WO2006104048A1 - Procédé de disposition de fines particules - Google Patents

Procédé de disposition de fines particules Download PDF

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Publication number
WO2006104048A1
WO2006104048A1 PCT/JP2006/305993 JP2006305993W WO2006104048A1 WO 2006104048 A1 WO2006104048 A1 WO 2006104048A1 JP 2006305993 W JP2006305993 W JP 2006305993W WO 2006104048 A1 WO2006104048 A1 WO 2006104048A1
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WIPO (PCT)
Prior art keywords
substrate
microparticles
laser
fine particles
arranging
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PCT/JP2006/305993
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English (en)
Japanese (ja)
Inventor
Hiroshi Masuhara
Yoichiroh Hosokawa
Yuji Hiraki
Chisa Shukunami
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Hamano Life Science Research Foundation
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Priority to JP2007510456A priority Critical patent/JPWO2006104048A1/ja
Publication of WO2006104048A1 publication Critical patent/WO2006104048A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00436—Maskless processes
    • B01J2219/00441—Maskless processes using lasers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00459—Beads
    • B01J2219/00468—Beads by manipulation of individual beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
    • B01J2219/00648—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of solid beads

Definitions

  • the present invention relates to a method for arranging fine particles.
  • biodevices also referred to as biodevices or biochips
  • Biomolecules such as DNA, proteins, sugar chains (also called biomolecules) or biodevices in which cells are immobilized on a support (substrate) are the above-mentioned immobilized biomolecules and other biomolecules or other It is possible to detect specific interactions with other compounds in large quantities simultaneously.
  • a protein chip in which an arbitrary antigen protein is placed on a substrate can be a new tool for early detection of diseases and allergies.
  • a biological device in which a physiologically active protein that controls cells is arranged on a substrate can be used as a cell culture substrate (see, for example, Patent Document 1). Application is expected.
  • biomolecules such as those described above may be arranged on a substrate by photolithography (Non-patent Document 1), inkjet printing (Patent Document 1, Non-Patent Document 2, and Technologies such as 3), micro contact printing (Non-patent document 4), laser direct writing (Non-patent document 5), and laser trapping (Patent document 2, Non-patent document 6) have been proposed.
  • a method for arranging proteins and cells using optical lithography is a method capable of patterning proteins with a high accuracy of 1 ⁇ m (Non-patent Document 1).
  • a protein placement method using ink jet printing is a method in which a fine droplet 132 of a protein solution is applied to a substrate 133 from an inkjet nozzle 131 to perform patterning.
  • a plurality of types of proteins can be patterned with an accuracy of several hundred / zm (Patent Document 1, Non-Patent Documents 2 and 3).
  • Patent Document 1 Non-Patent Documents 2 and 3
  • a protein placement method using microcontact printing is performed by attaching a microdroplet 132 of a protein solution to a needle tip or a fine structure (microstamp) 134 and attaching it to a substrate 133.
  • This is a transfer method (Non-patent Document 4).
  • Laser direct light For example, as shown in FIG. 13C, the protein is placed on the substrate 133 by directly irradiating the protein sample 135 with the laser 136 as shown in FIG. 13C.
  • multiple types of proteins can be panned with an accuracy of about 50 / zm (Non-patent Document 5).
  • the protein arrangement method using laser trapping is, for example, a method in which a polyhedron derived from an insect virus containing a protein in a crystalline state is arranged by a single laser trapping technique (Patent Document 2, Non-Patent Document 6).
  • Patent Document 1 Japanese Patent Laid-Open No. 2002-355026
  • Patent Document 2 JP 2003-155300 A
  • Non-Patent Literature 1 A. b. Blawas and W. M. Reichert, "Protein patterning. Biomatenals 19, 595-609 (1998)
  • Non-Patent Document 2 A. Roda et al., Protein microdeposition using a conventional ink-jet printer "BioTechniques 28, 492—496 (2000)
  • Non-Patent Document 3 B. T. Houseman et al., Peptide chips for the quantitative evaluation of protein kinase activity "Nat. Biotechnol. 20, 270 (2002)
  • Non-Patent Document 4 A. Bernard et al., "Microcontact printing of proteins” Adv. Mater. 12 1067-1070 (2000)
  • Patent Document 5 P. Serra et al., Laser direct writing of biomolecule microarrays, App 1. Phys. A 79, 949-952 (2004)
  • Non-Patent Document 6 Y. Hosokawa et al. "Protein Microarrays by Laser Patterning and Fixation of Single Protein Microcrystals: Implication for Highly Integrated Protein Chip” J. Appl. Phys. 96, 2945-2948 (2004)
  • the above technique also has a disadvantage when a biological device is manufactured by arranging biological microparticles (biomolecular microparticles) on a substrate.
  • biological microparticles such as biomolecule-immobilized carriers, biomolecule crystals, biomolecule aggregates, virus particles, and cells can be regarded as devices with biomolecule functions.
  • Establishment of a technique for manufacturing a biological device in which such biological microparticles are arranged and arranged non-destructively with high accuracy, high density, and high speed on a substrate is desired.
  • the inkjet printing technology is applied to fine particles, there is a risk that the fine particles will be clogged in the nozzles of the ink jet when trying to arrange with high accuracy.
  • an object of the present invention is to provide a technique for arranging fine particles, which is a technique for arranging fine particles, and which can be arranged with high accuracy, high density, and high speed.
  • a method for arranging microparticles includes a source substrate having a microparticle fixing surface, wherein the microparticles are immobilized on the microparticle fixing surface, and A substrate preparation step of preparing two substrates, a target substrate having a microparticle arrangement surface to be arranged with microparticles, and the source substrate and the target substrate, the microparticle fixing surface and the microparticle arrangement surface facing each other
  • the substrate placement step that faces the substrate through the liquid layer, the fine particle separation step that separates the fine particles from the fine particle fixing surface, and the source substrate force acting on the fine particles are also caused by the force toward the target substrate. Separating the separated microparticles from the fixed surface in the microparticle separation step. Is the induced physical Chikara ⁇ Byi ⁇ specific at least one by the arrangement method of the particulates carried out of power in the irradiation.
