonal space, the base hole 13 and the cylindrical fine domain 22 can be arranged 1: 1. Note that the shape of the space is not necessarily limited to a hexagon, and may be a square or a circle.
- the same graphoepitaxy effect can be obtained even if the space formed by the partition member is not a depression but only a wall surface.
- a hexagonal, quadrangular or circular space may be formed as described above.
- Other methods for controlling the arrangement of the fine domains include a method using an external field such as light and a method of chemically patterning the surface of the base material, which is optimal depending on the design of the DNA transport control device. Apply the method.
- the above arrangement control method can also be used in combination with the first embodiment of the present invention. In that case, the base hole and the cylindrical fine domain can be aligned with higher accuracy.
- the material of the partition member is not particularly limited, and a semiconductor material such as silicon, a metal material typified by gold or titanium, an oxide or nitride thereof, or an organic material such as a polymer or a resist is appropriately used. Can be used.
- self-alignment alignment or graphoepitaxy utilizing a partition member may be used.
- graphoepitaxy using a partition member may be used.
- parallel processing of DNA strand sequence reading can be realized, which is particularly effective for shortening the reading speed.
- Example 1 First embodiment
- the block copolymer includes polymethacrylate having a liquid crystalline side chain having a mesogenic group based on PEO as a hydrophilic polymer chain and azobenzene as a hydrophobic polymer chain.
- PEO-b-PMA (Az) consisting of a derivative (PMA (Az)) was used. Its chemical formula is shown below. (In the formula, m and n in the chemical formula are natural numbers and indicate the degree of polymerization of PEO and PMA (Az)).
- PEO-b-PMA (Az) was polymerized by an atom transfer radical polymerization method according to the method described in Y. Tian et al., Macromolecules 2002, 35, 3739-3747. The degree of polymerization of the obtained block copolymer was evaluated by 1H NMR and GPC. The chemical composition of the polymerized PEO-b-PMA (Az) is as shown in Table 1 below.
- the self-organized structure of the obtained PEO-b-PMA (Az) was evaluated.
- PEO-b-PMA (Az) was dissolved in toluene so as to have a concentration of 1.5% by weight, and spin-coated so that the film thickness was about 50 nm on a Si wafer piece on which a SiN thin film was formed.
- the film thickness of PEO-b-PMA (Az) was measured by an optical film thickness meter (F20 manufactured by Film Metrics), and the film thickness was adjusted by changing the number of revolutions during spin coating.
- the target film thickness was obtained at a rotational speed of about 3000 rpm.
- the obtained sample was introduced into a vacuum oven, and the PEO-b-PMA (Az) thin film was self-assembled by performing a thermal annealing treatment by the following method.
- the sample was left for 1 hour in a state heated to 140 ° C. in a vacuum. At this temperature, it was confirmed by observation with a polarizing microscope that PMA (Az) n formed an isotropic phase.
- the sample was cooled to 90 ° C., and PMA (Az) n was transferred from the isotropic phase to the smectic liquid phase. After standing in this state for 3 hours, it was naturally cooled to complete the self-organization process.
- the self-assembled structure of the obtained sample was observed with a scanning electron microscope (SEM, Hitachi High-Technologies S-4800) or a transmission electron microscope (TEM, Hitachi High-Technologies HF2200). Observation was carried out after staining the PEO phase by exposing the sample to Ru vapor. An example of the obtained microscope image is shown in FIG.
- FIG. 7A is an SEM image of a PEO 114 -b-PMA (Az) 34 thin film in which PEO cylindrical fine domains stained with Ru form a hexagonal close-packed structure in a matrix composed of PMA (Az). I found that it was arranged. According to SEM, PEO stained with Ru generates more secondary electrons than PMA (Az), so that the PEO cylindrical fine domain is white and the matrix PMA (Az) is observed black.
- FIG. 7B is a TEM image of a PEO 40 -b-PMA (Az) 84 thin film, in which a PEO cylindrical fine domain stained with Ru forms a hexagonal close-packed structure in a matrix made of PMA (Az). I found that it was arranged. According to TEM, PEO stained with Ru is harder to transmit electrons than PMA (Az), so that the PEO cylindrical fine domain is observed in black and the matrix PMA (Az) is observed in white.
- FIGS. 8 (a) to 8 (d) A DNA transport control device was fabricated according to the process schematically shown in FIGS. 8 (a) to 8 (d).
- a SiN membrane window manufactured by forming a SiN thin film with a film thickness of 30 nm on a Si wafer as a support substrate and removing a part of the Si wafer by anisotropic etching was purchased as a base material.
