JPWO2006104048A1 - How to arrange fine particles - Google Patents

Abstract

Providing an arrangement technology that can arrange various types of fine particles in a liquid with high precision, high density, and high speed. The arrangement method of the present invention includes a source substrate 1 having a fine particle fixing surface on which the fine particles 3 are immobilized, and a target substrate 2 having a fine particle arrangement surface on which the fine particles 3 are to be arranged. A substrate preparation step of preparing two substrates, and a substrate arrangement in which the source substrate 1 and the target substrate 2 are opposed to each other through the liquid layer 4 with the fine particle fixing surface and the fine particle arrangement surface facing each other. A microparticle separation step for separating the microparticles 3 from the microparticle fixing surface, and a microparticle placement surface for separating the microparticles 3 by a force from the source substrate toward the target substrate applied to the microparticles 3. In the fine particle separation step, separation of the fine particles 3 from the fixed surface is caused by a physical force 6 or chemical force induced by the laser irradiation 5. Divide.

Description

  The present invention relates to a method for arranging fine particles.

  In recent years, biodevices (also referred to as biodevices or biochips) that partially mimic biofunctions have been attracting attention through the fusion of biotechnology and technologies in other fields. Biomolecules (also referred to as biomolecules) such as DNA, proteins, sugar chains, or biodevices in which cells are immobilized on a support (substrate) include the immobilized biomolecules and other biomolecules or other compounds. It is possible to detect a specific interaction with a large amount simultaneously. For example, a protein chip in which an arbitrary antigen protein is arranged on a substrate can be a new tool for early detection of diseases and allergies. In addition, 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), and for example, applied to cell culture technology in the field of regenerative medicine. Is expected.

  For the production of these biodevices, 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 Documents 2 and 3), micro Techniques such as 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 protein and cell arrangement method using optical lithography is a method capable of patterning a protein with a high accuracy of 1 μm (Non-patent Document 1). For example, as shown in FIG. 13A, the protein arrangement method using inkjet printing is a method of applying a pattern by applying fine droplets 132 of a protein solution from an inkjet nozzle 131 onto a substrate 133. In this method, generally, a plurality of types of proteins can be patterned with an accuracy of several hundred μm (Patent Document 1, Non-Patent Documents 2 and 3). For example, as shown in FIG. 13B, a protein placement method using microcontact printing attaches a microdroplet 132 of a protein solution to a needle tip or a fine structure (microstamp) 134 and transfers it to a substrate 133. It is a method (nonpatent literature 4). The protein arrangement method using laser direct writing is, for example, a method of transferring the protein solution 132 to the substrate 133 by directly irradiating the protein sample 135 with the laser 136 as shown in FIG. 13C. In this method, multiple types of proteins can be panned with an accuracy of about 50 μm (Non-Patent Document 5). The protein arrangement method using laser trapping is, for example, a method of arranging an insect virus-derived polyhedron containing a protein in a crystalline state by a laser trapping technique (Patent Document 2, Non-Patent Document 6).
JP 2002-355026 A JP 2003-155300 A A. S. Brawas and W.M. M.M. Reichert, “Protein patterning.” Biomaterials 19, 595-609 (1998). A. Roda et al. , "Protein microdeposition using a conventional ink-jet printer" BioTechniques 28, 492-496 (2000) B. T. T. Houseman et al. , "Peptide chips for the quantitative evaluation of protein kinase activity" Nat. Biotechnol. 20,270 (2002) A. Bernard et al. , “Microcontact printing of proteins” Adv. Mater. 121067-1070 (2000) P. Serra et al. "Laser direct writing of biomolecular microarrays," Appl. Phys. A 79,949-952 (2004) 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)

  However, any of the above techniques has a disadvantage in arranging many types of biomolecules with high accuracy, high density, and high speed. It is difficult to arrange many kinds of biomolecules by a method using optical lithography. In a method using techniques such as ink jet printing, micro contact printing, and laser direct writing, many types of biomolecules can be arranged. However, when a minute amount of droplet 132 containing the biomolecule is ejected into the air, and the substrate 133. The biomolecules contained in the droplet 132 may be deactivated due to drying.

  Furthermore, the above technique also has a disadvantage when a biological device is manufactured by arranging biological microparticles (biomolecular microparticles) on a substrate. For example, biomolecule-immobilized carriers, biomolecule crystals, biomolecule aggregates, viral particles, cells, and other biological microparticles can be regarded as devices that integrate biomolecule functions. Establishment of a technique for manufacturing a biological device in which biological microparticles are arranged and arranged on a substrate in a nondestructive manner with high accuracy, high density, and high speed is desired. However, for example, when the inkjet printing technique is applied to fine particles, there is a possibility that the fine particles are clogged in the ink jet nozzle when trying to arrange with high accuracy. In addition, it is extremely difficult to apply the micro contact printing technique to the arrangement of the fine particles because high control of the micro stamp and the adsorption characteristics of the substrate and the fine particles is required. On the other hand, if the laser trapping technique is used, it is possible to arrange fine particles with high accuracy. However, the arrangement speed is extremely slow, which is disadvantageous for manufacturing highly integrated biological devices.