  • the inventors of the present invention have made extensive studies on the arrangement technology of fine particles such as protein-immobilized carriers, and if the physical force or chemical force induced by laser irradiation is utilized, the fine particles If the substrate can be separated without causing significant photochemical / photothermal damage to the carrier, and if the separated microparticles can be placed directly on another substrate, for example, a single unit of several tens of meters or less The present inventors have found that the microparticles can be patterned on a cell scale.
  • the arrangement method of the microparticles of the present invention for example, by adjusting the position and size of the condensing portion of the laser to be used, it is possible to arrange the microparticles with high density, high accuracy, and high speed. Become. In addition, separation using a physical force or an ionic force induced by laser irradiation has little damage, so that, for example, fine particles can be arranged non-destructively. Furthermore, for example, when the microparticles to be arranged are biological microparticles, according to the method for arranging microparticles of the present invention, for example, the microparticles can be arranged in an aqueous solution, so that the physiological activity of biomolecules such as proteins can be maintained. It is.
  • microparticles containing multiple types of factors that control cell functions such as proliferation, differentiation, and death can be arranged with a single cell level accuracy.
  • a cell culture substrate capable of signal transmission to each cell to be cultured can be produced. If such a cell culture substrate is used, it will be possible to reproduce the minimum unit of the cell arrangement that forms the basis of tissue formation and to induce tissue induction.
  • the fine particle arrangement substrate produced by the fine particle arrangement method of the present invention can be used for applications such as protein chips, bioreactors, biosensors and bioassay test pieces.
  • FIGS. 1A to 1C are schematic views showing an example of a method for arranging fine particles of the present invention.
  • FIGS. 2A to 2E are schematic views showing an example of manufacturing a source substrate of the present invention.
  • FIG. 3 is a schematic diagram showing a configuration example of the arrangement of the source substrate and the target substrate in the present invention.
  • FIG. 4 is a schematic diagram showing a configuration example of a placement device according to the present invention.
  • FIG. 5 is a schematic diagram showing a configuration of an electric stage according to the present invention.
  • 6A to 6E are schematic views showing other examples of the arrangement method of the present invention.
  • FIG. 7A and FIG. 7B are examples of micrographs of polygons arranged on a substrate by the method for arranging microparticles of the present invention (Example 1).
  • FIGS. 8A and 8B are other examples of micrographs of polygons arranged on a substrate by the method for arranging fine particles of the present invention (Example 2).
  • FIGS. 9A to 9C are examples of micrographs of cells cultured on the cell culture substrate of the present invention (Example 3).
  • FIG. 10 is still another example of a photomicrograph of a polyhedron arranged on a substrate by the method for arranging fine particles of the present invention (Example 4).
  • FIG. 11 is a schematic diagram of an example of a mechanical force based on laser abrasion when a pulsed laser is focused in water.
  • FIG. 12A is a schematic diagram of the arrangement of two types of polygons arranged by the method for arranging microparticles of the present invention
  • FIG. FIG. 12C is an example of a transmission image of a micrograph of a polyhedron of a kind
  • FIG. 12C is an example of the fluorescence image thereof.
  • FIGS. 13A to 13C are schematic diagrams for explaining a conventional technique.
  • FIG. 13A is a schematic diagram of ink jet printing technology
  • FIG. 13B is a schematic diagram of micro contact printing technology
  • FIG. 13C is a schematic diagram of laser direct writing technology.
  • Pulsed laser irradiation device Pulsed laser irradiation device
  • the physical force or chemical force induced by laser irradiation is induced by condensing the laser by an optical system including a lens. It is preferably a physical force or a chemical force.
  • the position of the condensing part of the laser is preferably the fine particles to be separated, or the liquid layer or the source substrate in the vicinity of the fine particles to be separated.
  • the physical force induced by laser concentration is a mechanical force based on laser abrasion.
  • the source substrate force also separates the fine particles.
  • the laser ablation preferably includes at least one generation of shock waves, bubbles, and convection.
  • the chemical force induced by laser irradiation causes modification of the source substrate and modification of the portion of the fine particles in contact with Z or the source substrate.
  • the microparticles are arranged on the arrangement surface as at least one of arbitrary points and lines by repeating the microparticle separation step and the microparticle arrangement step. be able to. Further, in the substrate preparation step of the arrangement method of the present invention, by preparing source substrates on which different types of microparticles are immobilized, different types of microparticles are arranged on the same microparticle arrangement surface. be able to.
  • the force applied to the fine particles may be a force selected from the group consisting of gravity, magnetic force, electrostatic force, light force, and buoyancy. preferable.
  • the method for arranging fine particles of the present invention includes the substrate arranging step!
  • the fine particles are arranged on the fine particle arrangement surface by gravity in the fine particle arrangement step by arranging the source substrate force so as to be positioned on the target substrate. .
  • the laser light is preferably light selected from the group consisting of infrared light, visible light, and ultraviolet light.
  • the laser may be a nanosecond laser, a picosecond laser, and a femtosecond laser power, a group power selected by a pulse laser, or
  • a continuous wave laser is preferable.
  • the microparticles are preferably selected from the group consisting of a carrier on which a biomolecule is immobilized, a crystal of biomolecule, an aggregate of biomolecules, virus particles, and cells.
  • the carrier on which the biomolecule is immobilized may be a polyhedron derived from an insect virus, or a polymer, a metal, a semiconductor, an aggregate of biomolecules, a crystal thereof, or a combination force thereof. Fine particles for which a group force is also selected may be used.
  • the method for producing a microparticle-arranged substrate of the present invention is a production method including a step of arranging microparticles on the substrate by the method for arranging microparticles of the present invention.
  • the fine particle arrangement device of the present invention is a fine particle arrangement device used in the fine particle arrangement method of the present invention or the production method of the present invention, wherein the source substrate installation unit, the target substrate It is a device for arranging fine particles including an installation section, laser irradiation means, and laser focusing means.
  • the cell or tissue culturing method of the present invention comprises a cell or tissue on a cell culture substrate produced by placing microparticles containing biomolecules exhibiting physiological activity by the microparticle placement method of the present invention.
