WO2021024416A1 - Flow cell adjustment method - Google Patents

Flow cell adjustment method Download PDF

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Publication number
WO2021024416A1
WO2021024416A1 PCT/JP2019/031122 JP2019031122W WO2021024416A1 WO 2021024416 A1 WO2021024416 A1 WO 2021024416A1 JP 2019031122 W JP2019031122 W JP 2019031122W WO 2021024416 A1 WO2021024416 A1 WO 2021024416A1
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flow cell
biopolymer
adjusting
substrate
particles
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PCT/JP2019/031122
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French (fr)
Japanese (ja)
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奈良原　正俊
田村　輝美
小林　紀子
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株式会社日立ハイテク
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Priority to PCT/JP2019/031122 priority Critical patent/WO2021024416A1/en
Publication of WO2021024416A1 publication Critical patent/WO2021024416A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • the present invention relates to a flow cell adjusting technique for analyzing a biopolymer.
  • a new technology for determining the base sequence of DNA or RNA has been developed.
  • a cDNA fragment sample synthesized by reverse transcription reaction from a DNA fragment for sequencing or an RNA sample is prepared in advance, and after a dideoxy reaction by a well-known Sanger method.
  • Electrophoresis is performed, and the molecular weight separation and development pattern is measured to determine the base sequence.
  • Non-Patent Document 1 a large number of DNA probes having the same sequence are fixed on the substrate. After cutting the DNA sample, the DNA probe sequence and the adapter sequence of the complementary strand are added to the end of each DNA sample fragment. By hybridizing these on a substrate, sample DNA fragments are randomly immobilized one molecule at a time on the substrate.
  • a fluorescence method is disclosed in which a substrate with a fluorescent dye that does not cause a DNA elongation reaction on the substrate is incorporated, and then the unreacted substrate is washed or fluorescence is detected to obtain sequence information of sample DNA.
  • Patent Document 1 the spots to which the sample DNA binds are arranged in a grid on the substrate.
  • a silicon wafer is used as the substrate, and after forming a hydrophobic HMDS (Hexamethyldisilizane) layer on the silicon wafer, a positive resist is applied.
  • the opening is provided at a predetermined position using photolithography technology, the HMDS at the bottom of the opening is removed.
  • the wafer is held in the gas phase of aminosilane, and aminosilane is introduced into the bottom of the opening.
  • dicing is performed to cut out the substrate.
  • a cover glass is attached via a polyurethane adhesive to prepare a flow cell for nucleic acid analysis.
  • aminosilane which is highly hydrophilic and can fix DNA, as the DNA ball fixing spot
  • hydrophobic HMDS which prevents DNA adsorption in other regions, when a solution containing DNA balls is introduced onto the substrate, DNA naturally occurs. It makes it possible to fix the ball only on the spot.
  • Patent Document 1 As a method for fixing a DNA sample to a spot, in Patent Document 1, after introducing a solution in which DNA balls are dispersed onto a substrate, the substrate is held in a shaken state for a certain period of time, and then the substrate is rinsed. The unfixed DNA balls are washed away and the DNA balls are fixed on the spot.
  • the biopolymer at this time is a DNA ball or protein having a large number of amplified copies.
  • the number of fluorescent dyes taken in during the sequence reaction decreases, and the number of DNA balls effective for the sequence decreases.
  • the efficiency of the sequence reaction in one cycle is not necessarily 100%, so that the reaction efficiency of the sequence reaction becomes low, the fluorescent dye taken in during the cycle is further reduced, and the reading base length becomes long. There is also the issue of shortening.
  • a metal such as gold or iron oxide or a metal oxide is generally used as the fine particle, and these metals or metal oxidation Objects often absorb light in the visible region and emit autofluorescence, and the autofluorescence emitted by the fine particle carrier becomes noise during fluorescence detection, which is often a problem for fluorescence detection.
  • the present invention solves the above-mentioned problems, and by fixing a biopolymer having a large molecular weight on a substrate with a high fixation rate, the density of samples fixed on the substrate can be increased and the biopolymer can be analyzed per unit area. It is an object of the present invention to provide a flow cell having a substrate that increases the number of molecules, does not have a fine particle carrier that causes noise at the time of detection, and enables highly accurate analysis.
  • the present invention is a method for adjusting a flow cell for analyzing a biopolymer, in which a step of injecting a solution in which a biopolymer is dissolved into the flow cell and a substrate constituting the flow cell
  • a flow cell adjusting method consisting of a step of centrifuging a flow cell in which a solution in which a biopolymer is dissolved is injected and a step of washing out a biopolymer unfixed on a substrate from the flow cell in order to fix the biopolymer on the surface.
  • a flow cell adjusting method for analyzing a biopolymer in which a solution in which the biopolymer is dissolved and particles are dispersed is injected into the flow cell. And the step of holding the flow cell infused with the solution in which the biopolymer is dissolved and the particles are dispersed for a certain period of time in order to fix the biopolymer on the substrate constituting the flow cell, and the step of not being on the substrate.
  • a method for adjusting a flow cell which comprises a step of washing out fixed biopolymers and particles from the flow cell.
  • a flow cell adjusting method for analyzing a biopolymer in which a solution in which the biopolymer is dissolved and particles are dispersed is injected into the flow cell. And the step of centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed is centrifuged in order to fix the biopolymer on the substrate constituting the flow cell, and the unfixed bio-height.
  • a method for adjusting a flow cell which comprises a step of washing out molecules from the flow cell.
  • the present invention by fixing a biopolymer having a large molecular weight on a substrate with a high fixation rate, the density of samples fixed on the substrate is increased, and the number of biopolymers that can be analyzed per unit area is increased. Moreover, it is possible to provide a flow cell having a substrate that enables highly accurate analysis.
  • FIG. 1 It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1.
  • FIG. It is a figure for demonstrating an example of the flow cell which concerns on Example 1.
  • FIG. It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1.
  • FIG. It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1.
  • FIG. It is a figure for demonstrating an example of the flow cell adjustment method which concerns on Example 1.
  • the biopolymer in the solution comprises a step of centrifuging the flow cell in which the solution in which the biopolymer is dissolved is injected and a step of washing out the biopolymer unfixed on the substrate from the flow cell.
  • centrifugal force By applying centrifugal force, the probability that the biopolymer comes into contact with the spot is increased, and the proportion of the spot where the biopolymer is fixed is increased.
  • washing out the biopolymer unfixed on the substrate from the flow cell there is no fine particle carrier on the substrate that causes noise at the time of detection, so that highly accurate detection is possible by a fluorescence method or the like.
  • the biopolymer is a nucleic acid amplification product obtained from a cyclic template. Since the nucleic acid amplification product obtained from the cyclic template can increase the amplified copy number, the fluorescent dye incorporated in the sequence reaction can be increased, and the read base length can be lengthened. Moreover, the molecular weight can be increased by increasing the amplified copy number.
  • the biopolymer is fixed on the substrate via spots.
  • the spots By arranging the spots at a high density and fixing one biopolymer for each spot, the biopolymer can be fixed at a high density on the substrate.
  • the unfixed biopolymer and the particles are washed out from the flow cell.
  • the biopolymer is pressed against the particles that settle under the force of gravity, increasing the probability that the biopolymer and the spot come into contact with each other. it can.
  • the particle carrier that causes noise does not exist on the substrate, and highly accurate fluorescence detection becomes possible.
  • the unfixed biopolymer and the particles are washed out from the flow cell.
  • the biopolymer is a nucleic acid amplification product obtained from a cyclic template.
  • the unfixed biopolymer and the particles are washed out from the flow cell.
  • the biopolymer is fixed on the substrate via spots. Due to the effect of the particles pressing by gravity, the weight of the high molecular weight, and the effect of high-density fixation by arranging the spots at high density and fixing one biopolymer per spot, on the substrate.
  • the biopolymer can be fixed at a higher density.
  • the unfixed biopolymer and the particles are washed out from the flow cell.
  • the particles are characterized by being composed of two or more types of particles having different particle size distributions.
  • the biopolymer is dissolved, and at the same time, the flow cell in which the solution in which the particles are dispersed is injected is centrifuged, and then the unfixed biopolymer is washed out from the flow cell. ..
  • the biopolymer can be pressed against the spot by using the centrifugal force applied to the particles, and the probability of contact between the biopolymer and the spot can be further increased.
  • the unfixed biopolymer is washed out from the flow cell after centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed at the same time is injected, and the biopolymer is formed.
  • It is a nucleic acid amplification product obtained from a cyclic template.
  • the molecular weight can be increased by increasing the number of amplified copies, and the probability of contact between the substrate and the biological sample can be further increased by the large centrifugal force applied to the nucleic acid amplification product itself and the force pushed by the centrifugal force applied to the particles.
  • the unfixed biopolymer is washed out from the flow cell after centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed at the same time is injected, and the biopolymer is formed. It is characterized in that it is fixed on the substrate via a spot. Higher due to the centrifugal force applied to the biopolymer and nucleic acid amplification product itself, the force pushed by the centrifugal force applied to the particles, and the effect of high-density fixation by fixing one biopolymer per spot. Can be fixed to density.
  • the unfixed biopolymer is washed out from the flow cell, and the particle size distribution of the particles is distributed. It is characterized by being composed of two or more different types of particles.
  • the first embodiment is a method for adjusting a flow cell for analyzing a biopolymer, in which a step of injecting a solution in which the biopolymer is dissolved into the flow cell and fixing the biopolymer on a substrate constituting the flow cell. Therefore, this is an example of a flow cell adjusting method including a step of centrifuging the flow cell in which a solution in which a biopolymer is dissolved is injected and a step of washing out the biopolymer unfixed on the substrate from the flow cell.
  • FIG. 1 shows a conceptual diagram for explaining an example of the flow cell adjustment method of the first embodiment.
  • the flow cell 103 is placed on the movable holder 102 attached to the centrifuge plate 101 so that the sample fixing surface for fixing the sample, which is a biopolymer, faces down.
  • a solution in which a biopolymer is dissolved is injected into the flow cell 103.
  • the centrifuge plate 101 is centrifuged for a certain period of time to fix the sample on the sample fixing surface.
  • the fixing time of the sample there is no particular limitation on the fixing time of the sample, but in general, biopolymers have a small diffusion coefficient and take a long time to fix, so 10 minutes or more is desirable.
  • the acceleration during centrifugation is also not particularly limited, but 100 G or more is desirable in order to more effectively press the biopolymer against the sample fixing surface. Further, when heat is applied during centrifugation, the diffusion coefficient of the biopolymer in the solution can be increased, and more biopolymers can be fixed.
  • the biopolymer not fixed on the sample fixing surface is washed out from the flow cell. Further, when the particles are mixed in the solution, the biopolymer can be pressed against the sample fixing surface by the weight of the particles themselves, so that a method of fixing without centrifuging can also be used.
  • FIG. 2 shows a configuration example of the flow cell of the first embodiment.
  • the flow cell is made by laminating a sita substrate 204, a wafer substrate 205, and an intermediate material 206.
  • the portion surrounded by the hollow portion 207 formed in the sita substrate 204, the wafer substrate 205, and the intermediate material 206 serves as the flow cell flow path.
  • the solution in which the biopolymer is dissolved is injected from the injection port 208 provided on the Sita substrate 204, and the liquid is discharged from the discharge port 209.
  • a spot mounting portion 210 is provided on the Sita substrate 204.
  • the spots 211 which are the places and points where the biopolymers are bound, are densely arranged inside the spot mounting portion 210.
  • the density of the biopolymers that can be fixed on the Cita substrate 204 can be increased.
  • the material used for the sita substrate 204 is not particularly limited, and is an inorganic material such as silicon, glass, quartz, sapphire, ceramic, ferrite, alumina, diamond, a metal material such as aluminum, SUS, titanium, iron, or a phenol resin.