  Accordingly, an object of the present invention is to provide a technology for arranging microparticles, which is a technology for arranging microparticles, which enables high-precision, high-density, and high-speed placement.

  In order to achieve the above object, the method for arranging microparticles according to the present invention includes a source substrate having a microparticle fixing surface, the source substrate having the microparticles fixed on the microparticle fixing surface, and the microparticles. A substrate preparing step of preparing two target substrates having a fine particle arrangement surface to be arranged, and the source substrate and the target substrate in a state where the fine particle fixing surface and the fine particle arrangement surface face each other. Substrate placement step facing the liquid layer, microparticle separation step of separating the microparticles from the microparticle fixing surface, and the microparticles separated by the force from the source substrate toward the target substrate on the microparticles. A microparticle placement step of placing the microparticles on the microparticle placement surface, wherein the separation of the microparticles from the fixed surface is induced by laser irradiation in the microparticle separation step. A physical force and chemical least one by arranging method microparticles made of force.

  The present inventors have conducted extensive research on the arrangement technology of microparticles such as protein immobilization carriers, and the microparticles are immobilized by using physical and chemical forces induced by laser irradiation. If the substrate can be separated without causing significant photochemical and photothermal damage to the carrier, and if the separated microparticles can be directly placed on another substrate, for example, the microparticles can be obtained on a single cell scale of several tens of μm or less. The inventors have found that patterning of particles is possible, and have reached the present invention.

  According to the arrangement method of the microparticles of the present invention, for example, by adjusting the position and size of the condensing part of the laser to be used, it is possible to arrange the microparticles with high density, high accuracy, and high speed. In addition, separation using a physical force or a chemical 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 physiological activity of biomolecules such as proteins can be maintained. .

  Furthermore, according to the method for arranging microparticles of the present invention, for example, each of the microparticles containing a plurality of factors that control cell functions such as proliferation, differentiation, and death is arranged with a single cell level accuracy. A cell culture substrate capable of signal transmission to cells can be produced. If such a cell culture substrate is used, it is considered that the minimum unit of the cell arrangement that forms the basis of tissue formation can be reproduced and tissue induction can be achieved. In addition, the microparticle-arranged substrate produced by the method for arranging microparticles of the present invention can be used for, for example, protein chips, bioreactors, biosensors, bioassay test pieces, and the like.

1A to 1C are schematic views showing an example of a method for arranging fine particles according to the present invention. 2A to 2E are schematic views illustrating examples of manufacturing the 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 illustrating a configuration example of the arrangement device according to the present invention. FIG. 5 is a schematic diagram showing the configuration of the electric stage in the present invention. 6A to 6E are schematic views showing other examples of the arrangement method of the present invention. 7A and 7B are examples of micrographs of polygons arranged on a substrate by the method for arranging fine particles of the present invention (Example 1). 8A and 8B are other examples of micrographs of polygons arranged on a substrate by the method for arranging microparticles of the present invention (Example 2). 9A to 9C are examples of micrographs of cells cultured on the cell culture substrate of the present invention (Example 3). FIG. 10 shows still another example of micrographs of polygons 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 ablation 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, and FIG. 12B shows the two types of polygons arranged by the method for arranging microparticles of the present invention. FIG. 12C is an example of the fluorescence image. 13A to 13C are schematic diagrams for explaining a conventional technique. FIG. 13A is a schematic diagram of an ink jet printing technique, FIG. 13B is a schematic diagram of a microcontact printing technique, and FIG. 13C is a schematic diagram of a laser direct writing technique.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Source substrate 2 Target substrate 3 Fine particle 4 Liquid layer 5 Pulse laser 6 Mechanical force based on laser ablation 7 Dispersion liquid 8 Micropipette 9 Source substrate 10-10 '''Polyhedron 11 Weak alkaline liquid 12 Target substrate 13 Glass Substrate 14 Silicon rubber substrate 15 Spacer 16 Liquid layer 20 Arrangement form 21 of source substrate and target substrate Erecting microscope 22 Stage 23 Condenser lens 24 Objective lens 25 Light source lamp 26 CCD camera 27 Pulse laser irradiation device 28 λ / 2 plate 29 Polarizing plate 30 Collimator lens 31 Dichroic mirror 32 Pulse laser 40 Arrangement device 41, 42 Electric stage 43 Source substrate fixing support 44 Source substrate 45 Target substrate 46, 47 Arrangement region 48, 49 Polyhedron 50 Source substrate Droplet 133 substrate 134 micro stamp 135 protein samples 136 laser arrangement form 51 fluid layer 60 objective lens normal to 100 pulses laser 101 condensing unit 103 substrate 131 jet 132 protein solution fine target substrate

  In the fine particle separation step of the fine particle arrangement method of the present invention, a physical force or a chemical force induced by laser irradiation is a physical force induced by laser focusing by an optical system including a lens. Force or chemical force is preferred. 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.