  • the culture method of the present invention includes culturing cells on the microparticles arranged on the cell culture substrate, and the biomolecules exhibiting the physiological activity are selected as a group force of proliferation, differentiation, migration and death force. It is preferable that the culture method transmits the above signal to the cells.
  • the method for producing a cell or tissue of the present invention is a production method including culturing a cell or tissue by the culture method of the present invention.
  • the arrangement of microparticles includes an arrangement of microparticles.
  • microparticles can be arranged by continuously arranging 1 dot of microparticles by laser irradiation.
  • the fine particles arranged by the fine particle arrangement method of the present invention can be immobilized on a substrate and separated by physical force or chemical force induced by laser irradiation.
  • a carrier on which a biomolecule is immobilized a crystal of biomolecule, an aggregate of biomolecules, virus particles, a cell, and the like can be mentioned.
  • the biomolecule is not particularly limited, and examples thereof include proteins, peptides, DNA, RNA, sugars, lipids, organic compounds, complexes thereof, and derivatives thereof.
  • An aggregate of biomolecules is, for example, a protein or DNA that is agglutinated.
  • One or more kinds of fine particles can be appropriately selected according to the use of the fine particle arrangement substrate of the present invention produced by the arrangement method of the present invention.
  • the size of the microparticles is not particularly limited, and for example, in the case of a spherical shape, the diameter is 10 nm to 100 m, Preferably, lOOnm ⁇ : LO ⁇ m, and more preferably 100 nm ⁇ l ⁇ m. Further, the shape of the fine particles is not particularly limited.
  • the carrier is not particularly limited, but a carrier that can immobilize the biomolecule and can be immobilized on a substrate is preferable.
  • a conventionally known carrier can be used according to the biomolecule to be immobilized. Insect virus-derived polyhedrons, macromolecules, metals, semiconductors, aggregates of biomolecules, crystals of these, and the combined force of these, fine particles selected.
  • a carrier for immobilizing proteins a polyhedron derived from a conventionally known insect virus described later is preferable. This is because the polyhedron has an effect of maintaining the physiological activity of the protein.
  • a polyhedron is a polyhedral structure of 100 nm to 10 ⁇ m on one side formed by an insect virus, which is the original meaning, in an infected cell, and the polyhedrin is associated in a crystalline form.
  • V and so-called crystalline protein inclusion bodies in which any protein is incorporated into the polyhedron while maintaining its biological function using known genetic engineering techniques (for example, patents) (Ref. Literature 2, Japanese Patent Laid-Open No. 2003-319778, WO2002Z036785, Bumplets IV).
  • the physical force induced by laser irradiation includes, for example, the physical force induced by laser focusing by an optical system including a lens, and is not particularly limited. Preferably, it is a mechanical force based on laser abrasion.
  • the laser ablation is an explosive erosion phenomenon that occurs when a high-intensity laser is irradiated and condensed. For example, when a pulse laser is focused in water, This is a phenomenon in which shock waves, bubbles, convection, and the like are generated by multiphoton absorption of water in the water. The generation of the bubbles includes the generation of so-called air bubbles (cavitation bubbles).
  • the mechanical force based on laser ablation includes, for example, the mechanical force due to the generation of the shock wave, bubble, convection, etc., but is not limited thereto, and generally the explosive erosion phenomenon.
  • the shock wave is a pressure wave generated at the condensing part of the pulse laser.
  • Figure 11 shows a schematic diagram of an example of the mechanical force based on laser abrasion when a pulsed laser is focused in water. As shown in the figure, for example, when the femtosecond pulse laser 100 is irradiated toward the substrate 103 and condensed, multi-photon absorption of water and laser ablation occur at the condensing unit 101, and this occurs.
  • Laser ablation may use a molecule having one-photon absorption in visible light or ultraviolet light in addition to the multiphoton absorption of water. For example, visible light or A molecule having one-photon absorption with ultraviolet light is added, and laser ablation is induced on the substrate by irradiating a laser that emits visible light or ultraviolet light.
  • the chemical force induced by laser irradiation includes, for example, modification of the source substrate and modification of Z or the portion of the microparticles in contact with the source substrate.
  • the modification of the source substrate and part of the Z or microparticle is a change in the chemical state of the source substrate and part of the Z or microparticle that is observed when the source substrate is irradiated with a laser, for example,
  • the material of the source substrate and part of Z or microparticles or modified products contain photoreactive or photodegradable molecules, and the chemical state of the molecules changes by laser irradiation.
  • Such chemical forces are preferably induced by laser focusing by an optical system including a lens.
  • the laser used in the method for arranging fine particles of the present invention is not particularly limited as long as it can induce the physical force and the ionic force, and the laser light is, for example, Infrared light, visible light or ultraviolet light can be used.
  • a continuous wave laser or a pulse laser can be used as the laser to be used.
  • a pulse laser for example, a conventionally known laser such as a nanosecond laser, a picosecond laser, or a femtosecond laser is used. it can.
  • femtosecond titanium sapphire lasers femto fiber lasers, femtoseconds, ittribium lasers, femtoseconds, excimer lasers, and picosecond YAG lasers.
  • the fine particle arrangement method of the present invention includes a substrate preparation step, a substrate arrangement step, a fine particle separation step, and a fine particle arrangement step. Next, each step will be described.
  • the source substrate has a fine particle fixing surface on which fine particles are fixed.
  • the microparticles to be immobilized are as described above.
  • the fine particle fixing surface may be one side or both sides of the source substrate.
  • the material of the source substrate is not particularly limited, and glass, plastic, rubber and the like can be used, and can be appropriately selected according to the fine particles to be fixed.
  • the source substrate may be a laminate including two or more layers. For example, a layer suitable for fixing microparticles may be stacked as the microparticle fixing surface.
  • the fine particle fixing surface of the source substrate may be modified to fix the fine particles. . Examples of the modification include modification using an antigen-antibody reaction such as avidin modification, and modification using a chemical reaction such as alkanethiol modification.
  • a fine particle arrangement surface utilizing chemical force can be obtained.
  • the thickness and size of the source substrate are not particularly limited.
  • the source substrate is preferably optically transparent when it is necessary to view fine particles through the source substrate.
  • a molecule having one-photon absorption may be included in the irradiated laser light.