  • Poly Resin materials such as ether ether ketone, mixed materials thereof, glass fiber, and carbon fiber reinforced inorganic materials can also be used.
  • Glass, quartz, SUS, titanium and the like are particularly desirable.
  • Spot 211 is formed by using a spotting device as shown in Patent Document 2 or a Lift-off process which is a known method.
  • a material capable of forming a spot on the substrate via a covalent bond is preferable.
  • silane is used as such a material.
  • Coupling material is desirable.
  • silane coupling materials those having a highly reactive functional group capable of forming a coating film containing an amino group via a covalent bond are preferable, and such functional groups include vinyl groups and epoxys.
  • Examples thereof include ethoxysilane and methoxysilane having a group, a styryl group, a methacryl group, an acrylic group, an amino group, a ureido group, an isocyanate group, an isocyanurate group and a mercapto group in the molecule. Further, it may be formed by using silicon as a substrate and using alkenes or alkynes. Further, a cationic polymer such as polydiallyl diammonium or polylysine may be used.
  • the diameter of the spot 211 should be larger than half of the DNA sample so that only one DNA sample can be fixed at a high fixation rate, and such a size includes a diameter of 50 nm or more and 1000 nm or less.
  • the material used for the web substrate 205 is not particularly limited, and is an inorganic material such as silicon, glass, quartz, sapphire, ceramic, ferrite, alumina, diamond, a metal material such as aluminum, SUS, titanium, iron, or a phenol resin.
  • Polyether Resin materials such as ether ketone, mixed materials thereof, glass fiber, and carbon fiber reinforced inorganic materials can also be used.
  • a sample having high transmittance of light having an excitation wavelength or light having a fluorescence wavelength is desirable, and such materials include inorganic materials such as glass, quartz, and sapphire, and acrylic resins. And highly transparent resins such as cyclic polyolefins are desirable.
  • the material used for the intermediate material 206 is not particularly limited, and pressure-sensitive double-sided tape, adhesive, rubber sheet, etc. can be used. It is also possible to hollow out the portion forming the flow path of the web substrate or the weft substrate and directly attach the shita substrate 204 and the weft substrate 205 without using the intermediate material 206.
  • FIG. 3 shows a schematic view of a part of the cross section inside the flow cell during centrifugation. Due to the centrifugal force during the centrifugation process, the biopolymer 313 in the flow cell flow path 312 is pressed against the spot 311 on the Sita substrate 304, so that the probability that the biopolymer 313 comes into contact with the spot 311 can be increased. The proportion of spots 311 on which the biopolymer 313 is fixed can be increased.
  • the biopolymer used is not particularly limited, but the larger the molecular weight, the more centrifugal force and gravity can be obtained, so DNA and proteins with a large molecular weight are desirable.
  • DNA a nucleic acid amplification product obtained from the cyclic template shown in Patent Document 1 and Patent Document 2 is particularly desirable.
  • FIG. 4 shows a schematic view of a part of the cross section inside the flow cell during centrifugation when the particles are dispersed in a solution in which the biopolymer to be injected into the flow cell is dissolved as a modified example of this embodiment.
  • the particles used are not particularly limited, but the materials include metal materials such as gold, silver, copper and platinum, semiconductor materials such as zinc sulfide and cadmium selenium, magnetic materials such as iron oxide, and inorganic materials such as silica and zirconia. Materials, polystyrene, thermoplastic elastomers, polyether sulfone resins, polyvinylidene fluoride, epoxy resins, polylactic acid resins, ethyl cellulose resins and other polymer materials are used. Further, a composite material may be used in which a magnetic material, a polymer material, or the like is used as a core, and the polymer material, the magnetic material, or the like is covered with a single layer or a plurality of layers. Among these particles, particles containing a metal material or a magnetic material having a large specific gravity, which are susceptible to gravity or centrifugation, are particularly desirable. This is because the biopolymer is pressed against the sample fixing surface by the weight of the particles themselves.
  • particles whose surface is coated with an organic film such as carboxylic acid or polyethylene glycol are also desirable. These organic coatings have an effect of preventing the biopolymer from being adsorbed on the particle surface and an effect of preventing the particles from being non-specifically adsorbed on the sita substrate.
  • particles coated with functional groups such as avidin and amino groups are reacted with a biotinylated compound capable of reacting with these functional groups or a compound having an N-hydroxysuccinimide ester group to prevent adsorption. Effective compounds and functional groups may be introduced.
  • the shape of the particles used is not particularly limited, but in order to prevent adsorption to the sita substrate, a spherical shape that can minimize the contact area when in contact with the sita substrate is particularly desirable.
  • the size is not particularly limited, but it is particularly desirable that the particle size is 1 to 50 ⁇ m, which makes it difficult for particles to agglomerate while reducing the efficiency of flushing from the flow path.
  • FIG. 5 shows a schematic view of the cross section of the flow cell when two or more kinds of particle groups having different particle size distributions are dispersed in a solution in which a biopolymer is dissolved as another modification of this embodiment. Since the particle 515 smaller than the particle 514 enters between the particle 514 and the particle 514, the biomolecule 513 can be efficiently pushed in when centrifugally applied.
  • FIGS. 6A to 6E an example of one step for producing the Sita substrate 604 having the spots used in this example will be described.
  • the Cita substrate 604 of this example can be produced by using the Lift-off process described above. After coating the hydrophobic film 616 on the Cita substrate 604 (a), the resist 617 is further coated (b). Then, after forming the opening 618 using lithography (c), the material forming the spot 611 is deposited (d). Then, the resist 617 is removed (e).
  • an example using a photolithography technique is shown, but a nanoimprint lithography technique as shown in Non-Patent Document 2 may be used.
  • a flow cell as shown in FIG. 2 is produced using the Cita substrate 604 on which the spot 611 produced in this manner is formed, and then set in the nucleic acid analyzer shown in FIG. 7 as an example to perform nucleic acid analysis.
  • the nucleic acid analyzer 719 is a holder unit 721 and a flow cell 720 capable of fixing the flow cell 720 and the flow cell 720 adjusted by the adjustment method of the present embodiment described above and controlling the temperature of the flow cell 720.
  • the stage unit 722 that moves the holder unit 721, the reagent container 723 that contains multiple reagents and wash water, and the liquid feeding unit that is the driving source for sucking the reagents contained in the reagent container 723 and injecting them into the flow cell 720.
  • the detection unit 727 comprises a fluorescence detection device based on the fluorescence method, and can irradiate the flow cell 720 on the stage unit 722 with excitation light to detect the generated fluorescence.
  • the nucleic acid analyzer 719 shown in FIG. 7 operates as follows. First, the flow cell 720 holding the nucleic acid sample to be measured adjusted by the above-mentioned flow cell adjustment method is installed in the holder unit 721. Next, the nozzle 725 accesses the reagent container 723, and the reagent is sucked by the liquid feeding unit 724. The nozzle 725 is conveyed to the upper surface of the flow cell 720 by the nozzle transfer unit 726, and the reagent is injected into the flow cell 716. Then, in the holder unit 721, the reaction occurs by adjusting the temperature of the nucleic acid sample and the reagent contained in the flow path of the flow cell 720.
  • the flow cell 720 is moved by the stage unit 722 and irradiated with excitation light to detect the fluorescence of a plurality of nucleic acid samples in the detection region.
  • the fluorescence may be detected by irradiating the excitation light through the Sita substrate, which is the substrate on the side on which the nucleic acid sample is fixed.
  • the flow cell 720 is slightly moved and detected by the same method, and the operation of detecting is repeated a plurality of times.
  • a nucleic acid sample can be read by repeating the operation of injecting another reagent and detecting it a plurality of times.
  • the nucleic acid analyzer is controlled by a control unit such as a computer (not shown), and the analysis operation can be automatically performed.
  • DNA sequencing can be performed using the flow cells obtained in this example, and for example, DNA sequencing and hybridization can be performed.
  • DNA sequencing DNA sequencing
  • the method of DNA sequencing is not limited, but it can be used for a stepwise synthesis method (Sequencing by synthesis) using a reversible terminator as shown in Non-Patent Document 2. That is, after injecting the solution in which the DNA sample is dissolved into the flow cell for nucleic acid analysis, the DNA sample is fixed on the spot by holding it for a certain period of time. Then, after hybridizing the sequence primer to the fixed DNA sample, the nucleotide sequence is determined using the Sequencing by synthesis method. By this method, it is possible to perform decoding of several tens to several hundreds of bp in one cycle, and it is possible to analyze data of several tens of Gb in one run.
  • Example 2 details of a specific example of the above-mentioned flow cell adjustment method will be described.
  • nucleic acid analysis After injecting the sequence primer solution into the flow cell and holding it for a certain period of time, the inside of the flow cell was washed with a buffer solution to remove the unreacted sequence primer. Then, after installing the flow cell 720 in the nucleic acid analyzer 719, nucleic acid analysis was performed by the method shown in Non-Patent Document 2. After injecting a solution containing a synthetic nucleotide labeled with a dye for sequencing and a DNA synthase (9 ° N mutant DNA polymerase), the mixture was held in a holder unit at 68 ° C. for 10 minutes.
  • a synthetic nucleotide solution not labeled with a dye was injected and kept at 68 ° C. for 10 minutes.
  • the flow path in the flow cell 720 was then washed with SPSC buffer to remove unreacted nucleotides.
  • the detection unit 727 applied excitation light to detect the fluorescence introduced into a plurality of DNA samples in the detection region.
  • a Na 2 PdCl 2 / P (PhSO 3 Na) 3 aqueous solution was injected into the flow cell 720 for nucleic acid analysis and then kept at 60 ° C.
  • the DNA sequence was determined by repeating the cycle of the above-mentioned single nucleotide extension reaction, additional single nucleotide extension reaction, fluorescence detection, and removal of fluorescent substance.
  • the flow cell adjusting method of the present invention can also be used for a flow cell having no spot and having a film capable of fixing a biopolymer on the entire upper surface of the Sita substrate.
  • the nucleic acid amplification product was used in this example, it can also be used for other biopolymers such as proteins.

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Abstract

Provided is a flow cell adjustment method which is for increasing analysis throughput by fixing biopolymers having a high molecular weight to a flow cell at high density, the method involving: injecting a solution having biopolymers 313 dissolved therein to the flow cell comprising a lower substrate 304, an upper substrate 305, a flow channel 312, and the like; centrifuging the flow cell to which the biopolymer-dissolved solution is injected in order to fix the biopolymers 313 on the lower substrate 304; and thereafter washing out the biopolymers unfixed to the substrates from the flow cell.

Description

フローセルの調整方法How to adjust the flow cell
　本発明は、生体高分子を解析するためのフローセルの調整技術に関する。 The present invention relates to a flow cell adjusting technique for analyzing a biopolymer.
　DNAやRNAの塩基配列を決定する新しい技術が開発されてきている。現在、通常用いられている電気泳動を利用した方法においては、予め配列決定用のDNA断片又はRNA試料から逆転写反応を行い合成したcDNA断片試料を調製し、周知のサンガー法によるジデオキシ反応した後、電気泳動を行い、分子量分離展開パターンを計測して塩基配列決定する。 A new technology for determining the base sequence of DNA or RNA has been developed. In the currently commonly used method using electrophoresis, a cDNA fragment sample synthesized by reverse transcription reaction from a DNA fragment for sequencing or an RNA sample is prepared in advance, and after a dideoxy reaction by a well-known Sanger method. , Electrophoresis is performed, and the molecular weight separation and development pattern is measured to determine the base sequence.
　これに対し、近年、基板に試料となるDNA断片を数多く固定して、パラレルに数多くの断片の配列情報を決定する方法として蛍光法などが提案されている。 On the other hand, in recent years, a fluorescence method or the like has been proposed as a method of fixing a large number of sample DNA fragments on a substrate and determining the sequence information of a large number of fragments in parallel.