  In the fine particle separation step of the fine particle arrangement method of the present invention, the physical force induced by laser focusing is preferably a mechanical force based on laser ablation. The microparticles are separated from the source substrate by bringing the mechanical force based on the laser ablation into contact with the microparticles. The laser ablation preferably includes generation of at least one of shock waves, bubbles, and convection.

  In the fine particle separation step of the fine particle arrangement method of the present invention, the chemical force induced by laser irradiation is modification of the source substrate and / or modification of the portion of the fine particles in contact with the source substrate. It is preferable. The microparticles are separated from the source substrate by weakening or eliminating the force of fixing the microparticles to the source substrate by modifying the source substrate and / or the microparticles by laser irradiation.

  In the method for arranging microparticles according to the present invention, the microparticles can be 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. 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 can be arranged on the same microparticle arrangement surface. it can.

  In the fine particle arrangement step of the fine particle arrangement method of the present invention, the force applied to the fine particles is preferably a force selected from the group consisting of gravity, magnetic force, electrostatic force, light force, and buoyancy.

  In the method for arranging microparticles according to the present invention, in the substrate arranging step, the source substrate is arranged so as to be positioned on the target substrate. It is preferable to arrange on the fine particle arrangement surface.

  In the present invention, the laser light is preferably light selected from the group consisting of infrared light, visible light and ultraviolet light. The laser is preferably a pulse laser selected from the group consisting of a nanosecond laser, a picosecond laser, and a femtosecond laser, or a continuous wave laser.

  In the present invention, the microparticle is 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 an insect virus-derived polyhedron, or a group consisting of a polymer, a metal, a semiconductor, an aggregate of biomolecules, a crystal thereof, and a combination thereof. Fine particles selected from:

  The manufacturing method of the microparticle arrangement | positioning board | substrate of this invention is a manufacturing method including the process of arrange | positioning microparticles | fine-particles on a board | substrate with the arrangement | positioning method of microparticles of this 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 manufacturing method of the present invention, wherein the source substrate installation unit, the target substrate installation unit, A device for arranging fine particles including a laser irradiation means and a laser focusing means.

  The cell or tissue culturing method of the present invention comprises culturing a cell or tissue on a cell culture substrate produced by arranging microparticles containing a biomolecule exhibiting physiological activity by the microparticle arrangement method of the present invention. A culture method comprising The culture method of the present invention includes culturing cells on the microparticles arranged on the cell culture substrate, wherein the biomolecule exhibiting physiological activity is selected from the group consisting of proliferation, differentiation, migration and death. It is preferable that the culture method transmits a signal to the cells. Moreover, the manufacturing method of the cell or tissue of this invention is a manufacturing method including culture | cultivating a cell or tissue by the culture method of this invention.

  Next, the method for arranging fine particles of the present invention will be described.

  In the method for arranging microparticles of the present invention, the arrangement of microparticles includes an arrangement of microparticles. For example, the microparticles can be arranged by continuously arranging one dot of the microparticles by laser irradiation.

  The fine particles arranged by the fine particle arrangement method of the present invention are not particularly limited as long as they can be immobilized on a substrate and can be separated by a physical force or a chemical force induced by laser irradiation, Examples thereof include a carrier on which a biomolecule is immobilized, a crystal of biomolecule, an aggregate of biomolecules, a virus particle, and a cell. The biomolecule is not particularly limited, and examples thereof include proteins, peptides, DNA, RNA, sugars, lipids, organic compounds, complexes thereof, and derivatives thereof. The aggregate of biomolecules refers to, for example, a protein or DNA that is chemically aggregated. According to the use of the fine particle arrangement substrate of the present invention produced by the arrangement method of the present invention, one or more kinds of fine particles can be appropriately selected.

  In the method for arranging microparticles 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 μm, preferably 100 nm. It is 10-10 micrometers, More preferably, it is 100 nm-1 micrometer. Further, the shape of the fine particles is not particularly limited.

  When the microparticle is a carrier on which a biomolecule is immobilized, the carrier is not particularly limited, but it is preferable that the biomolecule can be immobilized and immobilized on a substrate. As the carrier, a conventionally known carrier can be used according to the biomolecule to be immobilized. For example, polymorphs derived from insect viruses, polymers, metals, semiconductors, aggregates of biomolecules, crystals thereof, and combinations thereof Fine particles selected from the group consisting of Among these, a polyhedron derived from a conventionally known insect virus described later is preferable as a carrier for immobilizing a protein. This is because the polyhedron has an effect of maintaining the physiological activity of the protein. In the present invention, a polyhedron is a polyhedron structure having a side of 100 nm to 10 μm formed in an infected cell by an insect virus, which is the original meaning, and is a crystal structure formed by associating polyhedrin in a crystalline form. In addition, a so-called crystalline protein inclusion body in which an arbitrary protein is incorporated into the polyhedron while maintaining its biological function using a known genetic engineering technique is included (for example, Patent Document 2, Japanese Patent Application Laid-Open No. 2003-319778). No. publication, WO2002 / 036785 brochure).