- the opening of the SiN thin film was 250 ⁇ m square.
- one base hole was formed in the SiN membrane in the opening by the following method (FIG. 8B).
- a focused ion beam processing apparatus (FIB, FB2200 manufactured by Hitachi High-Technologies Corporation) was used for opening.
- FIB focused ion beam processing apparatus
- the processing time and the number of repetitions thereof were changed, and the conditions under which holes of a desired size could be stably processed were applied.
- the confirmation of the aperture shape and the evaluation of the aperture diameter were carried out by TEM observation. An example of the obtained base hole is shown in FIG.
- the opening is performed by irradiating an electron beam focused on the SiN membrane using a scanning transmission electron microscope (STEM, HD2700 manufactured by Hitachi High-Technologies Corporation) with an acceleration voltage of 200 kV. It was.
- the aperture diameter was adjusted by changing the electron beam irradiation time. The state of opening was confirmed by observing a bright field image using the STEM used for processing.
- PEO-b-PMA (Az) was formed on the surface of the base material where the base holes were opened (FIG. 8C).
- PEO-b-PMA (Az) was dissolved in toluene so as to have a concentration of 1.5% by weight, and spin-coated so that the film thickness was about 40 nm on a Si wafer piece on which a SiN thin film was formed.
- the film thickness of PEO-b-PMA (Az) was measured by an optical film thickness meter (F20 manufactured by Film Metrics), and the film thickness was adjusted by changing the number of revolutions during spin coating.
- the target film thickness was obtained by making rotation speed into about 3000 rpm.
- the obtained sample was thermally annealed using a vacuum oven to self-assemble the PEO-b-PMA (Az) thin film.
- the sample was allowed to stand for 1 hour in a state heated to 140 ° C. in a vacuum, and then cooled to 90 ° C. to transfer PMA (Az) n from the isotropic phase to the smectic liquid phase. After being left for 3 hours in this state, it was naturally cooled to complete the self-organization process (FIG. 8D).
- DNA transport control devices 1-1 to 1-9 were produced.
- the obtained DNA transport control device was observed using STEM (Hitachi High-Technologies HD2700) to confirm the arrangement of the base holes and the cylindrical fine domains.
- STEM Hitachi High-Technologies HD2700
- a bright field image by a transmission electron detector is observed and an image by a secondary electron detector is obtained at the same time, and an image (overlay image) obtained by superimposing these images can be produced.
- the information on the ground hole is obtained from the bright field image, and the information on the arrangement of the cylindrical fine domains is obtained from the secondary electron image. be able to.
- FIG. 1 An example of the obtained overlay image is shown in FIG. This figure is an observation of what was manufactured in accordance with the specifications of sample No. 1-2 in Table 2. From the obtained image, it was confirmed that cylindrical fine domains were arranged in a self-aligned relationship of 1: 1 at the center of the base hole (indicated by a dotted line for clarity). The other samples are also evaluated in the same manner, the arrangement state of the cylindrical fine domains composed of the base holes and the PEO is observed, the positional relationship between the base holes and the cylindrical fine domains, and the cylindrical fine domains arranged on the base holes. The number of evaluated.
- DNA transport control device prepared by the above method was installed in a flow cell made of acrylic resin.
- the flow cell had a solution cell (capacity 90 ⁇ l) on both sides of the DNA transport control device, and a flow path for introducing a liquid was provided inside the solution cell.
- Each solution cell was provided with an Ag / AgCl electrode.
- a DNA sample having a concentration of 2 nM dissolved in a buffer solution was introduced into one side of the solution cell through a flow path, and a buffer solution was introduced into the other side.
- a buffer solution was introduced into the other side.
- double-stranded DNA dsDNA, base length 1k, NoLimits, manufactured by Fermentas
- ssDNA single-stranded DNA
- buffer a mixed solution of 1M KCl, 10 mM Tris-HCl and 1 mM EDTA was used after adjusting to pH 7.5.
- the voltage was applied between the electrodes with a patch clamp amplifier (Axopatch® 200B, manufactured by Axon Instruments), and the time change of the ionic current flowing between the electrodes was measured.
- the signal was digitized and recorded at a sampling frequency of 50 kHz using an AD converter (NI USB-6281, manufactured by National Instruments) after removing high frequency components by a low pass filter (cutoff frequency: 2 kHz).
- FIG. 10 is a graph plotting ion current values observed with respect to time when a buffer solution containing a dsDNA strand was used as a sample.
- the voltage applied between the electrodes was 700 mV.
- spike-like peaks were observed in the ion current that steadily flows as a base. This spike originates from the fact that the ionic current changes when the dsDNA strand passes through the passage of the DNA transport control device.