　例えば、非特許文献1では、基板上に、同一配列を有する多数のDNAプローブを固定しておく。また、DNA試料を切断後、DNAプローブ配列と相補鎖のアダプター配列を各DNA試料断片の端に付加させる。これらを基板上でハイブリダイゼーションさせることにより、基板上にランダムに一分子ずつ試料DNA断片を固定化させている。この場合、基板上でDNA伸長反応を起こない、蛍光色素付き基質を取り込ませた後、未反応基質の洗浄や，蛍光検出を行い、試料DNAの配列情報を得る蛍光法を開示している。 For example, in Non-Patent Document 1, a large number of DNA probes having the same sequence are fixed on the substrate. After cutting the DNA sample, the DNA probe sequence and the adapter sequence of the complementary strand are added to the end of each DNA sample fragment. By hybridizing these on a substrate, sample DNA fragments are randomly immobilized one molecule at a time on the substrate. In this case, a fluorescence method is disclosed in which a substrate with a fluorescent dye that does not cause a DNA elongation reaction on the substrate is incorporated, and then the unreacted substrate is washed or fluorescence is detected to obtain sequence information of sample DNA.
　以上のように、基板上に、核酸断片試料を数多く固定することにより、パラレルに数多くの断片の配列情報を決定する方法が開発され、実用化されつつある。 As described above, a method for determining the sequence information of a large number of fragments in parallel by immobilizing a large number of nucleic acid fragment samples on a substrate has been developed and is being put into practical use.
　これらに使われる基板が、特許文献1や特許文献2に開示されている。特許文献１では、サンプルDNAが結合するスポットが基板上に格子配置されている。基板にはシリコンウェハが用いられ、シリコンウェハ上に疎水性のHMDS（Hexamethyldisilizane）層を形成した後、ポジ型レジストを塗布する。フォトリソグラフィ技術を用いて所定の位置に開口部を設けた後、開口部底のHMDSを除去する。その後、ウェハをアミノシラン気相中に保持し、開口部底にアミノシランを導入する。ウェハ上にレジスト塗布した後ダイシング加工し基板を切り出す。切り出された基板上のレジストを有機溶剤用いた超音波洗浄で除去した後、ポリウレタン製接着材を介してカバーガラスを貼りつけ核酸分析用のフローセルを作製する。親水性が高くDNA固定可能なアミノシランをDNAボール固定スポットに用い、その他の領域にDNAの吸着を防ぐ疎水性のHMDSを用いることで、DNAボール含む溶液を基板上に導入した時、自然にDNAボールをスポット上にのみ固定することを可能にしている。 The substrates used for these are disclosed in Patent Document 1 and Patent Document 2. In Patent Document 1, the spots to which the sample DNA binds are arranged in a grid on the substrate. A silicon wafer is used as the substrate, and after forming a hydrophobic HMDS (Hexamethyldisilizane) layer on the silicon wafer, a positive resist is applied. After the opening is provided at a predetermined position using photolithography technology, the HMDS at the bottom of the opening is removed. After that, the wafer is held in the gas phase of aminosilane, and aminosilane is introduced into the bottom of the opening. After applying resist on the wafer, dicing is performed to cut out the substrate. After removing the resist on the cut-out substrate by ultrasonic cleaning using an organic solvent, a cover glass is attached via a polyurethane adhesive to prepare a flow cell for nucleic acid analysis. By using aminosilane, which is highly hydrophilic and can fix DNA, as the DNA ball fixing spot, and by using hydrophobic HMDS, which prevents DNA adsorption in other regions, when a solution containing DNA balls is introduced onto the substrate, DNA naturally occurs. It makes it possible to fix the ball only on the spot.
　特許文献2では、スライドガラス上にアミノシランをコ－ティングし、2価性の架橋試薬1,4-diphenylen-diisothiocyanateで活性化した後、カスタムスポッティング装置を用いて、5’末端がアミノ化されたDNAオリゴマーを格子状に配置している。この後、DNAオリゴマーとDNAサンプルをハイブリダイズさせてDNAサンプルを格子状に固定している。 In Patent Document 2, aminosilane was coated on a slide glass, activated with a divalent cross-linking reagent 1,4-diphenylen-diisothiocyanate, and then the 5'end was aminated using a custom spotting apparatus. The DNA oligomers are arranged in a grid pattern. After that, the DNA oligomer and the DNA sample are hybridized to fix the DNA sample in a grid pattern.
　スポットへのDNAサンプルの固定方法としては、特許文献1では、DNAボールを分散させた溶液を基板上に導入した後、基板を振とうさせた状態で一定時間保持し、その後に、基板をリンスして固定されていないDNAボールを洗い流して、スポット上にDNAボールを固定している。 As a method for fixing a DNA sample to a spot, in Patent Document 1, after introducing a solution in which DNA balls are dispersed onto a substrate, the substrate is held in a shaken state for a certain period of time, and then the substrate is rinsed. The unfixed DNA balls are washed away and the DNA balls are fixed on the spot.
米国特許出願公開第US2009/0270273号明細書U.S. Patent Application Publication No. US2009 / 0270273 米国特許出願公開第US2009/0018024号明細書U.S. Patent Application Publication No. US2009 / 0018024
　スポット上に固定される生体高分子の分子量が大きい場合、生体高分子が固定されるスポットの割合が高まらずに、結果として、基板上に固定される生体高分子の密度が高まらず、パラレルで一度に数多くの生体高分子を分析することができなくなる課題が生じる。この時の生体高分子とは、増幅コピー数が多いDNAボールやタンパク質などで当てはまる。 When the molecular weight of the biopolymer fixed on the spot is large, the proportion of the spot where the biopolymer is fixed does not increase, and as a result, the density of the biopolymer fixed on the substrate does not increase, and in parallel. There is a problem that it becomes impossible to analyze many biopolymers at one time. The biopolymer at this time is a DNA ball or protein having a large number of amplified copies.
　一方、コピー数が少なく分子量が小さいDNAボールを用いた場合は、シーケンス反応時に取り込まれる蛍光色素が少なくなり、シーケンスに有効なDNAボールの数が減ってしまう課題が生じる。一般に、シーケンス反応のサイクルが進むと、必ずしも1サイクルのシーケンス反応の効率が100%ではないため、シーケンス反応の反応効率が低くなり、サイクル中に取り込まれる蛍光色素がさらに減って、読み取り塩基長が短くなる課題も生じる。 On the other hand, when a DNA ball having a small copy number and a small molecular weight is used, the number of fluorescent dyes taken in during the sequence reaction decreases, and the number of DNA balls effective for the sequence decreases. In general, as the sequence reaction cycle progresses, the efficiency of the sequence reaction in one cycle is not necessarily 100%, so that the reaction efficiency of the sequence reaction becomes low, the fluorescent dye taken in during the cycle is further reduced, and the reading base length becomes long. There is also the issue of shortening.
　また、DNA断片を保持する微粒子担体を基板上に固定して蛍光で検出する場合、一般に微粒子は金や酸化鉄のような金属、または金属酸化物などが用いられるが、これらの金属や金属酸化物は可視領域の光を吸収し、自家蛍光を発光することが多く、微粒子担体が発光する自家蛍光が蛍光検出時のノイズとなって蛍光検出の課題となることが多い。 Further, when a fine particle carrier holding a DNA fragment is fixed on a substrate and detected by fluorescence, a metal such as gold or iron oxide or a metal oxide is generally used as the fine particle, and these metals or metal oxidation Objects often absorb light in the visible region and emit autofluorescence, and the autofluorescence emitted by the fine particle carrier becomes noise during fluorescence detection, which is often a problem for fluorescence detection.
　本発明は、上記の課題を解決し、分子量が大きい生体高分子を固定率高く基板上に固定することで、基板上に固定されるサンプルの密度を高めて、単位面積当たりに分析できる生体高分子の数を増やし、検出時にはノイズとなるような微粒子担体が検出時に存在することなく、且つ高精度の分析を可能とする基板を有するフローセルを提供することを目的とする。 The present invention solves the above-mentioned problems, and by fixing a biopolymer having a large molecular weight on a substrate with a high fixation rate, the density of samples fixed on the substrate can be increased and the biopolymer can be analyzed per unit area. It is an object of the present invention to provide a flow cell having a substrate that increases the number of molecules, does not have a fine particle carrier that causes noise at the time of detection, and enables highly accurate analysis.
　上記の目的を達成するため、本発明においは、生体高分子を解析するためのフローセルの調整方法であって、生体高分子が溶解した溶液をフローセルに注入する工程と、フローセルを構成する基板の上に生体高分子を固定するため、生体高分子が溶解した溶液を注入したフローセルを遠心する工程と、基板に未固定の生体高分子をフローセルから洗い出す工程、とからなるフローセルの調整方法を提供する。 In order to achieve the above object, the present invention is a method for adjusting a flow cell for analyzing a biopolymer, in which a step of injecting a solution in which a biopolymer is dissolved into the flow cell and a substrate constituting the flow cell Provided is a flow cell adjusting method consisting of a step of centrifuging a flow cell in which a solution in which a biopolymer is dissolved is injected and a step of washing out a biopolymer unfixed on a substrate from the flow cell in order to fix the biopolymer on the surface. To do.
　また、上記の目的を達成するため、本発明においては、生体高分子を解析するためのフローセルの調整方法であって、生体高分子が溶解し、且つ粒子が分散している溶液をフローセルに注入する工程と、フローセルを構成する基板の上に生体高分子を固定するため、生体高分子が溶解し、且つ粒子が分散している溶液を注入したフローセルを一定時間保持する工程と、基板に未固定の生体高分子と粒子をフローセルから洗い出す工程、とからなるフローセルの調整方法を提供する。 Further, in order to achieve the above object, in the present invention, a flow cell adjusting method for analyzing a biopolymer, in which a solution in which the biopolymer is dissolved and particles are dispersed is injected into the flow cell. And the step of holding the flow cell infused with the solution in which the biopolymer is dissolved and the particles are dispersed for a certain period of time in order to fix the biopolymer on the substrate constituting the flow cell, and the step of not being on the substrate. Provided is a method for adjusting a flow cell, which comprises a step of washing out fixed biopolymers and particles from the flow cell.
　更に、上記の目的を達成するため、本発明においては、生体高分子を解析するためのフローセルの調整方法であって、生体高分子が溶解し、且つ粒子が分散している溶液をフローセルに注入する工程と、フローセルを構成する基板の上に生体高分子を固定するため、生体高分子が溶解し、且つ粒子が分散している溶液を注入したフローセルを遠心する工程と、未固定の生体高分子をフローセルから洗い出す工程、とからなるフローセルの調整方法を提供する。 Further, in order to achieve the above object, in the present invention, a flow cell adjusting method for analyzing a biopolymer, in which a solution in which the biopolymer is dissolved and particles are dispersed is injected into the flow cell. And the step of centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed is centrifuged in order to fix the biopolymer on the substrate constituting the flow cell, and the unfixed bio-height. Provided is a method for adjusting a flow cell, which comprises a step of washing out molecules from the flow cell.
　本発明によれば、分子量が大きい生体高分子を固定率高く基板上に固定することで、基板上に固定されるサンプルの密度を高めて、単位面積当たりに分析できる生体高分子の数を増やし、且つ高精度の分析を可能とする基板を有するフローセルを提供することができる。 According to the present invention, by fixing a biopolymer having a large molecular weight on a substrate with a high fixation rate, the density of samples fixed on the substrate is increased, and the number of biopolymers that can be analyzed per unit area is increased. Moreover, it is possible to provide a flow cell having a substrate that enables highly accurate analysis.
