WO2005093416A1 - Substrat pour disposer des billes et méthode de disposition de billes utilisant le substrat - Google Patents

Substrat pour disposer des billes et méthode de disposition de billes utilisant le substrat Download PDF

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Publication number
WO2005093416A1
WO2005093416A1 PCT/JP2005/005339 JP2005005339W WO2005093416A1 WO 2005093416 A1 WO2005093416 A1 WO 2005093416A1 JP 2005005339 W JP2005005339 W JP 2005005339W WO 2005093416 A1 WO2005093416 A1 WO 2005093416A1
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WO
WIPO (PCT)
Prior art keywords
substrate
beads
magnetic
bead
micro
Prior art date
Application number
PCT/JP2005/005339
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English (en)
Japanese (ja)
Inventor
Takanori Ichiki
Hiroaki Aizawa
Original Assignee
Japan Science And Technology Agency
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency filed Critical Japan Science And Technology Agency
Priority to JP2006511486A priority Critical patent/JPWO2005093416A1/ja
Publication of WO2005093416A1 publication Critical patent/WO2005093416A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • the present invention relates to a substrate for arranging beads and a method of arranging beads using the same.
  • the present invention relates to a bead placing substrate capable of placing beads in a plurality of micro-reaction tanks of the bead placing substrate with high efficiency and achieving stable placement, and a bead placing method using the same.
  • DNA chips are made by directly immobilizing biomolecules on a substrate.
  • Non-Patent Document 1 discloses a technique in which a micro semiconductor block is separated from an original substrate, and is dispersed at an arbitrary position on the substrate by spraying the micro semiconductor block on a target substrate in a liquid. Has been disclosed. It is reported that by fixing a biomolecule to a small semiconductor block and using the method described in Non-Patent Document 1, the biomolecule can be arranged at an arbitrary position on a substrate.
  • Non-Patent Document l H. Yeh and J. Smith, IEEE Photon. Tech. Lett., Vol. 6, .706, 1994 Disclosure of the invention
  • an object of the present invention is to arrange beads capable of immobilizing biomolecules and the like in a short time and with high efficiency at an arbitrary place, and to stably arrange the beads without detachment from an arbitrary place. It is an object of the present invention to provide a substrate for arranging beads, and a method for arranging beads using the same.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the above object can be achieved by combining a magnetic bead with a bead arrangement substrate having a magnetic thin film. Was completed.
  • the bead disposing substrate of the present invention is a bead disposing substrate for disposing magnetic beads in a micro-reaction tank, and comprises a substrate material and a magnetic thin film disposed on the substrate material. And a plurality of minute reaction vessels disposed on the magnetic thin film.
  • the magnetic beads are attracted into the micro-reaction tank by the action of the magnetic force of the magnetic beads and the magnetic thin film, so that the magnetic beads can be easily arranged and a stable arrangement can be obtained.
  • the magnetic beads can maintain a stable arrangement since the magnetization of the magnetic thin film remains after the magnet is removed, the magnetic beads can maintain a stable arrangement.
  • a magnetic bead on which a biomolecule is immobilized can be controlled by an external magnetic field and can be easily and reliably filled in a microreactor. It becomes possible to form a large number of micro reaction vessels.
  • FIG. 1 is a schematic view showing a process for producing a substrate for fixing beads according to an embodiment of the present invention.
  • FIG. 2 is an enlarged photograph of a micro-reaction tank formed on a substrate for arranging beads.
  • FIG. 3 is a schematic view of an apparatus used for observing an array of magnetic beads using an optical microscope.
  • FIG. 4 is a schematic view showing a step of producing a substrate for fixing beads according to another embodiment of the present invention.
  • FIG. 5 is (a) a microphotograph of magnetic beads under irradiation of white light and (b) a microphotograph of magnetic beads under irradiation of green light in Example 4.
  • the substrate material that can be used for the substrate for arranging beads of the present invention is preferably a transparent glass or a plastic material, more preferably a material that does not emit fluorescence.
  • Transparent board By using the material, optical observation of the biopolymer arbitrarily adsorbed on the surface of the magnetic beads becomes possible. In addition, by using a material that does not emit fluorescence for the substrate material, the accuracy of the optical measurement can be improved.
  • the magnetic beads can be fixed in the minute reaction tank.
  • the material of the magnetic thin film metals such as nickel, nickel alloys, iron and iron alloys can be suitably used. In the present invention, it is preferable to use a magnetic material having a large remanent magnetization. .