  In the method for arranging microparticles according to the present invention, the physical force induced by laser irradiation includes, for example, a 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 ablation. The laser ablation is an explosive erosion phenomenon that is observed when a substance is irradiated with a high-intensity laser and condensed. For example, when a pulse laser is condensed in water, This refers to a phenomenon in which shock waves, bubbles, convection, etc. are generated by multiphoton absorption, and the generation of the bubbles includes the generation of so-called air bubbles (cavitation bubbles). In the present invention, 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 the dynamic based on the explosive erosion phenomenon in general. Inclusive power. Moreover, the said shock wave is a pressure wave generate | occur | produced in the condensing part of a pulse laser. FIG. 11 shows a schematic diagram of an example of a mechanical force based on laser ablation 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 in the condensing unit 101, and thereby, in the direction of the arrow. A mechanical force due to the generation of a shock wave, a bubble, convection, etc. propagates and reaches the range of the dotted line 102. Laser ablation may use a molecule having one-photon absorption in visible light or ultraviolet light, in addition to using the multiphoton absorption of water. For example, laser ablation can be induced on the substrate by adding a molecule having one-photon absorption by visible light or ultraviolet light to the substrate and irradiating a laser emitting visible light or ultraviolet light.

  In the method for arranging microparticles of the present invention, the chemical force induced by laser irradiation includes, for example, modification of the source substrate and / or modification of a portion of the microparticles in contact with the source substrate. The modification of a part of the source substrate and / or microparticles is a change in the chemical state of a part of the source substrate and / or microparticles that is observed when the source substrate is irradiated with a laser. This includes that the material and modification of a part of the substrate and / or microparticles include photoreactive or photodegradable molecules, and the chemical state of the molecules changes by laser irradiation. Such chemical forces are preferably induced by the focusing of the laser by an optical system including a lens.

  Therefore, the laser used in the method for arranging microparticles of the present invention is not particularly limited as long as it can induce the physical force and chemical force, and the laser light can be, for example, infrared light or visible light. Light or ultraviolet light can be used. As the laser to be used, for example, a continuous wave laser or a pulse laser can be used. As the pulse laser, for example, a conventionally known laser such as a nanosecond laser, a picosecond laser, and a femtosecond laser can be used. Specific examples include a femtosecond titanium sapphire laser, a femtosecond fiber laser, a femtosecond yttrium laser, a femtosecond excimer laser, and a picosecond YAG laser.

  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.

(Board preparation process)
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, or the like can be used, and can be appropriately selected according to the fine particles to be immobilized. 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. Further, by introducing a photoreactive or photodegradable molecule into the modifying group, it is possible to obtain a fine particle arrangement surface utilizing chemical force. 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. In addition, when the laser is focused on the source substrate to induce ablation, as described above, a molecule having one-photon absorption may be included in the irradiated laser light.

  Immobilization of the fine particles and the source substrate can be appropriately selected from conventionally known techniques such as a method using chemical bonds and a method using physical adsorption. In the case where the source substrate and the fine particles are separated by physical force, an immobilization method in which the coupling is released by physical impact of mechanical force based on laser ablation is preferable. When the separation of the source substrate and the microparticles is performed by chemical force, for example, the source substrate and the microparticles are immobilized via photoreactive or photodegradable molecules, and the source by laser irradiation is used. The immobilization method is preferably such that the bonding is released by modification of the substrate and / or modification of the part of the fine particles in contact with the source substrate. As will be described later, when a plurality of microparticles are arranged on one target substrate, the plurality of microparticles may be divided and fixed on the source substrate.

  The target substrate has a fine particle arrangement surface that is a fine particle arrangement target. The material of the target 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 arranged and fixed. Further, the target substrate may be a laminate including two or more layers. For example, when the target substrate and the microparticles need to be immobilized, the microparticle placement surface is used to fix the microparticles. A suitable layer may be laminated. Further, 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. As the target substrate, for example, when a polygon is used as the fine particles, a glass substrate coated with polydimethylsiloxane (PDMS) can be used. The coating by PDMS can be performed by coating with dimertylsiloxane (DMS) and polymerizing by appropriate heating (for example, about 80 ° C. for 1 minute).

(Substrate placement process)
The substrate placement step in the method for placing microparticles according to the present invention includes the step of placing the source substrate and the target substrate through a liquid layer in a state where the microparticle fixed surface of the source substrate and the microparticle placement surface of the target substrate face each other It is a 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, 100 nm to 1 mm, preferably 100 nm to 100 μm, and more preferably 100 nm to 10 μm.

  A liquid layer is disposed between the source substrate and the target substrate. Thereby, liquid laser ablation can be utilized. Examples of 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. As described above, in the biodevice, when the arranged microparticles are biomolecules such as proteins and cells, it is preferable that these biomolecules are arranged while maintaining the biofunction. However, in conventional techniques such as ink jet printing, micro contact printing, and laser direct writing (see FIG. 13), when biomolecules are injected and fixed, the biomolecules may dry. It is thought to be lost. Therefore, the merit of the present invention is that the microparticles of biomolecules that are vulnerable to drying can be arranged to be dried in the 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.