実施例１に係る、フローセル調整方法の一例を説明するための概念図である。It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1. FIG. 実施例１に係る、フローセルの一例を説明するための図である。It is a figure for demonstrating an example of the flow cell which concerns on Example 1. FIG. 実施例１に係る、フローセル調整方法の一例を説明するための概念図である。It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1. FIG. 実施例１に係る、フローセル調整方法の一例を説明するための概念図である。It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1. FIG. 実施例１に係る、フローセル調整方法の一例を説明するための概念図である。It is a conceptual diagram for demonstrating an example of the flow cell adjustment method which concerns on Example 1. FIG. 実施例１に係る、フローセルの一例を説明するための図である。It is a figure for demonstrating an example of the flow cell which concerns on Example 1. FIG. 実施例１に係る、フローセルを用いた解析の一例を説明するための図である。It is a figure for demonstrating an example of analysis using a flow cell which concerns on Example 1. FIG.
　本発明の一実施態様によれば、生体高分子が溶解した溶液を注入したフローセルを遠心する工程と、基板に未固定の生体高分子をフローセルから洗い出す工程を含み、溶液中の生体高分子に遠心力を付与することで、生体高分子がスポットに接触する確率を高めて、生体高分子が固定されるスポットの割合を高める。また、基板に未固定の生体高分子をフローセルから洗い出すことにより、検出時にノイズとなるような微粒子担体が基板上に存在しないため、蛍光法などにおいて高精度な検出が可能となる。 According to one embodiment of the present invention, the biopolymer in the solution comprises a step of centrifuging the flow cell in which the solution in which the biopolymer is dissolved is injected and a step of washing out the biopolymer unfixed on the substrate from the flow cell. By applying centrifugal force, the probability that the biopolymer comes into contact with the spot is increased, and the proportion of the spot where the biopolymer is fixed is increased. Further, by washing out the biopolymer unfixed on the substrate from the flow cell, there is no fine particle carrier on the substrate that causes noise at the time of detection, so that highly accurate detection is possible by a fluorescence method or the like.
　また、本発明の一態様によれば、生体高分子が、環状テンプレートより得られた核酸増幅産物であることを特徴とする。環状テンプレートより得られる核酸増幅産物は、増幅コピー数を増やすことができるのでシーケンス反応で取り込まれる蛍光色素を増やすことができ、読み取り塩基長を長くすることができる。また、増幅コピー数を増やすことで分子量を増やすことができる。 Further, according to one aspect of the present invention, the biopolymer is a nucleic acid amplification product obtained from a cyclic template. Since the nucleic acid amplification product obtained from the cyclic template can increase the amplified copy number, the fluorescent dye incorporated in the sequence reaction can be increased, and the read base length can be lengthened. Moreover, the molecular weight can be increased by increasing the amplified copy number.
　また、本発明の一態様によれば、生体高分子がスポットを介して基板上に固定されることを特徴とする。スポットを高密度に配置しておき、スポット1個当たりに1個の生体高分子を固定することで、基板上に生体高分子を高密度に固定することができる。 Further, according to one aspect of the present invention, the biopolymer is fixed on the substrate via spots. By arranging the spots at a high density and fixing one biopolymer for each spot, the biopolymer can be fixed at a high density on the substrate.
　更に、本発明の一態様によれば、生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを一定時間保持した後、未固定の生体高分子と粒子をフローセルから洗い出す。その結果、溶液注入後にスポットが付いている基板を下にして保持することで、重力を受けて沈降する粒子に生体高分子が押し付けられて、生体高分子とスポットが接触する確率を高めることができる。また、粒子は蛍光法による検出時に洗い流されているため、ノイズとなるような微粒子担体が基板上に存在しなく高精度な蛍光検出が可能となる。 Further, according to one aspect of the present invention, after holding the flow cell in which the biopolymer is dissolved and the solution in which the particles are dispersed is injected for a certain period of time, the unfixed biopolymer and the particles are washed out from the flow cell. As a result, by holding the substrate with spots down after injecting the solution, the biopolymer is pressed against the particles that settle under the force of gravity, increasing the probability that the biopolymer and the spot come into contact with each other. it can. Further, since the particles are washed away at the time of detection by the fluorescence method, the particle carrier that causes noise does not exist on the substrate, and highly accurate fluorescence detection becomes possible.
　また、本発明の一態様によれば、生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを一定時間保持した後、未固定の生体高分子と粒子をフローセルから洗い出すと共に、生体高分子が環状テンプレートより得られた核酸増幅産物であることを特徴とする。重力を受けて沈降する粒子に生体高分子が押される効果と増幅コピー数が多く分子量が大きい生体高分子の自重による組み合わせで、生体高分子とスポットが接触する確率をさらに高めることができる。 Further, according to one aspect of the present invention, after holding the flow cell in which the biopolymer is dissolved and the solution in which the particles are dispersed is injected for a certain period of time, the unfixed biopolymer and the particles are washed out from the flow cell. , The biopolymer is a nucleic acid amplification product obtained from a cyclic template. The combination of the effect of the biopolymer being pushed by the particles that settle under the force of gravity and the weight of the biopolymer having a large number of amplified copies and a large molecular weight can further increase the probability of contact between the biopolymer and the spot.
　また、本発明の一態様によれば、生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを一定時間保持した後、未固定の生体高分子と粒子をフローセルから洗い出すと共に、生体高分子がスポットを介して基板上に固定されることを特徴とする。重力により粒子が押し付ける効果、高分子量による自重、ならびにスポットを高密度に配置しておき、スポット1個当たりに1個の生体高分子を固定することによる高密度化固定による効果によって、基板上に生体高分子をさらに高密度に固定することができる。 Further, according to one aspect of the present invention, after holding the flow cell in which the biopolymer is dissolved and the solution in which the particles are dispersed is injected for a certain period of time, the unfixed biopolymer and the particles are washed out from the flow cell. , The biopolymer is fixed on the substrate via spots. Due to the effect of the particles pressing by gravity, the weight of the high molecular weight, and the effect of high-density fixation by arranging the spots at high density and fixing one biopolymer per spot, on the substrate. The biopolymer can be fixed at a higher density.
　また、本発明の一態様によれば、生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを一定時間保持した後、未固定の生体高分子と粒子をフローセルから洗い出すと共に、粒子が粒径の分布が異なる2種類以上の粒子群からなること特徴とする。粒径が大きい粒子と粒子の間に粒径が小さい粒子がはさまれることによって、自重によって生体高分子を押し付ける力をさらに高めることができる。 Further, according to one aspect of the present invention, after holding the flow cell in which the biopolymer is dissolved and the solution in which the particles are dispersed is injected for a certain period of time, the unfixed biopolymer and the particles are washed out from the flow cell. The particles are characterized by being composed of two or more types of particles having different particle size distributions. By sandwiching the particles having a small particle size between the particles having a large particle size, the force of pressing the biopolymer by its own weight can be further increased.
　更に、本発明の一態様によれば、生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを遠心した後、未固定の生体高分子をフローセルから洗い出すことを特徴とする。粒子にかかる遠心力を用いて生体高分子をスポットに押し付けることができ、生体高分子とスポットが接触する確率をさらに高めることができる。 Further, according to one aspect of the present invention, the biopolymer is dissolved, and at the same time, the flow cell in which the solution in which the particles are dispersed is injected is centrifuged, and then the unfixed biopolymer is washed out from the flow cell. .. The biopolymer can be pressed against the spot by using the centrifugal force applied to the particles, and the probability of contact between the biopolymer and the spot can be further increased.
　また、本発明の一態様は、前記生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを遠心した後、未固定の生体高分子をフローセルから洗い出すと共に、生体高分子が、環状テンプレートより得られた核酸増幅産物であることを特徴とする。増幅コピー数を増やすことで分子量を増やすことができ、核酸増幅産物自体にかかる大きな遠心力、粒子にかかる遠心力により押される力によって、基板と生体試料が接触する確率をさらに高めることができる。 Further, in one aspect of the present invention, the unfixed biopolymer is washed out from the flow cell after centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed at the same time is injected, and the biopolymer is formed. , It is a nucleic acid amplification product obtained from a cyclic template. The molecular weight can be increased by increasing the number of amplified copies, and the probability of contact between the substrate and the biological sample can be further increased by the large centrifugal force applied to the nucleic acid amplification product itself and the force pushed by the centrifugal force applied to the particles.
　また、本発明の一態様は、前記生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを遠心した後、未固定の生体高分子をフローセルから洗い出すと共に、生体高分子がスポットを介して基板上に固定されることを特徴とする。生体高分子や核酸増幅産物自体にかかる遠心力、粒子にかかる遠心力により押される力、ならびにスポット1個当たりに1個の生体高分子を固定することによる高密度化固定による効果によって、さらに高密度に固定することができる。 Further, in one aspect of the present invention, the unfixed biopolymer is washed out from the flow cell after centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed at the same time is injected, and the biopolymer is formed. It is characterized in that it is fixed on the substrate via a spot. Higher due to the centrifugal force applied to the biopolymer and nucleic acid amplification product itself, the force pushed by the centrifugal force applied to the particles, and the effect of high-density fixation by fixing one biopolymer per spot. Can be fixed to density.
　また、本発明は、前記生体高分子が溶解し、同時に粒子が分散している溶液を注入したフローセルを遠心した後、未固定の生体高分子をフローセルから洗い出すと共に、粒子が粒径の分布が異なる2種類以上の粒子からなること特徴とする。これにより上述の効果に加え、大きい粒径の粒子と粒子および間にはまる粒径の小さな粒子から押し付けられる力によって、生体高分子とスポットを接触する確率をさらに高めることができる。 Further, in the present invention, after centrifuging the flow cell in which the solution in which the biopolymer is dissolved and the particles are dispersed at the same time is centrifuged, the unfixed biopolymer is washed out from the flow cell, and the particle size distribution of the particles is distributed. It is characterized by being composed of two or more different types of particles. As a result, in addition to the above-mentioned effects, the probability of contact between the biopolymer and the spot can be further increased by the force pressed from the particles having a large particle size and the particles having a small particle size in between.
　以下、本発明の新規な特徴と効果について、図を参照して説明する。ここでは、本発明の十分な理解のために、特定の実施例について詳細な説明を行うが、本発明はそれに記載した内容に限定されるものではない。また、各実施例は適宜組み合せることが可能であり、当該組み合せ形態も本発明を実施するための形態である。 Hereinafter, the novel features and effects of the present invention will be described with reference to the drawings. Here, in order to fully understand the present invention, specific examples will be described in detail, but the present invention is not limited to the contents described therein. In addition, each embodiment can be combined as appropriate, and the combined form is also a mode for carrying out the present invention.
　実施例１は、生体高分子を解析するためのフローセルの調整方法であって、生体高分子が溶解した溶液をフローセルに注入する工程と、フローセルを構成する基板の上に生体高分子を固定するため、生体高分子が溶解した溶液を注入したフローセルを遠心する工程と、基板に未固定の生体高分子をフローセルから洗い出す工程、とからなるフローセルの調整方法の実施例である。 The first embodiment is a method for adjusting a flow cell for analyzing a biopolymer, in which a step of injecting a solution in which the biopolymer is dissolved into the flow cell and fixing the biopolymer on a substrate constituting the flow cell. Therefore, this is an example of a flow cell adjusting method including a step of centrifuging the flow cell in which a solution in which a biopolymer is dissolved is injected and a step of washing out the biopolymer unfixed on the substrate from the flow cell.
　図１に実施例１のフローセル調整方法の一例を説明するための概念図を示す。遠心板101に付いている可動ホルダ102に、生体高分子であるサンプルを固定するサンプル固定面が下になるようにフローセル103を設置する。フローセル103には生体高分子が溶解した溶液が注入されている。 FIG. 1 shows a conceptual diagram for explaining an example of the flow cell adjustment method of the first embodiment. The flow cell 103 is placed on the movable holder 102 attached to the centrifuge plate 101 so that the sample fixing surface for fixing the sample, which is a biopolymer, faces down. A solution in which a biopolymer is dissolved is injected into the flow cell 103.