- FIG. 11 (a) shows an example of measuring spikes in ion current with increased time resolution. From such measurement results, the duration of each spike was evaluated, and the time required for the DNA strand to pass through the passage was measured.
- FIG. 11B shows a distribution diagram produced by measuring the duration of the spikes in a large number of spikes. FIG. 11B shows that the spike duration is normally distributed. When the duration with the highest frequency was determined, the value was 18 msec, and it was found that the average time required for one dsDNA strand to pass through the passage of the DNA transport control device was 18 msec. Since the dsDNA strand used had a base length of 1 k, it was found that the transit time per base was 18 ⁇ sec / base.
- sample No. having a DNA passage of 9 nm in diameter is used.
- 1-2 having one cylindrical fine domain formed by a block copolymer
- Sample No. Comparing 2-1 without the block copolymer thin film layer
- the dsDNA strand passage times were 18 ⁇ sec / base and 0.1 ⁇ sec / base, respectively, and the passage of DNA was caused by the formation of cylindrical microdomains. It can be seen that the time has been increased by 180 times. Further, in the case of the ssDNA strand, it was found that a greater delay effect was obtained, and the passage time could be increased by 4 digits or more due to the cylindrical fine domain formed by the block copolymer. Similar effects are obtained in sample No. 2 having a DNA passage of 2 nm in diameter. 1-6 (having one cylindrical fine domain formed by a block copolymer) and Sample No. It is also clear by comparing 2-2 (without the block copolymer thin film layer).
- a DNA transport control device having a cylindrical fine domain formed of a block copolymer can significantly delay the transport speed of DNA strands compared with a device consisting only of a solid state pore. .
- Example 2 Another aspect of the first embodiment
- a DNA transport control device having a different structure was prepared by the same method as in Example 1 (2), and DNA chain transport evaluation was performed.
- a DNA transport control device was fabricated according to the process schematically shown in FIGS. 8 (a), (b), (c ′) and (d ′).
- Example 2 the obtained sample was thermally annealed in the same manner as in Example 1 to self-assemble the PEO 114 -b-PMA (Az) 34 thin film to form a cylindrical fine domain having a diameter of 9 nm (FIG. 8 (d ′)).
- the transport measure of the DNA strand can be greatly delayed as compared with the device consisting only of the solid state pore.
- Example 3 Second embodiment
- the DNA transport control device of the second embodiment in which the diameter D of the base hole is smaller than the diameter d of the cylindrical fine domain was prepared, and the DNA chain transport evaluation was performed.
- FIGS. 12 (a) to 12 (e) A DNA transport control device was fabricated according to the process schematically shown in FIGS. 12 (a) to 12 (e).
- a Si wafer was prepared by laminating a SiO 2 thin film (thickness 40 nm) and a SiN thin film (thickness 10 nm), and a resist was formed on both sides and patterned.
- the resist on the back side of the Si wafer was used for opening a window in the Si wafer by anisotropic etching of the Si wafer, and the resist on the upper SiO 2 surface was used for patterning the SiO 2 .
- FIG. 12A is a cross-sectional view showing a state in which a 50 ⁇ m square window is opened in a Si wafer with a KOH aqueous solution after patterning a resist on both sides.
- the SiO 2 thin film was etched with buffered hydrofluoric acid using the upper resist as a mask, and a partition member made of SiO 2 was formed on the SiN surface serving as the base material.
- the shape of the partition member was a regular hexagon having a side of 110 nm (a distance between opposing vertices is 220 nm) as schematically shown in FIG. After processing, the resist remaining on both sides was removed.
- FIG. 12C by using a STEM (HD2700, manufactured by Hitachi High-Technologies Corporation), an electron beam converged at an acceleration voltage of 200 kV is irradiated to form a base hole having a diameter of 2 nm in the SiN base material. A hole was opened.
- the processing conditions at this time were determined by optimizing the SiN membrane of the same thickness prepared separately by changing the irradiation conditions.
- the position of the base hole was the center position of a regular hexagon formed by the wall surface of the partition member.
- PEO-b-PMA (Az) was formed on the surface of the SiN base material in the same manner as in Example 1.
- PEO 272 -b-PMA (Az) 94 having a large molecular weight was used as PEO-b-PMA (Az).
- the film formation of PEO 272 -b-PMA (Az) 94 was performed by spin coating so that the film thickness was about 20 nm on the surface of SiO 2 as a partition member. At this time, the film thickness in the space surrounded by the partition members, that is, on the SiN surface was considered to be about 30 nm. Note that the distance 220 nm between the opposing vertices of the hexagonal space surrounded by the partition members was 5 times, that is, an odd multiple, 44 nm, which is the center distance of the cylindrical fine domains.