(Microparticle separation process)
As a first aspect of the fine particle separation step in the fine particle arrangement method of the present invention, there is a step of separating the fine particles from the fine particle fixing surface by a physical force induced by laser irradiation. As described above, the physical force includes, for example, a mechanical force based on laser ablation. Laser ablation may be induced by focusing on a source substrate, but it is preferable to use laser ablation of a liquid layer disposed between the source substrate and the target substrate. The condensing part of the laser is not particularly limited as long as the target fine particles can be separated by a mechanical force based on laser ablation. For example, the laser condensing part may be directly condensed on the fine particles. However, from the viewpoint of preventing the deterioration of the fine particles and the like, it is preferable that the condensing part is not brought into contact with the fine particles. That is, damage caused by separating the microparticles by contacting only mechanical forces such as shock waves, bubbles, and convection generated in the light collecting portion can be further reduced. In addition, the shock wave generated by laser ablation propagates as a pulse, but the pulse shape of the shock wave relaxes as the distance from the condensing part increases, so the force such as the shock wave is more than the square of the distance from the condensing part. Decreases in proportion to the function of. Therefore, since the mechanical force effect based on laser ablation decreases rapidly as the distance from the condensing part increases, for example, the size of the condensing part of the pulse laser, the condensing position, the laser intensity, the laser light density, etc. If selected appropriately, a certain range of fine particles can be separated with high accuracy. For example, when the size of the light condensing part is 1 μm, the coupling between the surrounding fine particles in the range of several tens of μm and the source substrate can be solved by mechanical perturbation due to shock waves, collision of bubbles or convection. (See FIG. 11).

  As a second aspect of the fine particle separation step in the fine particle arrangement method of the present invention, chemical force induced by laser irradiation, for example, chemical modification of the source substrate and / or fine contact with the source substrate. A step of separating the fine particles from the fine particle fixing surface may be mentioned by modifying the particle portion. The modification of the source substrate and / or a part of the microparticle is performed by irradiating the molecule with a laser when the microparticle is immobilized on the source substrate via a photoreactive or photodegradable molecule. Including changing the target state and weakening or neutralizing the fixing force. The modification of the source substrate and / or part of the microparticles can be induced, for example, by focusing a laser on the interface between the source substrate and the microparticles. The condensing part of the laser is not particularly limited as long as the target fine particles can be separated by modification of the source substrate and / or a part of the fine particles. It may be a source substrate near the fine particles to be separated. From the viewpoint of preventing deterioration of the fine particles, it is preferable not to bring the light condensing part into contact with the fine particles.

  The fine particle separation step in the fine particle arrangement method of the present invention, as a third aspect, uses both a physical force and a chemical force induced by laser irradiation, and the fine particle fixing surface. It may be a step of separating the fine particles from.

  In the fine particle separation step, 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. That is, when the microparticles are arranged at an arbitrary specific position on the microparticle arrangement surface of the target substrate, the microparticles immobilized at the position of the immobilization surface of the source substrate facing the specific position of the arrangement surface are irradiated by laser irradiation. What is necessary is just to separate. Therefore, when the relative position of the source substrate and the target substrate is fixed, the patterning of laser irradiation to the source substrate can be transferred to the target substrate as it is as an arrangement of fine particles.

  In the fine particle separation step, the laser condensing, the adjustment of the size of the condensing part, and the means for adjusting the condensing position are not particularly limited, but for example, in an optical system such as a lens or a diaphragm. It is adjustable and preferably a microscope can be used. The condensing part of the pulse laser may be in the form of a dot, a circle having a certain area, or a sphere having a certain volume, and when the condensing part is circular or spherical, the radius is, for example, , More than 0 and not more than 100 μm, preferably more than 0 and not more than 10 μm, more preferably more than 0 and not more than 1 μm.

  In the fine particle separation step, the condensing position for condensing the pulsed laser is preferably the source substrate or the liquid in the vicinity of the fine particles to be separated as described above, and can be appropriately adjusted according to the range of the fine particles to be separated. The distance between the condensing position of the pulse laser and the fine particles is, for example, more than 0 and 1 mm or less, preferably more than 0 and 100 μm or less, more preferably more than 0 and 1 μm or less.

In the first aspect of the fine particle separation step, the light density (photon flux: photon flux) of the pulse laser when used for arranging fine particles in a radius region within 50 μm by laser ablation is, for example, 5 × 10 ^5. ˜1 × 10 ¹² (watt), preferably 5 × 10 ⁵ to 1 × 10 ⁹ (watt), more preferably 5 × 10 ⁵ to 1 × 10 ⁷ (watt).