  • the fixation between the fine particles and the source substrate can be appropriately selected from conventionally known techniques such as a method using a chemical bond and a method using physical adsorption.
  • a method using a chemical bond In the case where the source substrate and the fine particles are separated by a physical force, it is preferable to use a fixed layer method in which the coupling is released by a physical impact of a mechanical force based on laser abrasion.
  • separation of the source substrate and microparticles is performed by chemical force, for example, the source substrate and microparticles are immobilized via photoreactive or photodegradable molecules, and laser irradiation is performed.
  • the fixing method is such that the bond is released by modification of the source substrate and modification of the portion of the fine particles in contact with Z or the source substrate.
  • the plurality of fine particles may be divided and fixed on the source substrate.
  • the target substrate has a fine particle arrangement surface on which fine particles are to be arranged.
  • the material of the target substrate is not particularly limited, and glass, plastic, rubber or the like can be used, and can be appropriately selected according to the fine particles to be placed and fixed.
  • the target substrate may be a laminate including two or more layers.
  • the microparticle placement surface may be used to fix the microparticles.
  • a layer suitable for the above may be laminated.
  • the fine particle arrangement surface of the target substrate may be modified to fix the fine particles.
  • the thickness and size of the target substrate are not particularly limited.
  • the target substrate is preferably optically transparent when it is necessary to visually recognize fine particles on the target substrate through the target substrate.
  • the glass substrate is made of polydimethylsiloxane (P DMS) coated can be used.
  • Coating with PDMS can be performed by coating with dimertyl siloxane (DMS) and polymerizing by appropriate heating (for example, about 80 ° C. for 1 minute).
  • DMS dimertyl siloxane
  • the substrate placement step in the fine particle placement method of the present invention includes a source substrate and a target substrate, with a fine particle fixed surface of the source substrate and a fine particle placement surface of the target substrate facing each other via a liquid layer. It is the process of arranging.
  • the distance between the source substrate and the target substrate is not particularly limited, but is preferably a distance that allows fine particles separated by the source substrate to be accurately arranged on the target substrate.
  • the distance is, for example, lOOnm to lmm, preferably 100 ⁇ to 100 / ⁇ ⁇ , more preferably 100 nm to 10 ⁇ m.
  • a liquid layer is disposed between the source substrate and the target substrate. This makes it possible to use liquid laser ablation.
  • the liquid layer include an aqueous solution layer.
  • the aqueous solution is not particularly limited, and for example, water, physiological saline, various buffers, and the like can be used as appropriate.
  • conventional technologies such as inkjet printing, micro contact printing, and laser direct writing (see Fig. 13) may cause the biomolecules to dry when they are injected and fixed. Is thought to be lost.
  • the advantage of the present invention is very large if the fine particles of biomolecules that are vulnerable to drying can be disposed in an aqueous solution layer.
  • the liquid in the liquid layer is not limited to an aqueous solution, and may be, for example, an organic solvent or oil.
  • the fine particle separation step in the fine particle arrangement method of the present invention there is a step of separating the fine particle fixing surface force and fine particles by a physical force induced by laser irradiation. It is done.
  • the physical force includes, for example, a mechanical force based on laser abrasion.
  • Laser ablation is the source group Although it may be induced by focusing on a plate, it is preferable to use laser ablation of a liquid layer disposed between the source substrate and the target substrate.
  • the laser condensing part is not particularly limited as long as the target fine particles can be separated by a mechanical force based on laser abrasion. For example, the laser condensing part may be directly focused on the fine particles. .
  • the damage caused can be further reduced by separating the microparticles by contacting only mechanical forces such as shock waves, bubbles, and convection generated in the light collecting portion.
  • shock waves generated by laser ablation propagate as pulses, but the pulse shape of the shock waves relaxes as the distance of the focusing force increases.
  • the force such as shock wave decreases in proportion to the function of the square or more of the distance from the condensing part. Therefore, the mechanical force effect based on laser ablation decreases rapidly as the distance from the condensing part increases.
  • the size of the condensing part of the pulse laser, the condensing position, the laser If the intensity and laser light density are appropriately selected, a small range of fine particles can be separated with high accuracy. For example, when the size of the condensing part is 1 ⁇ m, the mechanical perturbation due to shock waves, bubbles, convection collisions, etc. (See Fig. 11).
  • the second aspect of the fine particle separation step in the fine particle arrangement method of the present invention is a chemical force induced by laser irradiation, such as chemical modification of the source substrate and Z or source substrate.
  • Modification of the source substrate and part of the Z or microparticle is performed by laser irradiation when the microparticle is immobilized on the source substrate via a photoreactive or photodegradable molecule. This includes changing the chemical state and weakening or neutralizing the fixing force.
  • Modification of the source substrate and part of the Z or microparticles can be induced, for example, by focusing a laser on the interface between the source substrate and the microparticles.
  • the laser condensing part is not particularly limited as long as the target microparticle can be separated by modification of the source substrate and Z or a part of the microparticle.
  • the laser condensing part is directly focused on the microparticle.
  • it may be a source substrate in the vicinity of the fine particles to be separated.
  • the condensing part is in contact with the microparticles. Preferably not.
  • the fine particle separation step in the fine particle arrangement method of the present invention uses, as a third aspect, both physical force and chemical force induced by laser irradiation,
  • the fine particle fixing surface force may be a step of separating the fine particles.
  • the position of the fine particles to be separated on the source substrate corresponds to the position on the target substrate where the fine particles are arranged.
  • the microparticles when the microparticles are arranged at any specific position on the surface of the target substrate where the microparticles are arranged, the microparticles fixed at the position of the fixed surface of the source substrate facing the specific position of the arrangement surface are arranged.
  • the particles may be separated by laser irradiation. Therefore, when the relative position between the source substrate and the target substrate is fixed, the pattern of laser irradiation to the source substrate can be transferred to the target substrate as it is as an arrangement of fine particles.
  • the means for condensing the laser, adjusting the size of the condensing part, and adjusting the condensing position are not particularly limited in the fine particle separation step, but for example, a lens or a diaphragm.
  • the optical system can be adjusted, and a microscope can be preferably used.