　その後遠心板101を一定時間遠心してサンプル固定面にサンプルを固定する。サンプルの固定時間に特に制限はないが、一般に、生体高分子は拡散係数が小さく固定に時間を要するため、10分間以上が望ましい。また、遠心時の加速度も特に制限はないが、より効果的に生体高分子をサンプル固定面に押し付けるために100G以上が望ましい。また、遠心時に熱を加えると生体高分子の溶液中の拡散係数を高めることができ、より多くの生体高分子を固定することができる。 After that, the centrifuge plate 101 is centrifuged for a certain period of time to fix the sample on the sample fixing surface. There is no particular limitation on the fixing time of the sample, but in general, biopolymers have a small diffusion coefficient and take a long time to fix, so 10 minutes or more is desirable. The acceleration during centrifugation is also not particularly limited, but 100 G or more is desirable in order to more effectively press the biopolymer against the sample fixing surface. Further, when heat is applied during centrifugation, the diffusion coefficient of the biopolymer in the solution can be increased, and more biopolymers can be fixed.
　本実施例においては、上述の遠心の後、サンプル固定面に未固定の生体高分子をフローセルから洗い出す。また、溶液中に粒子を混合する場合は、粒子自身の自重で生体高分子をサンプル固定面に押し付けることができるため、遠心しないで固定する方法も用いることができる。 In this example, after the above-mentioned centrifugation, the biopolymer not fixed on the sample fixing surface is washed out from the flow cell. Further, when the particles are mixed in the solution, the biopolymer can be pressed against the sample fixing surface by the weight of the particles themselves, so that a method of fixing without centrifuging can also be used.
　図２に実施例１のフローセルの一構成例を示す。フローセルは、シタ基板204、ウエ基板205、ならびに中間材206を貼り合わせて作られる。シタ基板204、ウエ基板205、ならびに中間材206に形成された中抜き部207に囲まれた部分がフローセルの流路となる。シタ基板204に設けられた注入口208から生体高分子が溶解した溶液が注入されて、排出口209から液が排出される。シタ基板204上にスポット搭載部210を設けられている。拡大して示すように、スポット搭載部210の内部には、生体高分子が結合する場所、地点であるスポット211が高密度に配列されている。この時、スポット211の一つ一つに生体高分子を一つずつ固定させることで、シタ基板204上に固定できる生体高分子の密度を高めることができる。 FIG. 2 shows a configuration example of the flow cell of the first embodiment. The flow cell is made by laminating a sita substrate 204, a wafer substrate 205, and an intermediate material 206. The portion surrounded by the hollow portion 207 formed in the sita substrate 204, the wafer substrate 205, and the intermediate material 206 serves as the flow cell flow path. The solution in which the biopolymer is dissolved is injected from the injection port 208 provided on the Sita substrate 204, and the liquid is discharged from the discharge port 209. A spot mounting portion 210 is provided on the Sita substrate 204. As shown in an enlarged manner, the spots 211, which are the places and points where the biopolymers are bound, are densely arranged inside the spot mounting portion 210. At this time, by fixing the biopolymers one by one to each of the spots 211, the density of the biopolymers that can be fixed on the Cita substrate 204 can be increased.
　シタ基板204に用いられる材料としては、特に制限なく、シリコン、ガラス、石英、サファイア、セラミック、フェライト、アルミナ、ダイヤモンドなどの無機材料、またはアルミニウム、SUS、チタン、鉄などの金属材料、またフェノール樹脂、エポキシ樹脂、メラミン樹脂、不飽和ポリエステル樹脂、アルキド樹脂、ポリウレタン、熱硬化性ポリイミド、透明ポリイミド、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリスチレン、ポリ酢酸ビニル、ABS樹脂、AS樹脂、アクリル樹脂 、ポリアミド、ナイロン、ポリアセタール、ポリカーボネート、変性ポリフェニレンエーテル、ポリブチレンテレフタレート、ポリエチレンテレフタレート、グラスファイバー強化ポリエチレンテレフタレート、環状ポリオレフィン、ポリフェニレンスルファイド、ポリサルフォン、ポリエーテルサルフォン、非晶ポリアリレート、液晶ポリマー、ポリエーテルエーテルケトン、などの樹脂材料及びこれらの混合材料やガラス繊維や炭素繊維強化無機材料を用いることもできる。これらの材料のなかで、DNAサンプルを蛍光法で分析する場合や、分析中に温度の上げ下げが行われる場合などは、自家蛍光が低く、熱膨張係数が小さく、且つ分析溶液の耐性が高いシリコン、ガラス、石英、SUS、チタンなどが特に望ましい。 The material used for the sita substrate 204 is not particularly limited, and is an inorganic material such as silicon, glass, quartz, sapphire, ceramic, ferrite, alumina, diamond, a metal material such as aluminum, SUS, titanium, iron, or a phenol resin. , Epoxy resin, melamine resin, unsaturated polyester resin, alkyd resin, polyurethane, thermosetting polyimide, transparent polyimide, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic Resin, polyamide, nylon, polyacetal, polycarbonate, modified polyphenylene ether, polybutylene terephthalate, polyethylene terephthalate, glass fiber reinforced polyethylene terephthalate, cyclic polyolefin, polyphenylensulfide, polysulfon, polyethersulfon, acrylate polyarylate, liquid crystal polymer, poly Resin materials such as ether ether ketone, mixed materials thereof, glass fiber, and carbon fiber reinforced inorganic materials can also be used. Among these materials, silicon with low autofluorescence, low coefficient of thermal expansion, and high resistance to analytical solutions when the DNA sample is analyzed by the fluorescence method or when the temperature is raised or lowered during the analysis. , Glass, quartz, SUS, titanium and the like are particularly desirable.
　スポット211は、特許文献2に示される様なスポッティング装置や、公知の手法であるLift-offプロセスを用いて形成される。用いられる材料としては、基板上に共有結合を介してスポットを形成できるようなものが良い。このような材料としては、基板表面に酸化膜を持つシリコン、ガラス、石英、サファイア、セラミック、フェライト、アルミナなどの無機材料やアルミニウム、SUS、チタン、鉄などの金属材料を用いる場合は、特にシランカップリング材が望ましい。また、シランカップリング材のなかでも、共有結合を介してアミノ基を含むコーティング膜を形成できるような反応性が高い官能基を持つものが良く、この様な官能基としては、ビニル基、エポキシ基、スチリル基、メタクリル基、アクリル基、アミノ基、ウレイド基、イソシアネート基、イソシアヌレート基、メルカプト基を分子内に持つエトキシシランやメトキシシランが挙げられる。また、基板にシリコンを用いてアルケン類やアルキン類を用いて形成しても良い。また、ポリジアリルジアンモニウムやポリリジンの様なカチオン性高分子を用いても良い。 Spot 211 is formed by using a spotting device as shown in Patent Document 2 or a Lift-off process which is a known method. As the material used, a material capable of forming a spot on the substrate via a covalent bond is preferable. As such a material, especially when an inorganic material such as silicon, glass, quartz, sapphire, ceramic, ferrite, or alumina having an oxide film on the substrate surface or a metal material such as aluminum, SUS, titanium, or iron is used, silane is used. Coupling material is desirable. Further, among the silane coupling materials, those having a highly reactive functional group capable of forming a coating film containing an amino group via a covalent bond are preferable, and such functional groups include vinyl groups and epoxys. Examples thereof include ethoxysilane and methoxysilane having a group, a styryl group, a methacryl group, an acrylic group, an amino group, a ureido group, an isocyanate group, an isocyanurate group and a mercapto group in the molecule. Further, it may be formed by using silicon as a substrate and using alkenes or alkynes. Further, a cationic polymer such as polydiallyl diammonium or polylysine may be used.
　一つのスポット211に複数種類のDNAサンプルが固定されると、複数種類のDNAサンプルからの蛍光色素が検出されるので誤検出となる。一方、スポット211が小さいと溶液中のDNAサンプルとの接触確立が低下し、DNAサンプルが固定されていないスポットが増えてDNA塩基配列決定のスループットが低下する。従って、スポット211の直径は、DNAサンプルが一個のみが固定率高く固定できる様な、DNAサンプルの半分よりも大きいようなサイズが良く、この様なサイズとして、直径50nm以上1000nm以下が挙げられる。 If multiple types of DNA samples are fixed in one spot 211, fluorescent dyes from multiple types of DNA samples will be detected, resulting in an erroneous detection. On the other hand, if the spot 211 is small, the probability of contact with the DNA sample in the solution decreases, the number of spots where the DNA sample is not fixed increases, and the throughput of DNA sequencing decreases. Therefore, the diameter of the spot 211 should be larger than half of the DNA sample so that only one DNA sample can be fixed at a high fixation rate, and such a size includes a diameter of 50 nm or more and 1000 nm or less.
　ウエ基板205に用いられる材料に特に制限はなく、シリコン、ガラス、石英、サファイア、セラミック、フェライト、アルミナ、ダイヤモンドなどの無機材料、またはアルミニウム、SUS、チタン、鉄などの金属材料、またフェノール樹脂、エポキシ樹脂、メラミン樹脂、不飽和ポリエステル樹脂、アルキド樹脂、ポリウレタン、熱硬化性ポリイミド、透明ポリイミド、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリスチレン、ポリ酢酸ビニル、ABS樹脂、AS樹脂、アクリル樹脂 、ポリアミド、ナイロン、ポリアセタール、ポリカーボネート、変性ポリフェニレンエーテル、ポリブチレンテレフタレート、ポリエチレンテレフタレート、グラスファイバー強化ポリエチレンテレフタレート、環状ポリオレフィン、ポリフェニレンスルファイド、ポリサルフォン、ポリエーテルサルフォン、非晶ポリアリレート、液晶ポリマー、ポリエーテルエーテルケトン、などの樹脂材料及びこれらの混合材料やガラス繊維や炭素繊維強化無機材料を用いることもできる。これらの材料のなかで、蛍光検出を行う場合、励起波長の光や蛍光波長の光の透過率が高いサンプルが望ましく、この様な材料としては、ガラス、石英、サファイアなどの無機材料やアクリル樹脂や環状ポリオレフィンなどの透明性が高い樹脂が望ましい。 The material used for the web substrate 205 is not particularly limited, and is an inorganic material such as silicon, glass, quartz, sapphire, ceramic, ferrite, alumina, diamond, a metal material such as aluminum, SUS, titanium, iron, or a phenol resin. Epoxy resin, melamine resin, unsaturated polyester resin, alkyd resin, polyurethane, thermosetting polyimide, transparent polyimide, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic resin , Polyamide, Nylon, Polyacetal, Polycarbonate, Modified polyphenylene ether, Polybutylene terephthalate, Polyethylene terephthalate, Glass fiber reinforced polyethylene terephthalate, Cyclic polyolefin, Polyphenylen sulphide, Polysulfone, Polyethersulfone, Acrylate polyarylate, Liquid crystal polymer, Polyether Resin materials such as ether ketone, mixed materials thereof, glass fiber, and carbon fiber reinforced inorganic materials can also be used. Among these materials, when performing fluorescence detection, a sample having high transmittance of light having an excitation wavelength or light having a fluorescence wavelength is desirable, and such materials include inorganic materials such as glass, quartz, and sapphire, and acrylic resins. And highly transparent resins such as cyclic polyolefins are desirable.
