WO2021024416A1 - Procédé de réglage de cytométrie de flux - Google Patents

Procédé de réglage de cytométrie de flux 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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English (en)
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/fr
Publication of WO2021024416A1 publication Critical patent/WO2021024416A1/fr

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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.

Abstract

L'invention concerne un procédé de réglage de cytométrie de flux étant destiné à augmenter le débit d'analyse par fixation de biopolymères ayant un poids moléculaire élevé à un cytomètre de flux à haute densité, le procédé comprenant : l'injection d'une solution ayant des biopolymères (313) dissous dans cette dernière au cytomètre de flux comprenant un substrat inférieur (304), un substrat supérieur (305), un canal d'écoulement (312), et analogues ; la centrifugation du cytomètre de flux à laquelle la solution dissoute dans un biopolymère est injectée afin de fixer les biopolymères (313) sur le substrat inférieur (304) ; puis le lavage des biopolymères non fixés sur les substrats à partir du cytomètre de flux.
PCT/JP2019/031122 2019-08-07 2019-08-07 Procédé de réglage de cytométrie de flux WO2021024416A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007143551A (ja) * 2005-11-07 2007-06-14 Canon Inc 生体組織処理用の基板、処理装置、処理方法及び処理キット
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 (fr) * 2013-09-20 2015-03-26 株式会社日立ハイテクノロジーズ Dispositif de mesure de biomolécules
WO2016084489A1 (fr) * 2014-11-27 2016-06-02 株式会社日立ハイテクノロジーズ Substrat à réseau de points, procédé de fabrication de celui-ci, procédé et dispositif d'analyse de polymère d'acide nucléique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007143551A (ja) * 2005-11-07 2007-06-14 Canon Inc 生体組織処理用の基板、処理装置、処理方法及び処理キット
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 (fr) * 2013-09-20 2015-03-26 株式会社日立ハイテクノロジーズ Dispositif de mesure de biomolécules
WO2016084489A1 (fr) * 2014-11-27 2016-06-02 株式会社日立ハイテクノロジーズ Substrat à réseau de points, procédé de fabrication de celui-ci, procédé et dispositif d'analyse de polymère d'acide nucléique

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