  • the concentrating part of the nozzle is the shape of a dot, a circle with a certain area, or a sphere with a certain volume. If the condensing part is circular or spherical, its radius Is, for example, more than 0 and not more than 100 ⁇ m, preferably more than 0 and not more than 10 ⁇ m, and more preferably more than 0 and not more than 1 ⁇ m.
  • the condensing position for condensing the pulse laser is, as described above, the range of the fine particles that the source substrate or liquid in the vicinity of the fine particles to be separated is preferably separated. It can be adjusted appropriately according to The distance between the focused position of the pulse laser and the fine particles is, for example, more than 0 and less than 1 mm, more preferably more than 0 and less than 100 m, and more preferably more than 0 and less than 1 ⁇ m.
  • the light density (photon flux) of the pulse laser when used for the placement of fine particles in a radius region within 50 m by laser abrasion is: For example, 5 x 10 5 to 1 x 10 12 (watt), preferably 5 x 10 5 to 1 x 10 9 (watt), more preferably 5 x 10 5 to 1 x 10 7 (watt) It is. [0046] Here, the optical density for the arrangement of microparticles in the radius region within 50 ⁇ m is shown.
  • the region affected by the pulse laser is proportional to the square of the region radius, for example, within 10 / zm
  • the optical density of the pulsed laser when placing fine particles in the radius region is 1/25 of the above, and the optical density of the pulse laser when placing fine particles in the radius region within 100 m is 400 times the case.
  • the pulse width of the pulse laser is ⁇ t
  • the relationship between the pulse laser intensity (I) and the light density (D) of the pulse laser is expressed by the following equation.
  • the intensity of the pulse laser used in this way is a force that can be adjusted appropriately according to the distance between the microparticles and the focusing position, the range of the microparticles to be separated, etc.
  • the intensity of the pulse laser when separating the fine small particles by chemical modification of the source substrate for example, 1 X 10- 9 ⁇ : LO a CiZpu lse), preferably, a 1 X 10- 6 ⁇ 1 (jZpulse) , more preferably 1 X 10- 6 ⁇ 1 X 10- 3 CFZpulse).
  • the laser intensity is, for example, 1 X 10- 6 ⁇ 10 (watt ), preferably a 1 X 10- 4 ⁇ 1 (watt) , more preferably, a 1 X 10- 4 ⁇ 1 X 10- 2 (watt).
  • the wavelength of the pulse laser can be, for example, a laser having a wavelength of 190 nm to 20 ⁇ m.
  • the wavelength of the pulse laser when condensing in a liquid layer, is higher than that of ultraviolet light having a strong absorption directly. Because infrared light can generate a shock wave at the laser concentrator regardless of the substrate material used, the wavelength is 400 ⁇ ! More preferably, l lOOnm is more preferable, and 600 l: L lOOnm.
  • Irradiation times of pulsed laser for separating the fine particles of one dot with the fixed surface force The number is not particularly limited, and is, for example, 1 shot (single shot) to 10 million shots, preferably 1 shot to 1000 shots, more preferably 1 shot to 10 shots, and even more preferably a single shot.
  • one dot refers to a region of fine particles that are separated from the fixed surface of the source substrate by laser irradiation to a single condensing point and are disposed on the surface of the target substrate.
  • the repetition frequency of the laser in the case of repeated irradiation is, for example, 1 Hz to: L00 MHz, preferably 1 Hz to: LMHz, more preferably 1 Hz to: LkHz, and more preferably, 1 ⁇ to 20 ⁇ .
  • the fine particle arrangement step in the fine particle arrangement method of the present invention is such that the fine particles separated in the separation step are arranged on the arrangement surface of the target substrate by the source substrate force applied to the fine particles in the direction of the target substrate. It is a process. Examples of the force that moves the microparticles from the source substrate to the target substrate include gravity, magnetic force, electrostatic force, and buoyancy. As another embodiment, there is a step of capturing the fine particles separated in the separation step using a laser trapping method using light power (radiation pressure of light) by a laser and moving the particles to a target substrate. It is done.
  • a preferred embodiment of this step is to drop the microparticles from the source substrate onto the target substrate by gravity.
  • An example of this embodiment will be specifically described with reference to FIGS. 1A to 1C, the same portions are denoted by the same reference numerals.
  • a source substrate 1 on which fine particles 3 are fixed is placed on a target substrate 2 via a liquid layer 4.
  • the laser ablation is induced by collecting the laser 5 and a mechanical force 6 based on the laser ablation is generated, thereby causing the microparticle 3 to Separate from source substrate 1.
  • the separated fine particles 3 fall and are arranged on the target substrate 2.
  • the pattern of laser irradiation to the source substrate can be transferred as it is as an array of the fine particles on the target substrate.
  • a method of moving the microparticles from the source substrate to the target substrate by giving magnetism or electric charge for example, can be mentioned. It is. Furthermore, as another aspect, for example, a method in which a source substrate is disposed under a target substrate via a liquid layer and the buoyancy of fine particles in the liquid is used. The movement method of these aspects can be appropriately implemented by a conventionally known technique.
  • the method for fixing the arranged fine particles to the target substrate is not particularly limited, and may be performed using a conventionally known method using chemical bonds, a method using physical adsorption, or the like, if necessary. it can.
  • the polygon is fixed as it is, but after placing the polygon, for example, at 37 ° C. It can be hardened by heating for 6 hours.
  • the arrangement surface has arbitrary points and / or lines.
  • the microparticles can be arranged. If a source substrate on which different types of microparticles are immobilized is used, a plurality of types of microparticles can be arranged on the same target substrate, as will be described in the following example.
  • the method for arranging a polyhedron of the present invention using an insect virus-derived polyhedron as an example of microparticles will be specifically described.
  • the insect virus-derived polyhedron is a crystalline substance formed by the outer shell protein of the virus called polyhedrin as described above, and the desired protein is incorporated into the protein while maintaining its activity.
  • the fine particles of the present invention are not limited to the polygon.
  • FIG. 2A An example of a source substrate preparation process in the polygon arrangement method of the present invention will be described with reference to FIGS. In the figure, the same portions are denoted by the same reference numerals.