Here, the optical density for the arrangement of microparticles in a radius region within 50 μm is shown. However, since the region affected by the pulse laser is proportional to the square of the radius of the region, for example, microparticles in a radius region within 10 μm are used. The optical density of the pulse laser when it is desired to be arranged is 1/25 of the above case, and the optical density of the pulse laser when it is desired to arrange fine particles in a radius region within 100 μm is 400 times that of the above case. When the pulse width of the pulse laser is Δt, the relationship between the intensity (I) of the pulse laser and the light density (D) of the pulse laser is expressed by the following equation.
I = D × Δt
The intensity of the pulse laser used in this way can be appropriately adjusted according to the distance between the microparticles and the condensing position, the range of the microparticles to be separated, etc., but when a general-purpose femtosecond laser (Δt = 100 fs) is used Examples are shown in Table 1 below.

In the second aspect of the fine particle separation step, the intensity of the pulse laser when fine particles are separated by chemical modification of the source substrate is, for example, 1 × 10 ⁻⁹ to 10 (J / pulse), Preferably, it is 1 × 10 ⁻⁶ to 1 (J / pulse), more preferably 1 × 10 ⁻⁶ to 1 × 10 ⁻³ (J / pulse). Moreover, when using a continuous wave laser, the laser intensity is 1 * 10 < ^-6 > ^-10 (watt), for example, Preferably, it is 1 * 10 < ^-4 > -1 (watt), More preferably, 1 × 10 ⁻⁴ to 1 × 10 ⁻² (watt).

  In the present invention, the wavelength of the pulse laser can be, for example, a laser having a wavelength of 190 nm to 20 μm. Among them, for example, when condensing in a liquid layer, infrared light is more directly than ultraviolet light having strong absorption. The wavelength is preferably 400 nm to 1100 nm, more preferably 600 nm to 1100 nm, since a shock wave can be generated at the laser condensing part regardless of the material such as the substrate to be used.

  The number of times of irradiation with a pulse laser for separating fine particles of 1 dot from the immobilization surface is not particularly limited, and is, for example, from 1 shot (single shot) to 10 million shots, preferably from one shot to 1000 shots, more preferably Is a single shot to 10 shots, more preferably a single shot. Here, in the present invention, 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. Moreover, the repetition frequency of the laser in the case of repeated irradiation is, for example, 1 Hz to 100 MHz, preferably 1 Hz to 1 MHz, more preferably 1 Hz to 1 kHz, and still more preferably 1 Hz to 20 Hz. .

(Microparticle placement process)
The fine particle arrangement step in the fine particle arrangement method of the present invention is a step of arranging the fine particles separated in the separation step by the force in the direction of the target substrate from the source substrate applied to the fine particles on the arrangement surface of the target substrate. It is. 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 may be mentioned a step of capturing the fine particles separated in the separation step using a laser trapping method using light force (radiation pressure of light) by a laser and moving the particles to a target substrate.

  As a preferred embodiment of this step, it is possible to drop fine particles from the source substrate onto the target substrate by gravity. An example of this aspect will be specifically described with reference to FIGS. 1A to 1C, the same portions are denoted by the same reference numerals. First, as shown in the schematic diagram of FIG. 1A, the source substrate 1 on which the microparticles 3 are immobilized is disposed on the target substrate 2 via the liquid layer 4. Next, as shown in the schematic diagram of FIG. 1B, laser ablation is induced by focusing the laser 5 to generate a mechanical force 6 based on the laser ablation, whereby the microparticles 3 are separated from the source substrate 1. Separate. Then, as shown in the schematic diagram of FIG. 1C, the separated microparticles 3 fall and are arranged on the target substrate 2. Thus, according to the arrangement method of the present invention, the patterning of the laser irradiation on the source substrate can be transferred as it is as the array of the fine particles on the target substrate.

  In addition, as another aspect of the force for moving the microparticles, there is a method of moving the microparticles from the source substrate to the target substrate by, for example, giving magnetism or electric charge. 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 immobilization of the arranged microparticles on the target substrate is not particularly limited, and can be performed using a conventionally known method using chemical bonding, a method using physical adsorption, or the like, if necessary. Further, when a polygon is fixed to a target substrate coated with PDMS, the polygon is fixed as it is, but after placing the polygon, for example, heated at 37 ° C. for 6 hours. It can be hardened more by doing.

  In the fine particle arrangement method of the present invention described above, the fine particles can be arranged as arbitrary points and / or lines on the arrangement surface by repeating the fine particle separation step and the fine particle arrangement step. it can. 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.

  Next, the method for arranging a polyhedron of the present invention using an insect virus-derived polyhedron as an example of a microparticle will be specifically described. Here, as described above, the polyhedron derived from insect virus is a crystalline substance formed by the outer shell protein of polyhedrin, which allows any protein to be taken in while maintaining its activity. It is a substance that can be used as a carrier (see, for example, Patent Document 1, Japanese Patent Application Laid-Open No. 2003-319778, and WO 2002/036785). However, the fine particles of the present invention are not limited to the polygon.