  • the thickness of the magnetic thin film is preferably 0.2 to 2 x m for the purpose.
  • an adhesive layer of chromium or the like can be provided between the substrate material and the magnetic thin film provided thereon to enhance the adhesiveness between the two.
  • the plurality of micro-reaction tanks provided on the magnetic thin film can be formed by using a known photolithography technique, which is a fine processing technique cultivated in the semiconductor industry.
  • a transparent photoresist As a material for forming the micro-reaction tank, a transparent photoresist, polydimethylsiloxane (PDMS), or the like can be suitably used.
  • PDMS polydimethylsiloxane
  • the filling rate of the magnetic beads into the reaction vessel depends on the diameter of the reaction vessel, and the larger the diameter of the reaction vessel is slightly larger than the diameter of the magnetic beads, the higher the filling rate is. Is 112 times the diameter of the magnetic beads. Further, in filling one magnetic bead into one microreactor, the depth of the microreactor is preferably 112 times the diameter of the magnetic beads.
  • the surface of the substrate for arranging beads and the inner wall of the microreactor of the present invention may be coated with a blocking agent for preventing nonspecific adsorption of biomolecules, for example, polyethylene glycol (PEG) or 2-methacryloyloxetyl phosphorylcholine (MPC). ) Can be suitably coated.
  • a blocking agent for preventing nonspecific adsorption of biomolecules for example, polyethylene glycol (PEG) or 2-methacryloyloxetyl phosphorylcholine (MPC).
  • PEG polyethylene glycol
  • MPC 2-methacryloyloxetyl phosphorylcholine
  • the micro reaction tank is hydrophilized.
  • the liquid magnetic beads dispersed liquid
  • the magnetic thin film at the bottom of the microreactor may be removed by a known method such as wet etching. Observation becomes possible. For example, it becomes possible to determine the expression of cells and proteins by fluorescence.
  • the bead placement method of the present invention when placing the magnetic beads in the microreaction tank of the above-described bead placement substrate, a magnet is placed below the bead placement substrate, and the magnet is placed on the substrate. Then, a dispersion liquid in which magnetic beads are dispersed is dropped. Thereby, the magnetic beads are attracted to the micro-reaction tank by the attraction of the magnet. Furthermore, the magnetic beads are dispersed by appropriately moving the magnet in a direction parallel to the substrate, and the filling rate in the reaction tank is improved.
  • the strength of the magnetic field applied to the bead placement substrate by the magnet is preferably 100 to 10,000 gauss in order to obtain a desired effect.
  • the magnetic thin film is magnetized by the magnet disposed under the substrate, and the magnetic beads remain in the magnetic thin film even after the magnetic beads are attracted to the reaction tank and the magnet is removed. It is possible to keep it fixed.
  • a chromium film is formed as an adhesive layer 2 on a transparent glass substrate material 1 of 20 mm ⁇ 20 mm, and nickel film, which is a magnetic material, has a film thickness of 0.
  • a magnetic thin film 3 was obtained.
  • a negative photoresist SU-8 indicated by reference numeral 4 in the figure was applied so as to have a thickness of 3 ⁇ m (FIG. L (b)).
  • the pattern of 10,000 micro-reactors with a diameter of 3 ⁇ m was transferred by contact exposure, and the circular pattern was removed with a developer to form a micro-reactor 5 (FIG. 1 (c)).
  • Fig. 1 (d) shows an enlarged photograph of the prepared bead placement substrate.
  • a magnetic beam having a diameter of 2.8 / m A dispersion liquid 12 in which the particles 11 were dispersed was dropped.
  • a magnet 13 having a magnetic flux density of 2000 Gauss was arranged on the back surface of the substrate 10 in order to guide the magnetic beads 11 into the micro reaction vessel 5. Further, by appropriately moving the magnet 13 in a direction parallel to the substrate 10, the induction of the magnetic beads 11 into the minute reaction tank 5 was promoted.
  • the arrangement of the magnetic beads 11 is observed with an optical microscope 14 connected to an arithmetic processing unit 15. did.
  • the optical microscope 14 it was confirmed that the magnetic beads 11 were filled one by one into each micro reaction vessel 5 with a high probability of 95% or more.
  • a substrate 10 was prepared in the same manner as in Example 1 except that nickel was not formed as the magnetic thin film 3, and the same test was performed.As a result, the filling rate in the microreactor 5 was 10% or less. Lower and lower values were shown. From this, it was confirmed that the filling rate was improved by the effect of the nickele film.