  • a polyhedron dispersion liquid 7 in which a polyhedron is dispersed in water, physiological saline, or various buffers is used to fix fine particles on the glass substrate 9 using a micropipette 8. Drop on a fixed surface and leave at room temperature for several hours. Then, as shown in the schematic diagram of FIG. 2B, the polygon 10 is adsorbed to the fixed surface of the glass substrate 9 by natural drying.
  • the polygon 10 and the substrate 9 are bonded to each other by electrostatic force, and then the water Does not peel off when dripping.
  • a weak alkaline aqueous solution 11 is dropped using a micropipette on the polygon 10 adsorbed on the fixed surface of the glass substrate 9, and left for several minutes. Treat with alkali.
  • the polyhedrin crystalline polyhedron is dissolved in an alkaline solution, and this treatment activates the protein on the polyhedron surface that has lost its activity upon drying.
  • the weak alkaline aqueous solution is removed with a micropipette, and a source substrate is prepared in which the polygon 10 is fixed on the fixed surface of the substrate 9 as shown in the schematic diagram of FIG. 2D.
  • the weakly alkaline aqueous solution include ⁇ .3 canolesulfonate buffer.
  • the activity due to alkali treatment of the polyhedron surface can be determined by, for example, antigen-antibody reaction and light emission of green fluorescent protein from the polyhedron surface.
  • the size of the region to be fixed is not particularly limited, and for example, the diameter is 1 mm or less. In this case, the number of droplets dropped on the substrate is not particularly limited. For example, as shown in FIG.
  • a plurality of types of polygons 10 to: LO ′′ ′ are fixed on the fixed surface of the glass substrate 9. Therefore, for example, when a 1 cm square substrate is used and the size of one fixed area is about 1 mm in diameter, a polygon containing about 100 different proteins on one substrate. The body can be placed.
  • FIG. 3 shows a schematic diagram of an example of the arrangement form.
  • the arrangement form 20 of the source substrate and the target substrate is that the polygon 10 prepared in the preparation step is formed on the target substrate 12 via the liquid layer 16 formed by the spacer 15.
  • the fixed source substrate 9 is arranged.
  • a target substrate 12 shown in FIG. 3 is an example of a target substrate in which a silicon rubber sheet 14 is laminated on a glass substrate 13 as a fine particle arrangement surface.
  • the silicon rubber sheet 14 is preferable because the operation of fixing the polygon 10 placed after the separation to the target substrate 12 can be omitted.
  • the material of the spacer 15 is not particularly limited.
  • silicon rubber, a plastic sheet, a metal thin plate, or the like can be used, and the thickness is not particularly limited, and is 100 m, for example.
  • the thickness of the silicon rubber sheet 14 is not particularly limited, and is 100 m, for example.
  • the polygon separation process and the arrangement process are performed by, for example, irradiating the source substrate and the target substrate configured as in the arrangement form 20 with a laser using the arrangement apparatus of the present invention using a microscope. it can.
  • FIG. 4 shows an example of an arrangement device of the present invention that can be used in the polygon arrangement method of the present invention.
  • the arrangement device 40 of the present invention includes an upright microscope 21 and a pulse laser irradiation device 27 as main components.
  • the upright microscope 21 includes a stage 22, a condenser lens 23, an objective lens 24, a light source lamp 25, a CCD camera 26, and a dichroic mirror 31.
  • a source substrate and a target substrate are arranged as in the arrangement form 20.
  • a condenser lens 23 is disposed below the stage 22 of the upright microscope 21.
  • a light source lamp 25 is disposed below the condenser lens 23.
  • a CCD camera 26 for detecting this light is disposed above the microscope 21. .
  • a pulse laser irradiation device 27 is arranged outside the upright microscope 21, and an optical system is arranged between the upright microscope 21 and the pulse laser irradiation device 27.
  • the optical system includes a ⁇ 2 plate 28, a polarizer 29, and a collimator lens 30, and the emitted pulse laser 32 passes in this order.
  • the pulse laser 32 introduced into the upright microscope 21 is reflected by the dichroic mirror 31, and is irradiated to the source substrate and the target substrate configured as in the arrangement form 20 through the objective lens 24.
  • the intensity of the pulse laser 32 can be adjusted by the ⁇ 2 plate 28 and the polarizer 29, and the pulse laser 32 can be adjusted by the collimator lens 30 so as to be focused on the image plane of the microscope.
  • the focusing position of the pulse laser 32 can be adjusted by the stage 22.
  • an example of an apparatus that irradiates the upper force laser of the source substrate using an upright microscope is taken up. However, even if an inverted microscope is used to irradiate a laser from below the target substrate. Yo! /
  • the source substrate and the target substrate are arranged on the stage 22 of the upright microscope 21 as shown in the arrangement form 20 shown in FIG.
  • the CCD camera 26 observes the state of the source substrate and the target substrate, and determines the polygon to be separated.
  • the stage 22 is adjusted so that the focused position of the pulse laser becomes an appropriate position.
  • pulse train A pulse laser 32 is irradiated by the laser irradiation device 27, the intensity is adjusted by the optical system, and the source substrate and the target substrate are irradiated with the laser. Laser ablation is induced in the condensing part of the pulse laser. This state will be described with reference to FIG.
  • the pulsed laser 5 is focused in the liquid layer 4 between the source substrate 1 and the target substrate 3, laser ablation is induced, and a mechanical force 6 based on laser abrasion is applied.
  • fine particles (polyhedrons) 3 in the vicinity of the light condensing part are separated from the source substrate 1 and fall onto the target substrate 2 due to gravity.
  • the dropped microparticles (polyhedrons) 3 land on the target substrate 2 and are arranged as shown in FIG. 1C.
  • the silicon rubber is used as the fine particle arrangement surface on the target substrate 2, the landed polygon and the silicon rubber are bonded to each other by electrostatic force, and the subsequent fixing work is omitted. Is preferable.
  • the width of the writing line is not particularly limited, and as described above, a force that can be adjusted by the intensity of the pulse laser, for example, in the range of 1 ⁇ m to 50 cm, preferably in the range of 1 ⁇ m to lcm. More preferably, it can be in the range of 10 / ⁇ ⁇ to 1 ⁇ .