(Preparation of source substrate)
An example of the 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. First, as shown in the schematic diagram of FIG. 2A, a polyhedron dispersion liquid 7 in which a polyhedron is dispersed in water, physiological saline, or various buffers is applied to the fine particle fixing surface of the glass substrate 9 using a micropipette 8. Drop and leave at room temperature for several hours. Then, as shown in the schematic diagram of FIG. 2B, the polygon 10 is adsorbed on the immobilization surface of the glass substrate 9 by natural drying. Thus, by natural drying, the polygon 10 and the substrate 9 are bonded by electrostatic force, and then do not peel even when water or the like is dropped. Next, as shown in the schematic diagram of FIG. 2C, a weak alkaline aqueous solution 11 is dropped using a micropipette on the polygon 10 adsorbed on the immobilization surface of the glass substrate 9, and left for about several minutes. To process. 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. Finally, the weak alkaline aqueous solution is removed with a micropipette, and a source substrate in which the polygon 10 is fixed to the fixing surface of the substrate 9 as shown in the schematic diagram of FIG. 2D is prepared. Examples of the weak alkaline aqueous solution include a carbonate buffer having a pH of 10.3. The activation of the polyhedron surface by alkali treatment can be determined by, for example, antigen-antibody reaction or 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. 2E, a plurality of types of polygons 10 to 10 ′ ″ are fixed to the fixing surface of the glass substrate 9. Also good. Therefore, for example, when a 1 cm square substrate is used and the size of one immobilization region is about 1 mm in diameter, a polyhedron containing about 100 different proteins can be arranged on one substrate. It becomes.

(Arrangement of source substrate and target substrate)
Next, a configuration is constructed in which the source substrate and the target substrate are arranged via the liquid layer in a state where the fine particle fixing surface of the source substrate and the fine particle arrangement surface of the target substrate face each other. In FIG. 3, the schematic diagram of an example of an arrangement | positioning form is shown. As shown in the figure, in the arrangement form 20 of the source substrate and the target substrate, the polygon 10 prepared in the preparation step is fixed on the target substrate 12 via the liquid layer 16 formed by the spacer 15. In this configuration, the source substrate 9 is arranged. Note that the 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. As will be described later, the silicon rubber sheet 14 is preferable because the fixing operation of 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, and for example, silicon rubber, a plastic sheet, a metal thin plate, or the like can be used, and the thickness is not particularly limited, and is, for example, 100 μm. The thickness of the silicon rubber sheet 14 is not particularly limited, and is 100 μm, for example.

(Polygon separation and placement process)
The polygon separation process and the arrangement process can be performed, for example, by 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. 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. As shown in the figure, 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 on the stage 22 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, and a CCD camera 26 that detects 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 λ / 2 plate 28, a polarizer 29, and a collimator lens 30, and the emitted pulsed 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. In this apparatus, the intensity of the pulse laser 32 can be adjusted by the λ / 2 plate 28 and the polarizer 29, and the pulse laser 32 can be adjusted by the collimator lens 30 so as to be condensed on the image plane of the microscope. In addition, the focusing position of the pulse laser 32 can be adjusted by the stage 22. In this example, an example of an apparatus that irradiates a laser from above a source substrate using an upright microscope is taken up, but a laser may be irradiated from below a target substrate using an inverted microscope.

  Separation and arrangement of polygons using this device 40 are performed as follows, for example. First, the source substrate and the target substrate are arranged on the stage 22 of the upright microscope 21 as in the arrangement form 20 shown in FIG. Then, the CCD camera 26 observes the state of the source substrate and the target substrate, and determines a polygon to be separated. Then, the stage 22 is adjusted so that the condensing position of the pulse laser becomes an appropriate position. Next, the pulse laser 32 is irradiated by the pulse 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. 1B described above. As shown in the figure, when the pulse laser 5 is condensed in the liquid layer 4 between the source substrate 1 and the target substrate 3 and laser ablation is induced, and a mechanical force 6 based on laser ablation is generated, As a result, fine particles (polyhedrons) 3 in the vicinity of the condensing part are separated from the source substrate 1 and fall onto the target substrate 2 by gravity. The fine particles (polyhedrons) 3 that have fallen land on the target substrate 2 and are arranged as shown in FIG. 1C. Here, as described above, when silicon rubber is used for the target substrate 2 as the fine particle arrangement surface, the landed polygon and silicon rubber are bonded by electrostatic force, and subsequent fixing work can be omitted. ,preferable.

  By using the placement apparatus of the present invention and continuously irradiating a pulse laser while moving the stage, it is possible to write a polygonal array in an arbitrary shape on the target substrate. The width of the line to be written is not particularly limited, and can be adjusted by the intensity of the pulse laser as described above. For example, the width is 1 μm to 50 cm, preferably 1 μm to 1 cm, and more preferably. Can be in the range of 10 μm to 1 mm. The writing speed is not particularly limited, but is, for example, in the range of 1 μm / sec to 10 cm / sec, preferably in the range of 1 μm / sec to 1 mm / sec, and more preferably in the range of 10 μm / sec to 100 μm / sec. It can be in the range of sec. As described above, according to the fine particle arrangement method of the present invention using the arrangement device of the present invention, it is possible to arrange the fine particles with high accuracy, high density, and high speed. Note that the placement device of the present invention is not limited to the above example as long as it includes a source substrate placement portion, a target substrate placement portion, laser irradiation means, and laser focusing means.