　中間材206に用いられる材料にも特に制限はなく、感圧性の両面テープ、接着剤、ゴムシートなどを用いることができる。ウエ基板やシタ基板の流路を形成する部分をくり抜いておいて、中間材206を用いずにシタ基板204とウエ基板205を直接貼り付けても良い。 The material used for the intermediate material 206 is not particularly limited, and pressure-sensitive double-sided tape, adhesive, rubber sheet, etc. can be used. It is also possible to hollow out the portion forming the flow path of the web substrate or the weft substrate and directly attach the shita substrate 204 and the weft substrate 205 without using the intermediate material 206.
　図３に遠心中のフローセル内部の断面の一部の概略図を示す。遠心工程時の遠心力により、フローセルの流路312内の生体高分子313がシタ基板304上のスポット311に押し付けられることによって、生体高分子313がスポット311に接触する確率を高めることができ、生体高分子313が固定されたスポット311の割合を高めることができる。 FIG. 3 shows a schematic view of a part of the cross section inside the flow cell during centrifugation. Due to the centrifugal force during the centrifugation process, the biopolymer 313 in the flow cell flow path 312 is pressed against the spot 311 on the Sita substrate 304, so that the probability that the biopolymer 313 comes into contact with the spot 311 can be increased. The proportion of spots 311 on which the biopolymer 313 is fixed can be increased.
　用いられる生体高分子に特に制限はないが、分子量が大きいほど遠心力や重力を得ることができるので、分子量が大きいDNAやタンパク質が望ましい。このようなDNAとして、特許文献1や特許文献2に示される環状テンプレートより得られる核酸増幅産物が特に望ましい。 The biopolymer used is not particularly limited, but the larger the molecular weight, the more centrifugal force and gravity can be obtained, so DNA and proteins with a large molecular weight are desirable. As such DNA, a nucleic acid amplification product obtained from the cyclic template shown in Patent Document 1 and Patent Document 2 is particularly desirable.
　図４に、本実施例の変形例として、フローセルに注入する生体高分子が溶解した溶液に粒子を分散させた場合の遠心中のフローセル内部の断面の一部の概略図を示す。生体高分子413が遠心力を受けた粒子414に押されることで、生体高分子413とスポット411が接触する確率を高めることができる。 FIG. 4 shows a schematic view of a part of the cross section inside the flow cell during centrifugation when the particles are dispersed in a solution in which the biopolymer to be injected into the flow cell is dissolved as a modified example of this embodiment. By pushing the biopolymer 413 by the particles 414 subjected to the centrifugal force, the probability that the biopolymer 413 and the spot 411 come into contact with each other can be increased.
　用いられる粒子に特に制限はないが、材質としては、金、銀、銅、白金などの金属材料、硫化亜鉛、セレン化カドミウムなどの半導体材料、酸化鉄などの磁性材料、シリカ、ジルコニアなどの無機材料、ポリスチレン、熱可塑性エラストマー、ポリエーテルスルホン樹脂、ポリフッ化ビリニデン、エポキシ樹脂、ポリ乳酸樹脂、エチルセルロース樹脂などの高分子材料などが用いられる。また、磁性材料、高分子材料などをコアとして、高分子材料や磁性材料などが単層、または複数層覆われたような複合材料を用いても良い。これらの粒子の中で、重力や遠心を受けやすい、比重が大きい金属材料や磁性材料を含む粒子が特に望ましい。粒子自身の自重で生体高分子をサンプル固定面に押し付けられるためである。 The particles used are not particularly limited, but the materials include metal materials such as gold, silver, copper and platinum, semiconductor materials such as zinc sulfide and cadmium selenium, magnetic materials such as iron oxide, and inorganic materials such as silica and zirconia. Materials, polystyrene, thermoplastic elastomers, polyether sulfone resins, polyvinylidene fluoride, epoxy resins, polylactic acid resins, ethyl cellulose resins and other polymer materials are used. Further, a composite material may be used in which a magnetic material, a polymer material, or the like is used as a core, and the polymer material, the magnetic material, or the like is covered with a single layer or a plurality of layers. Among these particles, particles containing a metal material or a magnetic material having a large specific gravity, which are susceptible to gravity or centrifugation, are particularly desirable. This is because the biopolymer is pressed against the sample fixing surface by the weight of the particles themselves.
　また、表面をカルボン酸やポリエチレングリコールなどの有機膜でコーティングされた粒子も望ましい。これらの有機コーティングは、生体高分子が粒子表面に吸着するのを防止する効果や、粒子がシタ基板に非特異的に吸着するのを防止する効果が得られる。また、表面にアビジンやアミノ基などの官能基がコーティングされている粒子に、これらの官能基と反応可能なビオチン化された化合物やN-ヒドロキシスクシンイミドエステル基を持つ化合物を反応させて吸着防止に効果がある化合物や官能基を導入しても良い。
用いられる粒子の形状にも特に制限はないが、シタ基板への吸着を防ぐために、シタ基板へ接触した場合に接触する面積を最小化できるような球状が特に望ましい。大きさも特に制限はないが、流路から洗い流す効率が下がらすに粒子同士の凝集が起きづらい粒径1～50μmのものが特に望ましい。 Further, particles whose surface is coated with an organic film such as carboxylic acid or polyethylene glycol are also desirable. These organic coatings have an effect of preventing the biopolymer from being adsorbed on the particle surface and an effect of preventing the particles from being non-specifically adsorbed on the sita substrate. In addition, particles coated with functional groups such as avidin and amino groups are reacted with a biotinylated compound capable of reacting with these functional groups or a compound having an N-hydroxysuccinimide ester group to prevent adsorption. Effective compounds and functional groups may be introduced.
The shape of the particles used is not particularly limited, but in order to prevent adsorption to the sita substrate, a spherical shape that can minimize the contact area when in contact with the sita substrate is particularly desirable. The size is not particularly limited, but it is particularly desirable that the particle size is 1 to 50 μm, which makes it difficult for particles to agglomerate while reducing the efficiency of flushing from the flow path.
　図５に本実施例の他の変形例として、粒径の分布が異なる2種類以上の粒子群を生体高分子が溶解する溶液に分散させた場合のフローセル断面の概略図を示す。粒子514と粒子514の間に粒子514より小さい粒子515が入り込むので、遠心をかけた場合に生体分子513を効率よく押し込むことができる。 FIG. 5 shows a schematic view of the cross section of the flow cell when two or more kinds of particle groups having different particle size distributions are dispersed in a solution in which a biopolymer is dissolved as another modification of this embodiment. Since the particle 515 smaller than the particle 514 enters between the particle 514 and the particle 514, the biomolecule 513 can be efficiently pushed in when centrifugally applied.
　図６の(a)～(e)を用いて、本実施例で用いられるスポットを有するシタ基板604を作製するための一工程例を説明する。本実施例のシタ基板604は、上述したLift-offプロセスを用いて作製することができる。シタ基板604上に疎水膜616をコーティングした後（a）、さらにレジスト617をコーティングする（b）。その後、リソグラフィを用いて開口部618を形成した後（c）、スポット611を形成する材料を蒸着する（d）。その後、レジスト617を除去する（e）。本実施例では、フォトリソグラフィ技術を用いた例を示したが、非特許文献2に示される様な、ナノインプリントリソグラフィ技術を用いても良い。このようにして作製したスポット611が形成されたシタ基板604を用いて、図２に示すようなフローセルを作製した後、図７に一例を示した核酸分析装置にセットし核酸分析を行う。 Using FIGS. 6A to 6E, an example of one step for producing the Sita substrate 604 having the spots used in this example will be described. The Cita substrate 604 of this example can be produced by using the Lift-off process described above. After coating the hydrophobic film 616 on the Cita substrate 604 (a), the resist 617 is further coated (b). Then, after forming the opening 618 using lithography (c), the material forming the spot 611 is deposited (d). Then, the resist 617 is removed (e). In this embodiment, an example using a photolithography technique is shown, but a nanoimprint lithography technique as shown in Non-Patent Document 2 may be used. A flow cell as shown in FIG. 2 is produced using the Cita substrate 604 on which the spot 611 produced in this manner is formed, and then set in the nucleic acid analyzer shown in FIG. 7 as an example to perform nucleic acid analysis.
　図７に示すように、核酸分析装置719は、上述した本実施例の調整方法で調整したフローセル720、フローセル720を固定し、なおかつフローセル720の温度を制御することができるホルダユニット721、フローセル720とホルダユニット721を移動させるステージユニット722、複数の試薬や洗浄水が収容された試薬容器723、試薬容器723に収容された試薬を吸引しフローセル720に注入するための駆動源である送液ユニット724、試薬の吸引・吐出の際、実際に試薬容器723やフローセル720にアクセスするノズル725、ノズル725を搬送させるノズル搬送ユニット726、フローセル720に固定された試料を観察するための検出ユニット727、及び廃液を収容する廃液容器728等を有する。検出ユニット727は、蛍光法に基づく蛍光検出装置からなり、ステージユニット722上のフローセル720に励起光を照射し、発生した蛍光を検出することができる。 As shown in FIG. 7, the nucleic acid analyzer 719 is a holder unit 721 and a flow cell 720 capable of fixing the flow cell 720 and the flow cell 720 adjusted by the adjustment method of the present embodiment described above and controlling the temperature of the flow cell 720. And the stage unit 722 that moves the holder unit 721, the reagent container 723 that contains multiple reagents and wash water, and the liquid feeding unit that is the driving source for sucking the reagents contained in the reagent container 723 and injecting them into the flow cell 720. 724, Nozzle 725 that actually accesses the reagent container 723 and the flow cell 720 when sucking and discharging the reagent, Nozzle transfer unit 726 that conveys the nozzle 725, Detection unit 727 for observing the sample fixed to the flow cell 720, It also has a waste liquid container 728 and the like for storing the waste liquid. The detection unit 727 comprises a fluorescence detection device based on the fluorescence method, and can irradiate the flow cell 720 on the stage unit 722 with excitation light to detect the generated fluorescence.
　図７に示した核酸分析装置719は以下の通りに動作する。まず、上述したフローセルの調整方法で調整された測定対象の核酸試料を保持したフローセル720をホルダユニット721に設置する。次に、ノズル725が試薬容器723にアクセスし、試薬を送液ユニット724により吸引する。ノズル725は、ノズル搬送ユニット726によりフローセル720上面に搬送され、フローセル716に試薬を注入する。そして、ホルダユニット721にて、フローセル720の流路内に含まれた核酸試料と試薬を温度調整することで反応が起こる。 The nucleic acid analyzer 719 shown in FIG. 7 operates as follows. First, the flow cell 720 holding the nucleic acid sample to be measured adjusted by the above-mentioned flow cell adjustment method is installed in the holder unit 721. Next, the nozzle 725 accesses the reagent container 723, and the reagent is sucked by the liquid feeding unit 724. The nozzle 725 is conveyed to the upper surface of the flow cell 720 by the nozzle transfer unit 726, and the reagent is injected into the flow cell 716. Then, in the holder unit 721, the reaction occurs by adjusting the temperature of the nucleic acid sample and the reagent contained in the flow path of the flow cell 720.
　反応した核酸試料を観察するために、フローセル720をステージユニット722にて移動させ、励起光を当てて検出領域内にある複数の核酸試料の蛍光を検出する。この際、好ましくは、核酸試料が固定された側の基板であるシタ基板を通して励起光を当て、蛍光を検出すればよい。検出後、フローセル720を微小に動かし同様の方法で検出、という動作を複数回繰り返す。全ての検出領域で観察が終了したら、試薬容器723に収容された洗浄水を送液ユニット724により吸引し、フローセル720へ注入することで、フローセル720の流路内を洗浄する。また、別の試薬を注入し検出、という動作を複数回繰り返すことで、核酸試料を読み取ることが出来る。核酸分析装置は、図示を省略したコンピュータなどの制御部により制御され、上記分析動作を自動的に行うことができる。 In order to observe the reacted nucleic acid sample, the flow cell 720 is moved by the stage unit 722 and irradiated with excitation light to detect the fluorescence of a plurality of nucleic acid samples in the detection region. At this time, preferably, the fluorescence may be detected by irradiating the excitation light through the Sita substrate, which is the substrate on the side on which the nucleic acid sample is fixed. After the detection, the flow cell 720 is slightly moved and detected by the same method, and the operation of detecting is repeated a plurality of times. When the observation is completed in all the detection areas, the washing water contained in the reagent container 723 is sucked by the liquid feeding unit 724 and injected into the flow cell 720 to wash the inside of the flow path of the flow cell 720. In addition, a nucleic acid sample can be read by repeating the operation of injecting another reagent and detecting it a plurality of times. The nucleic acid analyzer is controlled by a control unit such as a computer (not shown), and the analysis operation can be automatically performed.