- a thermal annealing process is performed in the same manner as in Example 1, and PEO 272 -b-PMA (Az) 94 is self-organized, so that Cylindrical fine domains were formed inside the square space.
- Example 4 Another aspect of the second embodiment
- a DNA transport control device having a different structure was produced by the same method as in Example 3 (1), and DNA chain transport evaluation was performed.
- the same PEO 272 -b-PMA (Az) 94 as in Example 3 is formed on the surface of the SiO 2 that is the partition member so that the film thickness is twice that of Example 3.
- the transport measure of the DNA strand can be greatly delayed as compared with the device consisting only of the solid state pore.
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Abstract
Description
図1は、本発明のDNA搬送制御デバイスを用いたDNAシーケンシング装置の断面構造を示す模式図である。電解質水溶液33を含有する2つの溶液セル30が、DNA搬送制御デバイス10の通過経路14(ナノポア)を介して連通している。ここで、片方の溶液セル30には配列を読み取るべき試料であるDNA鎖31が含まれている。DNA搬送制御デバイス10のDNA鎖の通過経路14は、下地孔13とシリンダ状微細ドメイン22から構成される。また、各溶液セル30には電極32が設置されており、両極に電圧を印加することにより、DNA搬送制御デバイス10中の通過経路14をDNA鎖31が通過する。
本発明のDNA搬送制御デバイス10は、下地孔13を開孔した下地材11の上にブロック共重合体薄膜20が製膜された構造を有する。なお、本明細書において、下地材11の上にブロック共重合体薄膜20を有するとは、下地材11の上面もしくは下面またはその両面の平面上にブロック共重合体薄膜20が存在する場合のみならず、下地孔13の中にのみブロック共重合体薄膜20が存在する場合や、下地材11の上面もしくは下面または両面の平面上に加えて下地孔13の中にもブロック共重合体薄膜20が存在する場合をも含む概念である。ブロック共重合体薄膜20は、ミクロ相分離により自己組織化することで形成された薄膜を貫通するシリンダ状微細ドメイン22を有する。なお、微細ドメイン22の形状は図示するようにシリンダ状であることが望ましいが、薄膜を貫通するように形成されている限りシリンダ状でなくてもよい。
図2は、ブロック共重合体薄膜20の構造を示す概略図である。図2(a)および(b)に示すように、ブロック共重合体薄膜20には、ミクロ相分離により、マトリックス21(連続相)の中にシリンダ状微細ドメイン22が形成された構造を有する。ブロック共重合体薄膜は、下地孔13を有する下地材11の表面に形成されている。なお、図2(a)では、シリンダ状微細ドメイン22の一部を、下地孔13とシリンダ状微細ドメイン22との位置関係を明瞭に示すために省略して図示している。図2(a)ではブロック共重合体薄膜20は下地材11の表面のみに製膜されているが、後述するように、下地材11の両面や下地孔13の内部にブロック共重合体薄膜20が形成されていてもよい。
図3に示したように、ブロック共重合体薄膜20には親水性高分子鎖42からなるシリンダ状微細ドメイン22が、ブロック共重合体薄膜20を貫通するように配列している。シリンダ状微細ドメイン22の直径dは、用いるブロック共重合体の分子量により、最小で1nm、最大で100nmの範囲で制御することが可能である。ブロック共重合体薄膜20は、疎水性、すなわち水に溶解しないマトリックス21中に、親水性、すなわち水溶液を含むことができるシリンダ状微細ドメイン22が配列した構造を有している。
シリンダ状微細ドメイン22が貫通するように配列したブロック共重合体薄膜20は以下の方法により製造することができる。
図2に模式的に示したように、ブロック共重合体40が自己組織化することで形成する微細ドメインは多数のドメインが一定周期で配列した周期構造となる。本周期構造はブロック共重合体分子が分子オーダーで相分離するというミクロ相分離の原理に由来している。
まず、第一の実施形態について図5(a)~(d)を適宜参照しながら説明する。本実施形態によれば、本発明の第一の効果であるDNA鎖の搬送遅延化に加え、下地孔のサイズより微細な通過経路を得ることができるという第二の効果を同時に得ることができる。
式1: d < D < L
次に、本発明の第二の実施形態について図5(e)~(g)を適宜参照しながら説明する。第二の実施形態は、第一の実施形態とは異なり、下地孔13の直径Dがシリンダ状微細ドメイン22の直径dより小さいことを特徴とする。