  • the writing speed is not particularly limited, but is, for example, in the range of: mZsec to 10 cmZsec, preferably in the range of 1 ⁇ mZsec to: LmmZsec, and more preferably in the range of 10 mZsec lOO / z mZsec. It can be a range.
  • the placement device of the present invention is not limited to the above example as long as the placement device includes a source substrate placement portion, a target substrate placement portion, laser irradiation means, and laser focusing means.
  • the polyhedron can carry various proteins by a conventionally known genetic manipulation. Therefore, as shown in Fig. 2E, multiple types of polyhedrons are arbitrarily arranged on the target substrate using a source substrate on which polygons 10 ⁇ : LO '"carrying different types of proteins are arranged.
  • An arrangement method according to the present invention will be described in accordance with an arrangement form of the source substrate and the target substrate shown in the developed schematic diagram of FIG. This can be done by using 0.
  • the arrangement form 50 has two electric stages 41 and 42 as main components, and the electric substrate 41 can fix the source substrate 44 via the fixing support 43 on the electric stage 41 that can fix the target substrate 45. Stage 42 is placed. In the arrangement form 50, when the electric stage 41 is moved, the electric stage 42 is also moved without changing its relative position, and when the electric stage 42 is moved, only the electric stage 42 is moved. To do.
  • the polygons are separated and arranged using the source substrate and the target substrate arranged as in the arrangement form 50, for example, as shown in FIGS.
  • the placement device on which the electric stage is placed can be selected as appropriate, for example, an upright microscope as shown in FIG. 4 or an inverted microscope.
  • FIG. 6 the same parts as those in FIG. 6A to 6E are schematic views showing a process of arranging the polygon 48, the region 47, and the polygon 49 in the liquid layer 51 in the region 46 of the target substrate 45, respectively.
  • the target substrate 45 is fixed to the electric stage 41 in FIG. 5, and the source substrate 44 and the fixing support 43 are fixed to the electric stage 42 in FIG.
  • the broken line 60 indicates the irradiation position of the laser, that is, the normal line of the objective lens of the microscope that is the placement device.
  • the immobilization region of each type of polygon is less than lmm
  • a polyhedron carrying about 100 kinds of proteins can be fixed in a 1 cm 2 region of the source substrate 44.
  • the electric stage can be controlled with an accuracy of a few zm.
  • polygons carrying different proteins can be arranged on the same target substrate at arbitrary positions with high accuracy, high density, and high speed. is there.
  • the force described above as an example of the polygon arrangement method of the present invention is not limited to polygons, It can also be used in the method for arranging microparticles of the present invention for microparticles.
  • the fine particle arrangement substrate of the present invention is such that the fine particles are arranged on the substrate using the fine particle arrangement method of the present invention, and the production method uses the fine particle arrangement method of the present invention.
  • Others are not particularly limited.
  • the microparticle-arranged substrate of the present invention uses, for example, a cell culture substrate, a protein chip, a DNA chip, a protein, peptide, DNA, RNA, sugar, lipid, organic compound, etc.
  • the present invention can be used without being particularly limited to various conventionally known applications such as bioreactors, biosensors, and bioassay test pieces.
  • the cell culture substrate of the present invention is a cell culture substrate in which physiologically active substances are arranged on a substrate, and microparticles containing a physiologically active substance or a physiologically active substance-immobilized carrier are arranged on the substrate by the arrangement method of the present invention.
  • the physiologically active substance include conventionally known proteins that control functions such as proliferation, differentiation, and death. By arranging multiple types of these, various proteinaceous intercellular signaling substances can act at the individual cell level.
  • the physiologically active substance include, but are not limited to, site force-in, hormones, growth factors and the like. It should be noted that the cell culture substrate of the present invention may additionally have no physiological activity! / Microparticles may be arranged.
  • the cell or tissue culture method of the present invention is a method of culturing cells or tissues using the cell culture substrate of the present invention, and the method of producing a cell or tissue of the present invention is a cell or tissue of the present invention.
  • cells or tissues are obtained by culturing according to a tissue culture method.
  • Cells to be cultured in the method for culturing and producing cells or tissues of the present invention are not particularly limited. According to the cell / tissue culture and production method of the present invention, it is possible to arrange and control a high level of cells that induce organization, which has been difficult with the conventional method, and the minimum cell sequence that forms the basis of tissue formation. The unit can be reproduced.
  • a protein-immobilized carrier supporting only a polyhedron which is a crystalline medium formed by an insect virus-derived protein (polyhedrin) was prepared as follows. First, the recombinant virus vector AcCP -H (Mori et al. (1993) J. Gen. Virol. 74, 99—102) was infected with IPLB—Sf21—AE (Sf21) cells derived from Spodoptera Fru giperda. Next, a cubic polyhedron was collected from the infected cells on the 4th day, and PBS (20 mM NaH PO, 20 mM Na HPO) was collected.
  • the polyhedron is dispersed in water and dropped onto a glass substrate (thickness: 100 m) using a micropipette so as to form a droplet of about 1 mm, left at 24 ° C for several hours, and then naturally dried. . Thereafter, a weakly alkaline aqueous solution ( ⁇ .3) is dropped on the surface of the polyhedron and allowed to stand for about several minutes to dissolve the surface of the polyhedron, and the alkaline aqueous solution is removed using a pipette. A substrate was prepared.
  • a silicon rubber sheet (thickness 100 ⁇ m) was laminated on the glass substrate as the target substrate. A thing was used. A spacer made of a silicon rubber sheet (thickness: 100 m) is placed on the target substrate, and the hollowed portion is filled with water. The source substrate was placed so as to be sealed and sealed to prepare an array plate. This was placed on a stage 22 of an upright microscope 21 as shown in FIG. 4, and the process of arranging the polygonal bodies and the state after the arrangement were observed with a CCD camera 26. A high-power femtosecond laser irradiation device (120 fs, 800 nm, 20 Hz, 10 mW) was used as the laser irradiation device 27.
  • FIG. 7 shows an example of the result of arranging the polygons.
  • 7A and B are photomicrographs of the target substrate on which polygons are arranged
  • FIG. 7B is an enlarged view of the portion surrounded by the square in FIG. 7A.
  • the portions that appear to be linear are polygons arranged
  • each that appears as a square is a polygon.