　本実施例で得られたフローセルを用いて種々の核酸分析を実施することができ、例えば、DNA配列決定やハイブリダイゼーションを行うことができる。特に本実施例のフローセルを用いてDNA配列決定（DNAシークエンス）を行うことができる。DNAシークエンスの方法は限定されないが、非特許文献2に示される様な、可逆的なターミネータを用いた段階的合成法（Sequencing by synthesis)に用いることができる。すなわち、核酸分析用フローセルにDNAサンプルが溶解した溶液を注入した後、一定時間保持してスポット上にDNAサンプルを固定する。その後、固定されたDNAサンプルにシーケンスプライマをハイブリダイゼーションさせた後にSequencing by synthesis法を用いて塩基配列を決定する。該方法により、１サイクルで数十～数百bpの解読を行うことができ、１ランで数十Gbのデータを解析することが可能である。 Various nucleic acid analyzes can be performed using the flow cells obtained in this example, and for example, DNA sequencing and hybridization can be performed. In particular, DNA sequencing (DNA sequencing) can be performed using the flow cell of this example. The method of DNA sequencing is not limited, but it can be used for a stepwise synthesis method (Sequencing by synthesis) using a reversible terminator as shown in Non-Patent Document 2. That is, after injecting the solution in which the DNA sample is dissolved into the flow cell for nucleic acid analysis, the DNA sample is fixed on the spot by holding it for a certain period of time. Then, after hybridizing the sequence primer to the fixed DNA sample, the nucleotide sequence is determined using the Sequencing by synthesis method. By this method, it is possible to perform decoding of several tens to several hundreds of bp in one cycle, and it is possible to analyze data of several tens of Gb in one run.
  実施例２として、上述したフローセルの調整方法の一具体例の詳細を説明する。 As Example 2, details of a specific example of the above-mentioned flow cell adjustment method will be described.
　（１）シタ基板の作製
  上述したLift-offプロセスに準じた。シリコンウェハ上にHMDSをコーティングした後、ベーキングして疎水膜を導入した。ポジ型EBレジスト(ZEP-520A7)を塗布した後、EBフォトリソグラフィ装置（Elionix ELS-7500）を用いて露光した。その後、酢酸アミルを用いて現像処理を行い直径500nmの開口部を形成した。3-アミノプロピルトリメトキシシランを蒸着した後、有機溶剤中に基板を浸漬しながら超音波処理によりポジ型EBレジストを除去した。 (1) Preparation of Sita substrate According to the Lift-off process described above. After coating HMDS on a silicon wafer, it was baked to introduce a hydrophobic film. After applying a positive EB resist (ZEP-520A7), it was exposed using an EB photolithography apparatus (Elionix ELS-7500). Then, development treatment was carried out using amyl acetate to form an opening having a diameter of 500 nm. After vapor deposition of 3-aminopropyltrimethoxysilane, the positive EB resist was removed by ultrasonic treatment while immersing the substrate in an organic solvent.
　（２）フローセルの作製
  得られたシタ基板とレーザ加工により中抜き部が形成された両面テープとウエ基板を貼り合わせてフローセルを形成した。 (2) Preparation of Flow Cell A flow cell was formed by laminating the obtained sita substrate, a double-sided tape having a hollow portion formed by laser processing, and a wafer substrate.
　（３）環状テンプレートより得られる核酸増幅産物の作製
  特許文献2に示されている方法に従い環状DNAテンプレートより得られた増幅DNAサンプルを作製した。2種類の90塩基長の合成オリゴマーにポリヌクレオチドキナーゼを用いてリン酸化した後、26塩基長のオリゴヌクレオチドとAmpLigaseを用いて環状化した。環状化反応は、94℃/2分と40℃/10分のサイクル反応を3サイクル行った。その後、ExonucleaseIとExonucleaseIIIを加えて残っている直鎖のテンプレートを分解した。得られた環状テンプレートにPhi29とdNTPを加えた後30℃に70時間保持し、増幅DNAサンプルを作製した。得られた増幅DNAサンプルはMicrocon YM-50フィルターを用いて精製した。 (3) Preparation of Nucleic Acid Amplification Product Obtained from Circular Template An amplified DNA sample obtained from a circular DNA template was prepared according to the method shown in Patent Document 2. Two types of 90-base-long synthetic oligomers were phosphorylated with polynucleotide kinases and then cyclized with 26-base-long oligonucleotides and Amp Ligase. As for the cyclization reaction, three cycles of 94 ° C./2 minutes and 40 ° C./10 minutes were performed. Then Exonuclease I and Exonuclease III were added to disassemble the remaining linear template. After adding Phi29 and dNTP to the obtained circular template, the mixture was kept at 30 ° C. for 70 hours to prepare an amplified DNA sample. The obtained amplified DNA sample was purified using a Microcon YM-50 filter.
　（４）フローセル調整方法
  得られた核酸増幅産物が溶解した溶液を注入口を介してフローセルに注入した後、注入口と排出口をテープで封止した。その後、遠心機を用いてフローセルを1000Gで二時間遠心し、スポット上に核酸増幅産物を固定した。その後、封止に用いたテープを剥がした後、注入口より緩衝溶液を注入して未固定の核酸増幅産物を洗い出した。 (4) Flow cell adjustment method After the solution in which the obtained nucleic acid amplification product was dissolved was injected into the flow cell through the injection port, the injection port and the discharge port were sealed with tape. Then, the flow cell was centrifuged at 1000 G for 2 hours using a centrifuge, and the nucleic acid amplification product was fixed on the spot. Then, after peeling off the tape used for sealing, a buffer solution was injected from the injection port to wash out the unfixed nucleic acid amplification product.
　（５）核酸分析
  フローセル内にシーケンスプライマ溶液を注入して一定時間保持した後に、フローセル内を緩衝溶液で洗浄して未反応のシーケンスプライマを除去した。その後、核酸分析装置719にフローセル720を設置した後、非特許文献2に示す方法で核酸分析を行った。配列決定用の色素でラベル化された合成ヌクレオチドとDNA合成酵素(9°N mutant DNA polymerase)を含む溶液を注入した後、ホルダユニットで68℃で10分保持した。その後、未反応で残っているDNAサンプルの1塩基伸長反応を終わらせるために、色素でラベル化されていない合成ヌクレオチド溶液を注入して、さらに68℃で10分間保持した。その後、SPSCバッファーでフローセル720内の流路を洗浄し、未反応のヌクレオチドを除去した。フローセル720をステージユニット722にて移動させた後、検出ユニット727にて励起光を当てて検出領域内にある複数のDNAサンプルに導入された蛍光を検出した。その後、DNAサンプルに導入された蛍光物質を除去するため、核酸分析用フローセル720にNa2PdCl2/P(PhSO3Na)3水溶液を注入した後60℃で5分保持した。その後、フローセル720に緩衝溶液を注入して洗浄した。前述の一塩基伸長反応、追加の一塩基伸長反応、蛍光検出、蛍光物質除去のサイクルを繰り返しDNA配列を決定した。 (5) Nucleic acid analysis After injecting the sequence primer solution into the flow cell and holding it for a certain period of time, the inside of the flow cell was washed with a buffer solution to remove the unreacted sequence primer. Then, after installing the flow cell 720 in the nucleic acid analyzer 719, nucleic acid analysis was performed by the method shown in Non-Patent Document 2. After injecting a solution containing a synthetic nucleotide labeled with a dye for sequencing and a DNA synthase (9 ° N mutant DNA polymerase), the mixture was held in a holder unit at 68 ° C. for 10 minutes. Then, in order to end the 1-base extension reaction of the unreacted DNA sample, a synthetic nucleotide solution not labeled with a dye was injected and kept at 68 ° C. for 10 minutes. The flow path in the flow cell 720 was then washed with SPSC buffer to remove unreacted nucleotides. After moving the flow cell 720 by the stage unit 722, the detection unit 727 applied excitation light to detect the fluorescence introduced into a plurality of DNA samples in the detection region. Then, in order to remove the fluorescent substance introduced into the DNA sample, a Na 2 PdCl 2 / P (PhSO 3 Na) 3 aqueous solution was injected into the flow cell 720 for nucleic acid analysis and then kept at 60 ° C. for 5 minutes. Then, the buffer solution was injected into the flow cell 720 and washed. The DNA sequence was determined by repeating the cycle of the above-mentioned single nucleotide extension reaction, additional single nucleotide extension reaction, fluorescence detection, and removal of fluorescent substance.
　本実施例ではスポットを持つフローセルを用いたが、本発明のフローセルの調整方法は、スポットがなくシタ基板上面全面に生体高分子を固定可能な膜を持つフローセルにも用いることができる。また、本実施例では、核酸増幅産物を用いたが、タンパクのようなその他の生体高分子にも用いることができる。 Although a flow cell having a spot was used in this embodiment, the flow cell adjusting method of the present invention can also be used for a flow cell having no spot and having a film capable of fixing a biopolymer on the entire upper surface of the Sita substrate. In addition, although the nucleic acid amplification product was used in this example, it can also be used for other biopolymers such as proteins.
  実施例３として、上述したフローセルの調整方法のさらなる具体例の一つを詳細に説明する。核酸増幅産物が溶解した溶液中に表面がカルボキシル化されたビーズ（DynabeadsTM M-270 Carboxylic acid、Invitrogen社）を分散させた後、得られた溶液をフローセルに注入した。その他は実施例２と同様に行った。 As the third embodiment, one of further specific examples of the above-mentioned flow cell adjusting method will be described in detail. Beads having a carboxylated surface (Dynabeads TM M-270 Carboxylic acid, Invitrogen) were dispersed in a solution in which a nucleic acid amplification product was dissolved, and then the obtained solution was injected into a flow cell. Others were the same as in Example 2.
  実施例４として、上述したフローセルの調整方法のさらなる具体例の一つを詳細に説明する。核酸増幅産物が溶解した溶液中に表面がカルボキシル化された粒径2.7umのビーズ（DynabeadsTM M-270 Carboxylic acid、Invitrogen社）と粒径1.0umのビーズ（DynabeadsTM MyOneTM Carboxylic acid、Invitrogen社）の両方を分散させた後、得られた溶液をフローセルに注入した。その他は実施例３と同様に行った。 As the fourth embodiment, one of further specific examples of the above-mentioned flow cell adjusting method will be described in detail. 2.7 um particle size beads (Dynabeads TM M-270 Carboxylic acid, Invitrogen) and 1.0 um particle size beads (Dynabeads TM MyOne TM Carboxylic acid, Invitrogen) whose surface was carboxylated in a solution in which the nucleic acid amplification product was dissolved. ) Was dispersed, and then the obtained solution was injected into the flow cell. Others were the same as in Example 3.