式2: d > D
前述したように、本発明の第一の実施形態および第二の実施形態のいずれにおいても、下地孔13とシリンダ状微細ドメイン22を1:1で配置することが可能である。この特徴を活用すれば任意の位置にDNA鎖を搬送する複数の通過経路14が配置されたマルチポアDNA搬送制御デバイスを得ることができる。具体的には任意の位置に複数の下地孔13を設け、その表面にブロック共重合体薄膜を製膜し、シリンダ状微細ドメイン22が下地孔に対して1:1で配置されるようにする。その際、下地孔13とシリンダ状微細ドメイン22の位置合わせには上述した各種方法を適用すればよい。すなわち、本発明の第一の実施形態の場合には自己整合的な位置合わせもしくは仕切り部材を活用したグラフォエピタキシーを利用すればよい。また、第二の実施形態の場合には、仕切り部材を活用したグラフォエピタキシーを利用すればよい。マルチポアDNA搬送制御デバイスによれば、DNA鎖の配列読取の並列処理を実現でき、読取り速度の短縮化に特に効果的である。
(1)液晶性ブロック共重合体の合成と評価
ブロック共重合体には、親水性高分子鎖としてPEOを、疎水性高分子鎖としてアゾベンゼンに基づくメソゲン基を有する液晶性側鎖を有するポリメタクリレート誘導体(PMA(Az))からなるPEO-b-PMA(Az)を用いた。その化学式を以下に示す。
図8(a)~(d)に模式的に示したプロセスに従ってDNA搬送制御デバイスを製作した。まず、サポート基板であるSiウエハ上に、膜厚が30nmのSiN薄膜を製膜し、Siウエハの一部を異方エッチングにより除去して製造されたSiNメンブレンウインドウを購入し、下地材とした(図8(a))。SiN薄膜の開口部は250μm角であった。次に、開口部のSiNメンブレンに下地孔を1つ、以下の方法で開孔した(図8(b))。
上記方法で作製したDNA搬送制御デバイスをアクリル樹脂で作製したフローセル内に設置した。フローセルはDNA搬送制御デバイスの両側に溶液セル(容量90μl)を有し、溶液セル内には内部に液体を導入するための流路を設けた。また、各溶液セルにはAg/AgCl電極を設置した。
実施例1(2)と同等の方法で、異なる構造を有するDNA搬送制御デバイスを作製し、DNA鎖搬送評価を行った。
まず、図8(a)、(b)、(c’)および(d’)に模式的に示したプロセスに従ってDNA搬送制御デバイスを製作した。表2のサンプルNo.1-2と同じ下地材(下地孔直径D=21nm)を用意し(図8(a)および(b))、その下地材の上にPEO114-b-PMA(Az)34薄膜を実施例1よりも薄く、5nmの厚さで製膜した(図8(c’))。次に、得られたサンプルを実施例1と同様に熱アニール処理して、PEO114-b-PMA(Az)34薄膜を自己組織化させ、直径9nmのシリンダ状微細ドメインを形成させた(図8(d’))。
上記のように作製したデバイスを用い、実施例1と同様にDNA鎖搬送評価を行った。その結果、実施例1と同様に、dsDNA鎖(塩基長1k)の一塩基あたりの通過時間は18μsec/塩基、ssDNA鎖(塩基長5k)の一塩基あたりの通過時間は7μsec/塩基であり、これらの値はソリッドステートポアのみからなるDNA搬送制御デバイス(サンプルNo2-1および2-2)と比較して非常に大きな値となった。
第一の実施形態とは異なり、下地孔の直径Dがシリンダ状微細ドメインの直径dよりも小さい第二の実施形態のDNA搬送制御デバイスを作製しDNA鎖搬送評価を行った。
図12(a)~(e)に模式的に示したプロセスに従ってDNA搬送制御デバイスを製作した。SiO2薄膜(厚み40nm)とSiN薄膜(厚み10nm)が積層されたSiウエハを準備し、その両面にレジストを製膜しパターニングした。ここで、Siウエハ裏面側のレジストはSiウエハの異方エッチングによりSiウエハにウインドウを開孔するために用い、上部SiO2表面のレジストはSiO2をパターニングするために用いた。図12(a)は、レジストを両面にパターニングした後にKOH水溶液によりSiウエハに50μm角のウインドウを開孔した状態の断面図を示している。
上記方法で作製したデバイスを用い、実施例1および2と同様にDNA鎖搬送評価を行った。その結果、ssDNA鎖(塩基長5k)の一塩基あたりの通過時間は9μsec/塩基であった。一方、図12(c)までの手順で得られた下地孔のみからなるサンプルを用いて測定を行ったところ、ssDNA鎖(塩基長5k)の一塩基あたりの通過時間は1×10-4μsec/塩基であった。すなわち、同一サイズの孔であっても、シリンダ状微細ドメインが1:1で配置されたことにより通過時間を9×104倍に大幅に遅延化したことがわかった。
実施例3(1)と同等の方法で、異なる構造を有するDNA搬送制御デバイスを作製し、DNA鎖搬送評価を行った。
図12(a)~(c)、(d’)および(e’)に模式的に示したプロセスに従ってDNA搬送制御デバイスを製作した。図12(c)までは実施例3と同様にして、直径D=2nmの下地孔を厚み10nmのSiN薄膜に開孔した。次に、図12(d’)に示すように、実施例3で同じPEO272-b-PMA(Az)94を、仕切り部材であるSiO2表面上に、膜厚が実施例3の倍である40nmとなるようスピンコート法により製膜した。その後、実施例3と同様に熱アニールを実施し、PEO114-b-PMA(Az)34薄膜を自己組織化させて、直径d=20nmのシリンダ状微細ドメインを形成させた。