  • Example 1 As in Example 1, first, a chromium film was formed as an adhesive layer 2 on a transparent glass substrate material 1 of 20 mm ⁇ 20 mm, and nickel was used as a magnetic material so that the film thickness became 0.5 ⁇ . (Fig. L (a)). A negative photoresist SU-8 indicated by reference numeral 4 in the figure was applied to the magnetic thin film 3 of the nickele so as to have a thickness of 3-10 x m (FIG. 1 (b)). Next, the pattern of the circular microreactor 5 having a diameter of 3, 4, 5, 6, 7, and 10 zm was transferred by contact exposure, and the circular pattern was removed with a developer to form the microreactor 5 ( Figure 1 (c)).
  • the surface was subjected to hydrophilization treatment by irradiating the surface with oxygen plasma at 100 mTorr and 200 W for 5 minutes to prepare a substrate for bead placement (Fig. 1 (d) )).
  • a dispersion liquid 12 in which 4 ⁇ 10 6 magnetic beads 11 having a diameter of 2.8 ⁇ m are dispersed on the manufactured substrate 10 is dropped.
  • a magnet 13 was fixed to the back surface of the substrate 10 to guide the magnetic beads 11 into the reaction tank 5. Further, by moving the magnet 13, the induction of the magnetic beads 11 into the microreactor 5 was promoted.
  • Example 1 After cleaning the surface with pure water with the magnet 13 fixed to the back surface of the substrate 10, the same as in Example 1 was performed.
  • the state of the arrangement of the magnetic beads 11 was observed with the optical microscope 14 connected to the arithmetic processing unit 15 as described above. Observation with an optical microscope 14 revealed that the filling rate of the magnetic beads 11 increased in proportion to the diameter of the microreactor 5.
  • a micro-reaction tank having a large diameter was filled with a plurality of magnetic beads 11.
  • the depth of the microreactor 5 was 6 ⁇ m or more, a plurality of magnetic beads 11 were mixed in the longitudinal direction.
  • the diameter of the microreactor 5 was 6 ⁇ m or less, one magnetic bead 11 was filled in one microreactor 5.
  • the magnetic beads 11 are detached from the micro-reaction tank 5 having a diameter substantially the same size as the force of the magnetic beads 11 detached during washing. Not observed. From the above, it was clarified that the depth and diameter of the microreactor 5 were suitably 6 ⁇ m or less.
  • FIG. 4 (a) As in the case of Examples 1 and 2, first, as shown in FIG. 4 (a), on a transparent glass substrate material 1 of 20 mm ⁇ 20 mm, a nickel material, which is a magnetic material, was formed to a thickness of 0.5 ⁇ . Then, a magnetic thin film 3 was obtained. Thereafter, a negative photoresist SU-8 indicated by reference numeral 4 in the figure was applied to a thickness of 3 / im (FIG. 4 (b)). Further, the patterns of 10,000 circular microreactors 5 having a diameter of 3 / im were transferred by contact exposure, and the circular patterns were removed with a developer to form microreactors 5 (FIG. 4 (c)).
  • the magnetic thin film 3 (nickel film) at the bottom of the microreactor 5 was removed by wet etching (Fig. 4 (d)).
  • the surface was subjected to a hydrophilic treatment by irradiating the surface with oxygen plasma at 100 mTorr and 200 W for 5 minutes using a flat-plate inductively coupled plasma generator, and a substrate for bead placement was fabricated (Fig. 4 (e)). ).
  • a substrate for arranging beads was prepared by the method shown in Example 1.
  • Base Quartz glass which does not emit fluorescence in the visible light region with high UV transmittance, was used as the substrate material of the plate.
  • a PEG (polyethylene glycol) film was coated on the substrate surface to prevent nonspecific adsorption of proteins.
  • the obtained bead placement substrate was filled with magnetic beads whose surface was modified with biotin using PEG, and an aqueous solution in which streptavidin serving as a target molecule was dispersed was poured.
  • streptavidin used was fluorescently labeled with Texas Red.
  • the aqueous solution in which streptavidin was dispersed in pure water was washed.
  • Figure 5 shows the results.
  • Figure 5 (a) is a micrograph of magnetic beads under white light irradiation, and (b) is a micrograph of magnetic beads under green light irradiation. Strong red fluorescence was observed from the magnetic beads, confirming that streptavidin, a target molecule, was placed on the magnetic beads fixed in the microreaction tank.
  • the present invention provides a substrate structure and arrangement suitable for immobilizing biomolecules on a magnetic bead surface and then disposing the biomolecules on a substrate instead of directly immobilizing the biomolecules on a substrate like a DNA chip.
  • a large amount of biomolecules can be arranged on a substrate by self-assembly in a short time, and it is also easy to collect a biomolecule at a desired position from the substrate after the arrangement. .
  • This technology can be applied to large-scale parallel screening that is required for evolutionary molecular engineering that rapidly evolves biopolymers to create useful molecules. Therefore, the present invention is expected to contribute to a wide range of industrial fields, such as new drug development, the environment, bioprocesses, and foods, as basic elemental technologies of advanced molecular reactors that realize the theory of advanced molecular engineering.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