  実施例５として、上述したフローセルの調整方法のさらなる具体例の一つを詳細に説明する。表面がカルボキシル化されたビーズ（DynabeadsTM M-270 Carboxylic acid、Invitrogen社）を分散させた後、得られた溶液をフローセルに注入した。その後、遠心は行わずに、シタ基板を下にして２時間保持してスポット上に核酸増幅産物を固定した。その他は実施例3と同様に行った。 As the fifth embodiment, one of further specific examples of the above-mentioned flow cell adjusting method will be described in detail. After dispersing beads having a carboxylated surface (Dynabeads TM M-270 Carboxylic acid, Invitrogen), the obtained solution was injected into a flow cell. Then, without centrifuging, the nucleic acid amplification product was fixed on the spot by holding the Cita substrate down for 2 hours. Others were the same as in Example 3.
  実施例６として、上述したフローセルの調整方法のさらなる具体例の一つを詳細に説明する。核酸増幅産物が溶解した溶液中に表面がカルボキシル化された粒径2.7umのビーズ（DynabeadsTM M-270 Carboxylic acid、Invitrogen社）と粒径1.0umのビーズ（DynabeadsTM MyOneTM Carboxylic acid、Invitrogen社）の両方を分散させた後、得られた溶液をフローセルに注入した。その後、遠心は行わずに、シタ基板を下にして２時間保持してスポット上に核酸増幅産物を固定した。その他は実施例３と同様に行った。 As the sixth embodiment, one of further specific examples of the above-mentioned flow cell adjusting method will be described in detail. 2.7 um particle size beads (Dynabeads TM M-270 Carboxylic acid, Invitrogen) and 1.0 um particle size beads (Dynabeads TM MyOne TM Carboxylic acid, Invitrogen) whose surface was carboxylated in a solution in which the nucleic acid amplification product was dissolved. ) Was dispersed, and then the obtained solution was injected into the flow cell. Then, without centrifuging, the nucleic acid amplification product was fixed on the spot by holding the Cita substrate down for 2 hours. Others were the same as in Example 3.
　以上詳述した本発明のフローセルの調整方法を用いて、種々の生体高分子を解析することができる。本発明は上記した実施例に限定されるものではなく、様々な変形例が含まれる。例えば、上記した実施例は本発明のより良い理解のために詳細に説明したのであり、必ずしも説明の全ての構成を備えるものに限定されるものではない。 Various biopolymers can be analyzed by using the flow cell adjusting method of the present invention described in detail above. The present invention is not limited to the above-described examples, and includes various modifications. For example, the above-mentioned examples have been described in detail for a better understanding of the present invention, and are not necessarily limited to those having all the configurations of the description.
101　遠心板
102　可動ホルダ
103　フローセル
204,304,404,504,605　シタ基板 
205,305,405,505　ウエ基板
206　中間材 
207  中抜き部
208　注入口
209　排出口
210　スポット搭載部
211,311,411,511,611　スポット
312,412,512　流路
313,413,513　生体高分子
414,514　粒子
515　小さい粒子
616　疎水性膜
617　レジスト
618　開口部
719　核酸分析装置
720　フローセル
721　ホルダユニット
722　ステージユニット
723　試薬容器
724　送液ユニット
725　ノズル
726　ノズル搬送ユニット
727　検出ユニット
728　廃液容器 101 Centrifugal plate
102 Movable holder
103 flow cell
204,304,404,504,605 Sita board
205,305,405,505 Ue board
206 Intermediate material
207 hollow part
208 inlet
209 outlet
210 Spot mounting part
211,311,411,511,611 Spots
312,412,512 channels
313,413,513 Biopolymers
414,514 particles
515 small particles
616 Hydrophobic membrane
617 resist
618 opening
719 Nucleic acid analyzer
720 flow cell
721 holder unit
722 Stage unit
723 Reagent container
724 Liquid transfer unit
725 nozzle
726 Nozzle transfer unit
727 Detection unit
728 Waste liquid container

Claims (14)

  1. 生体高分子を解析するためのフローセルの調整方法であって、
    前記生体高分子が溶解した溶液をフローセルに注入する工程と、
    前記フローセルを構成する基板の上に前記生体高分子を固定するため、前記生体高分子が溶解した溶液を注入した前記フローセルを遠心する工程と、
    前記基板に未固定の前記生体高分子を前記フローセルから洗い出す工程、とからなる、
    ことを特徴とするフローセルの調整方法。     A flow cell adjustment method for analyzing biopolymers.
    The step of injecting the solution in which the biopolymer is dissolved into the flow cell, and
    In order to fix the biopolymer on the substrate constituting the flow cell, a step of centrifuging the flow cell in which the solution in which the biopolymer is dissolved is injected and
    A step of washing out the biopolymer unfixed on the substrate from the flow cell.
    A method of adjusting the flow cell, which is characterized in that.
  2. 請求項１に記載のフローセルの調整方法であって、
    前記生体高分子が、環状テンプレートより得られた核酸増幅産物である、
    ことを特徴とするフローセルの調整方法。     The flow cell adjusting method according to claim 1.
    The biopolymer is a nucleic acid amplification product obtained from a cyclic template.
    A method of adjusting the flow cell, which is characterized in that.
  3. 請求項1または2に記載のフローセルの調整方法であって、
    前記生体高分子がスポットを介して前記基板の上に固定される、
    ことを特徴とするフローセルの調整方法。     The flow cell adjustment method according to claim 1 or 2.
    The biopolymer is fixed onto the substrate via spots,
    A method of adjusting the flow cell, which is characterized in that.
  4. 請求項1乃至3のいずれか１項に記載のフローセルの調整方法であって、
    前記フローセルは、励起光が照射され、発生した蛍光が検出される、
    ことを特徴とするフローセルの調整方法。     The flow cell adjusting method according to any one of claims 1 to 3.
    The flow cell is irradiated with excitation light, and the generated fluorescence is detected.
    A method of adjusting the flow cell, which is characterized in that.
  5. 生体高分子を解析するためのフローセルの調整方法であって、
    前記生体高分子が溶解し、同時に粒子が分散している溶液をフローセルに注入する工程と、
    フローセルを構成する基板上に前記生体高分子を固定するために、溶液を注入した前記フローセルを一定時間保持する工程と、未固定の生体高分子と前記粒子をフローセルから洗い出す工程、からなることを特徴とするフローセルの調整方法。     A flow cell adjustment method for analyzing biopolymers.
    The step of injecting the solution in which the biopolymer is dissolved and the particles are dispersed at the same time into the flow cell,
    In order to fix the biopolymer on the substrate constituting the flow cell, the process comprises holding the flow cell infused with the solution for a certain period of time, and washing out the unfixed biopolymer and the particles from the flow cell. A characteristic flow cell adjustment method.
  6. 請求項5に記載のフローセルの調整方法であって、
    前記生体高分子が、環状テンプレートより得られた核酸増幅産物である、
    ことを特徴とするフローセルの調整方法。     The flow cell adjustment method according to claim 5.
    The biopolymer is a nucleic acid amplification product obtained from a cyclic template.
    A method of adjusting the flow cell, which is characterized in that.
  7. 請求項5または6に記載のフローセルの調整方法であって、
    前記生体高分子がスポットを介して前記基板上に固定される、
    ことを特徴とするフローセルの調整方法。     The flow cell adjustment method according to claim 5 or 6.
    The biopolymer is fixed onto the substrate via spots.
    A method of adjusting the flow cell, which is characterized in that.
  8. 請求項5乃至7のいずれか１項に記載のフローセルの調整方法であって、
    前記粒子が粒径の分布が異なる2種類以上の粒子群からなる,
    こと特徴とするフローセルの調整方法。     The flow cell adjusting method according to any one of claims 5 to 7.
    The particles consist of two or more types of particles with different particle size distributions.
    The characteristic flow cell adjustment method.
  9. 請求項5乃至8のいずれか１項に記載のフローセルの調整方法であって、
    前記フローセルは、励起光が照射され、発生した蛍光が検出される、
    ことを特徴とするフローセルの調整方法。     The flow cell adjusting method according to any one of claims 5 to 8.
    The flow cell is irradiated with excitation light, and the generated fluorescence is detected.
    A method of adjusting the flow cell, which is characterized in that.
  10. 生体高分子を解析するためのフローセルの調整方法であって、
    前記生体高分子が溶解し、同時に粒子が分散している溶液をフローセルに注入する工程と、
    フローセルを構成する基板上に前記生体高分子を固定するために、溶液を注入した前記フローセルを遠心する工程と、未固定の生体高分子をフローセルから洗い出す工程、からなる、
    ことを特徴とするフローセルの調整方法。     A flow cell adjustment method for analyzing biopolymers.
    The step of injecting the solution in which the biopolymer is dissolved and the particles are dispersed at the same time into the flow cell,
    In order to fix the biopolymer on the substrate constituting the flow cell, the step of centrifuging the flow cell in which the solution is injected and the step of washing out the unfixed biopolymer from the flow cell.
    A method of adjusting the flow cell, which is characterized in that.
  11. 請求項10に記載のフローセルの調整方法であって、
    前記生体高分子が、環状テンプレートより得られた核酸増幅産物である、
    ことをフローセルの調整方法。     The flow cell adjusting method according to claim 10.
    The biopolymer is a nucleic acid amplification product obtained from a cyclic template.
    That is how to adjust the flow cell.
  12. 請求項10または11に記載のフローセルの調整方法であって、
    前記生体高分子がスポットを介して前記基板上に固定される,
    ことを特徴とするフローセルの調整方法。     The flow cell adjusting method according to claim 10 or 11.
    The biopolymer is fixed onto the substrate via spots,
    A method of adjusting the flow cell, which is characterized in that.
  13. 請求項10乃至12のいずれか１項に記載のフローセルの調整方法であって、
    前記粒子が粒径の分布が異なる2種類以上の粒子群からなる、
    ことを特徴とするフローセルの調整方法。     The flow cell adjusting method according to any one of claims 10 to 12.
    The particles consist of two or more types of particles having different particle size distributions.
    A method of adjusting the flow cell, which is characterized in that.
  14. 請求項10乃至13のいずれか１項に記載のフローセルの調整方法であって、
    前記フローセルは、励起光が照射され、発生した蛍光が検出される、
    ことを特徴とするフローセルの調整方法。     The flow cell adjusting method according to any one of claims 10 to 13.
    The flow cell is irradiated with excitation light, and the generated fluorescence is detected.
    A method of adjusting the flow cell, which is characterized in that.
PCT/JP2019/031122 2019-08-07 2019-08-07 Flow cell adjustment method WO2021024416A1 (en)

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Citations (5)

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JP2007143551A (en) * 2005-11-07 2007-06-14 Canon Inc Substrate, apparatus, method, and kit for treating biotissue
US20090018024A1 (en) * 2005-11-14 2009-01-15 President And Fellows Of Harvard College Nanogrid rolling circle dna sequencing
US20090270273A1 (en) * 2008-04-21 2009-10-29 Complete Genomics, Inc. Array structures for nucleic acid detection
WO2015040930A1 (en) * 2013-09-20 2015-03-26 株式会社日立ハイテクノロジーズ Biomolecule measuring device
WO2016084489A1 (en) * 2014-11-27 2016-06-02 株式会社日立ハイテクノロジーズ Spot array substrate, method for producing same, and nucleic acid polymer analysis method and device

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007143551A (en) * 2005-11-07 2007-06-14 Canon Inc Substrate, apparatus, method, and kit for treating biotissue
US20090018024A1 (en) * 2005-11-14 2009-01-15 President And Fellows Of Harvard College Nanogrid rolling circle dna sequencing
US20090270273A1 (en) * 2008-04-21 2009-10-29 Complete Genomics, Inc. Array structures for nucleic acid detection
WO2015040930A1 (en) * 2013-09-20 2015-03-26 株式会社日立ハイテクノロジーズ Biomolecule measuring device
WO2016084489A1 (en) * 2014-11-27 2016-06-02 株式会社日立ハイテクノロジーズ Spot array substrate, method for producing same, and nucleic acid polymer analysis method and device

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