上記方法で作製したデバイスを用い、実施例1~3と同様にDNA鎖搬送評価を行った。その結果、ssDNA鎖(塩基長5k)の一塩基あたりの通過時間は12μsec/塩基であった。この値は、実施例3のデバイスと比較して若干大きなものとなったが、これは実施例4のデバイス構成では実施例3と比較して通過経路を構成しているシリンダ状微細ドメインの高さが大きく、より大きな遅延効果が得られたためであると考えられる。
11 下地材
12 サポート基板
13 下地孔
14 通過経路
15 仕切り部材
16 ノッチ
20 ブロック共重合体薄膜
21 マトリックス
22 シリンダ状微細ドメイン
30 溶液セル
31 DNA鎖
32 電極
33 電解質溶液
40 ブロック共重合体
41 疎水性高分子鎖
42 親水性高分子鎖
43 固定点
51 SiN薄膜
52 シリコンウエハ
53 PEO-b-PMA(Az)薄膜
54 PMA(Az)マトリックス
55 PEOシリンダ状微細ドメイン
56 PEO-b-PMA(Az)残膜
57 SiO2薄膜
58 レジスト
Claims (15)
- 一分子のDNA鎖のみが通過可能なサイズのナノポアを有するDNA搬送制御デバイスであって、
開孔を有する下地材と、前記下地材の上に形成されたブロック共重合体の薄膜とを有し、
前記薄膜は、前記ブロック共重合体の自己組織化により形成された、前記薄膜を貫通する微細ドメインと、その周囲のマトリックスとから構成され、
前記ナノポアが、前記下地材の開孔の一つと、単一の前記微細ドメインとから構成されていることを特徴とする、前記デバイス。 - ブロック共重合体が親水性高分子鎖と疎水性高分子鎖からなり、微細ドメインが親水性高分子鎖により、その周囲のマトリックスが疎水性高分子鎖によりそれぞれ形成されている、請求項1に記載のDNA搬送制御デバイス。
- 疎水性高分子鎖が液晶性側鎖を有する、請求項2に記載のDNA搬送制御デバイス。
- 微細ドメインがシリンダ状である、請求項1~3のいずれか1項に記載のDNA搬送制御デバイス。
- 下地材の開孔の直径Dが、シリンダ状微細ドメインの直径dよりも大きく、かつシリンダ状微細ドメインの中心間距離Lよりも小さい、請求項4に記載のDNA搬送制御デバイス。
- 下地材の開孔の直径Dが、シリンダ状微細ドメインの直径dよりも小さい、請求項4に記載のDNA制御デバイス。
- さらに下地材の上に設けられた仕切り部材を有し、微細ドメインが前記仕切り部材の形状に対応する配列を有する、請求項1~3のいずれか1項に記載のDNA制御デバイス。
- 親水性高分子鎖がポリエチレンオキシド、ポリ乳酸、ポリヒドロキシアルキルメタクリレート、または核酸を含む、請求項2または3に記載のDNA制御デバイス。
- 疎水性高分子鎖が、ポリアルキルメタクリレートにおけるアルキル部分が部分的にまたは全て液晶性側鎖に置換された構造を有する、請求項3に記載のDNA制御デバイス。
- 一分子のDNA鎖のみが通過可能なサイズのナノポアを有するDNA搬送制御デバイスであって、
開孔を有する下地材と、前記下地材の上に形成された薄膜とを有し
前記薄膜は、前記薄膜を貫通する微細ドメインと、その周囲のマトリックスとから構成され、
前記微細ドメインには親水性高分子鎖が充填されており、かつ前記親水性高分子鎖は前記微細ドメインとマトリックスの界面に固定されており、
前記ナノポアが、前記下地材の開孔の一つと、単一の前記微細ドメインとから構成されていることを特徴とする、前記デバイス。 - 一分子のDNA鎖のみが通過可能なサイズのナノポアを有するDNA搬送制御デバイスの製造方法であって、
開孔を有する下地材を用意する工程、
前記下地材の上にブロック共重合体の薄膜を製膜する工程、および
前記ブロック共重合体を自己組織化させて、前記薄膜を貫通する微細ドメインとその周囲のマトリックスとを形成させることにより、前記下地材の開孔の一つと単一の前記微細ドメインとから構成されるナノポアを形成する工程
を含む、前記方法。 - ブロック共重合体の自己組織化により形成される微細ドメインがシリンダ状であり、その直径および中心間距離を予め測定し、その値に基づいて下地材の開孔の大きさを決定することを含む、請求項11に記載の方法。
- 下地材の上に仕切り部材を設ける工程をさらに含み、仕切り部材に沿って微細ドメインを形成させることにより、下地材の開孔と微細ドメインの位置合わせを行うことを含む、請求項11または12に記載の方法。
- 請求項1~3のいずれか1項に記載のDNA搬送制御デバイスと、前記DNA搬送制御デバイスのナノポアを介して連通する二つの溶液セルと、各溶液セルに設けられた前記二つの溶液セルの間に電圧を印加するための電極とを有するDNAシーケンシング装置。
- DNA搬送制御デバイスの内部または近傍にナノポアを通過するDNA鎖を読み取るためのセンサを有する、請求項14に記載のDNAシーケンシング装置。
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016038998A1 (ja) * | 2014-09-12 | 2016-03-17 | 株式会社日立ハイテクノロジーズ | 生体ポリマ分析デバイス及び分析システム |
WO2017104398A1 (ja) * | 2015-12-17 | 2017-06-22 | 株式会社日立ハイテクノロジーズ | 生体分子測定装置 |
JP2017116379A (ja) * | 2015-12-24 | 2017-06-29 | 株式会社日立ハイテクノロジーズ | 生体ポリマを分析するための測定試薬及び分析デバイス |