Un substrat pour disposer des billes et une méthode de disposition de billes utilisant le substrat permettant aux billes capables de fixer avec efficacité des biomolécules à n’importe quel endroit en peu de temps à être disposées de façon stable sur celles-ci pour que ces billes ne soient pas enlevées de cet endroit. Le substrat (10) pour disposer les billes pour fixer les billes magnétiques (11) dans des chambres de réaction minuscules (5) comprend un matériau de substrat (1),un film fin de corps magnétique (3) disposé sur le matériau du substrat (1), et la pluralité de chambres de réaction minuscules (5) disposées sur le film fin de corps magnétique (3). La méthode de disposition des billes comprend les étapes consistant à disposer un aimant (13) sous le substrat (10) pour disposer les billes et laisser tomber une dispersion (12) goutte à goutte, dans lequel les billes magnétiques (11) sont dispersées sur le substrat (10) pour fixer les billes magnétiques (11) dans les chambres de réaction minuscules (5) du substrat (10) pour disposer les billes.
PCT/JP2005/005339 2004-03-26 2005-03-24 Substrat pour disposer des billes et méthode de disposition de billes utilisant le substrat WO2005093416A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006511486A JPWO2005093416A1 (ja) 2004-03-26 2005-03-24 ビーズ配置用基板およびそれを用いたビーズ配置方法