  • FIGS. 8A and B are photomicrographs of the target substrate on which polygons are arranged
  • FIG. 8B is an enlarged view of the portion surrounded by the square in FIG. 8A.
  • the time required for the arrangement of the three characters “JST” was about 1 minute, and the polyhedron could be arranged at a speed of about 10 / z mZsec.
  • the line width is almost the same as the size of animal cells. Therefore, by culturing animal cells on this polyhedron array, the polyhedron can act on specific individual cells.
  • the cell culture substrate prepared in Example 2 was used as a culture solution (medium: Dulbecco's modification of Eagl e's medium (DMEM) containing 5% fetal bovine serum), and NIH3T3 strain was added thereto. After a few minutes, the added cells are deposited on the cell culture substrate, and the CO incubator
  • FIGS. 9A to C are photomicrographs of the cell culture substrate
  • FIG. 9B is an enlarged view of the portion enclosed by the lower square in FIG. 9A
  • FIG. 9C is the portion enclosed by the upper square in FIG. Is an enlargement of
  • the droplet-shaped pattern is a proliferated cell, and the cell proliferated remarkably on the polyhedron as compared with the cell culture substrate. This is considered to be because the environment on the polyhedron composed of proteins is more prone to cell growth than on the cell culture substrate (target substrate).
  • it can be said that cell proliferation can be controlled.
  • VP3ZEGFP and a set that expresses the polyhedron protein used in Example 1.
  • the crystalline polyhedrin inclusion body in which the EGFP protein was embedded was recovered in the same manner as in Example 1 except that Sf21 cells were double-infected with the replacement virus vector AcCP-H.
  • FIG. 6 shows an arrangement apparatus according to the present invention including the source substrate and the target substrate that are arranged as shown in the arrangement form 50 of FIG.
  • the results are shown in Fig. 10.
  • This figure is a fluorescence micrograph of the target substrate.
  • the circled part is the place where the polyhedron with EGFP introduced is placed, and the partial force EGFP enclosed by the square is introduced. Place a strong polyhedron It is a place. Since luminescence caused by EGFP was observed from the polyhedron into which EGFP was introduced, it was shown that according to the arrangement method of the present invention, the protein-immobilized carrier can be arranged while maintaining the protein activity.
  • a source substrate in which a polyhedron containing EGFP and a polyhedron containing no EGFP were immobilized on the same fine particle fixing surface was prepared.
  • a target substrate was prepared by coating a glass substrate with dimethyl siloxane (DMS) and then heating at 80 ° C. for 1 minute to form a polydimethylsiloxane (PDMS) coat having a thickness of about 20 ⁇ m.
  • DMS dimethyl siloxane
  • the arrangement device of the present invention is used to form a pine pattern by the same manner as in Example 4. Patter jung. That is, 40 m ⁇ 40 m blocks were placed using EGPF-containing polyhedra and EGFP-free polyhedra alternately. Each block was placed by laser scanning with straight lines spaced 5 m. The interval between each block was 5 m.
  • FIG. 12A A schematic diagram of this arrangement is shown in FIG. 12A, and an example of a transmission image of the target substrate actually arranged is shown in FIG. 12B. The arrangement shown in Figure 12B could be done in about 30 minutes. When this target substrate was observed with a fluorescence microscope, a checkered fluorescent image was obtained as shown in FIG. 12C.
  • the method for arranging fine particles of the present invention is an arrangement method using laser abrasion.
  • a sequencing technique capable of maintaining a physiological activity of a carrier to which a physiologically active factor is immobilized at high accuracy, high density, and high speed, and further, for example, a protein to be sequenced. It is useful in fields that use various biochips.
  • the present invention is useful, for example, in the field of regenerative medicine. That is, by using the cell culture substrate produced by the arrangement method of the present invention, it becomes possible to cause various immobilized cell signal transduction factors to act at the single cell level, and to induce organization and induction. Advanced cell arrangement and control becomes possible.
  • the present invention provides tissue formation It is intended to contribute to tissue and organ regeneration technology by reproducing the minimum unit of cell arrangement that is the basis of the above and by inducing tissue guidance.

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  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)

Abstract

La présente invention concerne un procédé de disposition de fines particules permettant de disposer divers types de fines particules dans un liquide avec grande précision à haute densité et grande vitesse. Le procédé de disposition comprend la phase de fourniture de deux substrats, c’est-à-dire un substrat source (1), ayant une face de fixation de fines particules et à laquelle les fines particules (3) ci-dessus sont fixées, et un substrat cible (2) ayant une face de disposition de fines particules sur laquelle les fines particules (3) doivent être disposées (phase de fourniture de substrat), la phase de disposition du substrat source (1) et du substrat cible (2) de telle sorte que la face de fixation de fines particules se trouve face à la face de disposition de fines particules à travers une couche liquide (4) (phase de disposition de substrat), la phase de séparation des fines particules (3) à partir de la face de fixation de fines particules (phase de séparation de fines particules), et la phase de disposition des fines particules séparées (3) sur la phase de disposition de fines particules par une force dans une direction allant du substrat source au substrat cible appliquée aux fines particules (3) (phase de disposition de fines particules). Dans la phase de séparation de fines particules, la séparation des fines particules (3) à partir de la face de fixation est réalisée par une force physique (6) ou une force chimique induite par irradiation laser (5).
PCT/JP2006/305993 2005-03-25 2006-03-24 Procédé de disposition de fines particules WO2006104048A1 (fr)

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CN103641112A (zh) * 2013-12-24 2014-03-19 华南师范大学 一种利用飞秒激光制备纳米金刚石的方法
CN103641112B (zh) * 2013-12-24 2016-03-23 华南师范大学 一种利用飞秒激光制备纳米金刚石的方法
CN106660004A (zh) * 2014-05-08 2017-05-10 公立大学法人大阪府立大学 聚集装置及聚集方法、微小物体聚集结构体的制造装置、微生物的聚集除去装置、被检测物质的检测装置、被分离物质的分离装置以及被导入物质的导入装置
JP2020514823A (ja) * 2017-03-15 2020-05-21 ユニベルシテ ドゥ ボルドー ターゲット上に粒子を堆積するための装置および方法

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