WO2018016117A1 (ja) * | 2016-07-19 | 2018-01-25 | 株式会社日立製作所 | 生体分子分析用電解質溶液,生体分子分析用デバイス及び生体分子分析装置 |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009057519A (ja) * | 2007-09-03 | 2009-03-19 | Tokyo Institute Of Technology | ミクロ相分離構造膜、及びその製造方法 |
WO2013012881A2 (en) * | 2011-07-20 | 2013-01-24 | The Regents Of The University Of California | Dual-pore device |
WO2013011879A1 (ja) * | 2011-07-19 | 2013-01-24 | 株式会社日立製作所 | 分析装置及び分析システム |
WO2013140316A2 (en) * | 2012-03-22 | 2013-09-26 | Koninklijke Philips N.V. | Manufacturing method of an apparatus for the processing of single molecules |
WO2014208184A1 (ja) * | 2013-06-28 | 2014-12-31 | 株式会社 日立ハイテクノロジーズ | 分析装置 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08248198A (ja) | 1995-03-13 | 1996-09-27 | Nikon Corp | 酸化シリコンメンブレンの作製方法 |
WO2001081896A1 (en) * | 2000-04-24 | 2001-11-01 | Eagle Research & Development, Llc | An ultra-fast nucleic acid sequencing device and a method for making and using the same |
US6503409B1 (en) * | 2000-05-25 | 2003-01-07 | Sandia Corporation | Lithographic fabrication of nanoapertures |
SG173398A1 (en) * | 2006-07-19 | 2011-08-29 | Bionanomatrix Inc | Nanonozzle device arrays: their preparation and use for macromolecular analysis |
US9034637B2 (en) * | 2007-04-25 | 2015-05-19 | Nxp, B.V. | Apparatus and method for molecule detection using nanopores |
EP3196645B1 (en) | 2009-09-18 | 2019-06-19 | President and Fellows of Harvard College | Bare single-layer graphene membrane having a nanopore enabling high-sensitivity molecular detection and analysis |
JP5988258B2 (ja) * | 2011-07-29 | 2016-09-07 | 凸版印刷株式会社 | 微細構造体の製造方法、複合体の製造方法 |
CN102901763B (zh) * | 2012-09-25 | 2014-06-11 | 清华大学 | 基于石墨烯纳米孔-微腔-固态纳米孔的dna测序装置及制作方法 |
CN103193189B (zh) * | 2013-02-21 | 2015-08-26 | 东南大学 | 一种用于dna检测的多电极纳米孔装置及其制造方法 |
JP6472208B2 (ja) * | 2014-10-24 | 2019-02-20 | 株式会社日立ハイテクノロジーズ | 核酸搬送制御デバイス及びその製造方法、並びに核酸シーケンシング装置 |
-
2014
- 2014-11-04 DE DE112014004341.9T patent/DE112014004341B4/de not_active Expired - Fee Related
- 2014-11-04 GB GB1606151.7A patent/GB2534737B/en not_active Expired - Fee Related
- 2014-11-04 WO PCT/JP2014/079162 patent/WO2015068673A1/ja active Application Filing
- 2014-11-04 CN CN201480049886.7A patent/CN105531360B/zh active Active
- 2014-11-04 US US15/029,712 patent/US10253362B2/en active Active
- 2014-11-04 JP JP2015546634A patent/JP6208253B2/ja not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009057519A (ja) * | 2007-09-03 | 2009-03-19 | Tokyo Institute Of Technology | ミクロ相分離構造膜、及びその製造方法 |