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JP2004093655 2004-03-26
JP2004-093655 2004-03-26

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009025085A (ja) * 2007-07-18 2009-02-05 Kobe Univ 標的分子のセンシングチップの作製方法
WO2014007034A1 (fr) * 2012-07-06 2014-01-09 株式会社 日立ハイテクノロジーズ Dispositif et procédé d'analyse
JP2017138306A (ja) * 2016-01-31 2017-08-10 アークレイ株式会社 分析用具および分析装置
WO2019050017A1 (fr) * 2017-09-07 2019-03-14 三菱瓦斯化学株式会社 Substrat pour biopuce, biopuce, procédé de fabrication de biopuce et procédé de conservation de biopuce

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000249706A (ja) * 1999-02-26 2000-09-14 Hokuto Kagaku Sangyo Kk 新規の生物学的チップ及び分析方法
WO2002043855A1 (fr) * 2000-11-29 2002-06-06 Comissariat A L'energie Atomique Micro reseau statique de sondes biologiques ou chimiques, immobilisees sur un support par attraction magnetique
JP2002525579A (ja) * 1998-09-11 2002-08-13 トラスティーズ・オブ・タフツ・カレッジ 微小球を利用する標的分析物センサー
JP2004037338A (ja) * 2002-07-05 2004-02-05 Yokogawa Electric Corp 磁気ビーズを用いて生体高分子を基板へ固定する方法およびその方法を用いた生体高分子測定装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002525579A (ja) * 1998-09-11 2002-08-13 トラスティーズ・オブ・タフツ・カレッジ 微小球を利用する標的分析物センサー
JP2000249706A (ja) * 1999-02-26 2000-09-14 Hokuto Kagaku Sangyo Kk 新規の生物学的チップ及び分析方法
WO2002043855A1 (fr) * 2000-11-29 2002-06-06 Comissariat A L'energie Atomique Micro reseau statique de sondes biologiques ou chimiques, immobilisees sur un support par attraction magnetique
JP2004037338A (ja) * 2002-07-05 2004-02-05 Yokogawa Electric Corp 磁気ビーズを用いて生体高分子を基板へ固定する方法およびその方法を用いた生体高分子測定装置

Non-Patent Citations (1)

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Title
ICHIWARA ET AL: "Jiki Beads Sosa no tame no Micro Jiki Device no Sakusei.", OYO BUTSURIGAKU KANKEI RENGO KOENKAI KOEN YOKOSHU., no. 3, 2003, pages 1398, XP002998318 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009025085A (ja) * 2007-07-18 2009-02-05 Kobe Univ 標的分子のセンシングチップの作製方法
WO2014007034A1 (fr) * 2012-07-06 2014-01-09 株式会社 日立ハイテクノロジーズ Dispositif et procédé d'analyse
CN104471380A (zh) * 2012-07-06 2015-03-25 株式会社日立高新技术 分析装置以及分析方法
JPWO2014007034A1 (ja) * 2012-07-06 2016-06-02 株式会社日立ハイテクノロジーズ 分析装置及び分析方法
CN104471380B (zh) * 2012-07-06 2017-11-17 株式会社日立高新技术 分析装置以及分析方法
US9964539B2 (en) 2012-07-06 2018-05-08 Hitachi High-Technologies Corporation Analysis device and analysis method
EP2871464B1 (fr) * 2012-07-06 2021-07-28 Hitachi High-Tech Corporation Dispositif et procédé d'analyse
JP2017138306A (ja) * 2016-01-31 2017-08-10 アークレイ株式会社 分析用具および分析装置
WO2019050017A1 (fr) * 2017-09-07 2019-03-14 三菱瓦斯化学株式会社 Substrat pour biopuce, biopuce, procédé de fabrication de biopuce et procédé de conservation de biopuce
JPWO2019050017A1 (ja) * 2017-09-07 2020-10-22 三菱瓦斯化学株式会社 バイオチップ用基体、バイオチップ、バイオチップの製造方法およびその保存方法

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