WO2013011879A1 (ja) * | 2011-07-19 | 2013-01-24 | 株式会社日立製作所 | 分析装置及び分析システム |
WO2013012881A2 (en) * | 2011-07-20 | 2013-01-24 | The Regents Of The University Of California | Dual-pore device |
WO2013140316A2 (en) * | 2012-03-22 | 2013-09-26 | Koninklijke Philips N.V. | Manufacturing method of an apparatus for the processing of single molecules |
WO2014208184A1 (ja) * | 2013-06-28 | 2014-12-31 | 株式会社 日立ハイテクノロジーズ | 分析装置 |
Non-Patent Citations (3)
Title |
---|
HAQUE, F. ET AL.: "Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA", NANO TODAY, vol. 8, no. 1, February 2013 (2013-02-01), pages 56 - 74 * |
YAMAMOTO, T. ET AL.: "Block Copolymer Permeable Membrane with Visualized High-Density Straight Channels of Poly(ethylene oxide", ADV. FUNCT. MATER., vol. 21, no. ISSUE, 2011, pages 918 - 926 * |
YANG, SY. ET AL.: "DNA-functionalized nanochannels for SNP detection", NANO LETT., vol. 11, no. 3, 2011, pages 1032 - 1035 * |
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GB2549187B (en) * | 2014-09-12 | 2021-05-19 | Hitachi High Tech Corp | Biopolymer analysis device and analysis system |
WO2016038998A1 (ja) * | 2014-09-12 | 2016-03-17 | 株式会社日立ハイテクノロジーズ | 生体ポリマ分析デバイス及び分析システム |
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GB2560668A (en) * | 2015-12-17 | 2018-09-19 | Hitachi High Tech Corp | Biomolecule measurement apparatus |
WO2017104398A1 (ja) * | 2015-12-17 | 2017-06-22 | 株式会社日立ハイテクノロジーズ | 生体分子測定装置 |
GB2560668B (en) * | 2015-12-17 | 2022-08-24 | Hitachi High Tech Corp | Biomolecule measurement apparatus |
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WO2017110226A1 (ja) * | 2015-12-24 | 2017-06-29 | 株式会社日立ハイテクノロジーズ | 生体ポリマを分析するための測定試薬及び分析デバイス |
JP2017116379A (ja) * | 2015-12-24 | 2017-06-29 | 株式会社日立ハイテクノロジーズ | 生体ポリマを分析するための測定試薬及び分析デバイス |
US10996211B2 (en) | 2015-12-24 | 2021-05-04 | Hitachi High-Tech Corporation | Measuring reagent and analysis device for analyzing biopolymer |
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WO2018016117A1 (ja) * | 2016-07-19 | 2018-01-25 | 株式会社日立製作所 | 生体分子分析用電解質溶液,生体分子分析用デバイス及び生体分子分析装置 |
JP2018011532A (ja) * | 2016-07-19 | 2018-01-25 | 株式会社日立製作所 | 生体分子分析用電解質溶液,生体分子分析用デバイス及び生体分子分析装置 |
WO2018196307A1 (zh) * | 2017-04-28 | 2018-11-01 | 京东方科技集团股份有限公司 | 基因测序芯片、装置以及方法 |
WO2019000158A1 (zh) * | 2017-06-26 | 2019-01-03 | 武汉科技大学 | 一种基于隧道识别技术的纳米检测装置及方法 |
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