  As described above, the polyhedron can carry various proteins by a conventionally known genetic manipulation. Therefore, a plurality of types of polygons are arbitrarily arranged on the target substrate using a source substrate on which polygons 10 to 10 ′ ″ carrying different types of proteins are arranged as shown in FIG. 2E. The arrangement method of the present invention will be described. This arrangement method can be implemented, for example, by using the arrangement form 50 of the source substrate and the target substrate shown in the developed schematic diagram of FIG. In the drawing, the arrangement form 50 has two electric stages 41 and 42 as main components, and an electric motor that can fix the source substrate 44 via the fixing support 43 on the electric stage 41 that can fix the target substrate 45. A stage 42 is arranged. 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.

  Separation and arrangement of the polygons using the source substrate and the target substrate arranged as in the arrangement form 50 are performed, for example, as shown in FIGS. The placement device on which the electric stage is installed may be an upright microscope as shown in FIG. 4 or an inverted microscope, for example, and can be selected as appropriate. In FIG. 6, the same portions as those in FIG. 6A to 6E are schematic views showing a process of arranging a polygon 48 in a region 46 of the target substrate 45, a polygon 49 in a region 47, and a polygon 49 in the liquid layer 51, respectively. Although not shown, 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 laser irradiation position, that is, the normal line of the objective lens of the microscope that is the placement device. First, as shown in FIG. 6A, the target substrate 45 and the source substrate 44 are respectively moved on the broken line 60 of the laser irradiation position in order to arrange the polygon 48 in the region 46, and laser irradiation is performed as shown in FIG. 6B. A polygon 48 is arranged on the region 46 of the target substrate 45. Next, as shown in FIG. 6C, the electric stage 41 (see FIG. 5) to which the target substrate 45 is fixed is moved, and the region 47 is positioned on the broken line 60. However, in this state, there is no polygon 49 in the region of the source substrate 44 on the broken line 60. Therefore, as shown in FIG. 6D, only the source substrate 44 is moved by the electric stage 42 (see FIG. 5), and the polygonal body 49 is positioned on the broken line 60. Thereby, as shown in FIG. 6E, different types of polygons 48 and 49 can be arranged on the regions 46 and 47 of the target substrate 45, respectively. Here, the liquid layer 51 may be sealed as shown in FIG. 3, but can be held between the source substrate 44 and the target substrate 45 by surface tension without sealing. Further, even when the source substrate 44 and the target substrate 45 are moved, it is possible to keep the sample portion always covered with the liquid layer 51.

In the source substrate 44 of FIG. 6, if the immobilization region of each type of polygon is less than 1 mm, the polyhedron carrying about 100 types of proteins is immobilized on the 1 cm ² region of the source substrate 44. Can do. Further, the electric stage can be controlled with an accuracy of several μm. As described above, according to the polygon arrangement method of the present invention, it is possible to arrange polygons carrying different proteins on the same target substrate at any position with high accuracy, high density, and high speed. is there.

  As mentioned above, although the example of the arrangement | positioning method of the polygon of this invention was demonstrated, the arrangement | positioning form, arrangement | positioning apparatus, and arrangement method of the source substrate and target substrate of this invention in this example are not limited to a polygon, Other microparticles Can also be used in the method for arranging microparticles of the present invention.

  Next, the fine particle arrangement substrate of the present invention will be described. The microparticle-arranged substrate of the present invention is such that microparticles are arranged on the substrate using the method of arranging microparticles of the present invention, and the manufacturing method thereof is other than the method of arranging microparticles of the present invention. Is not particularly limited. The microparticle-arranged substrate of the present invention uses, as a microparticle to be immobilized, a protein, peptide, DNA, RNA, sugar, lipid, organic compound, etc., for example, cell culture substrate, protein chip, DNA chip, bioreactor , Biosensors, test specimens for bioassays, and the like, and can be used without particular limitation for various known applications.

  Among these, the cell culture substrate of the present invention will be described. The cell culture substrate of the present invention is a cell culture substrate in which physiologically active substances are arranged on the 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. Is manufactured. Examples of the physiologically active substance include conventionally known proteins that control functions such as proliferation, differentiation, and death. By arranging a plurality of these, various proteinaceous intercellular signaling substances can act at the individual cell level. As described above, by culturing the cells on the microparticles on which the cell culture substrate of the present invention is arranged, it becomes possible to control proliferation and differentiation for each cell. Specific examples of the physiologically active substance include, but are not limited to, cytokines, hormones, growth factors, and the like. In addition, in addition to the cell culture substrate of the present invention, fine particles having no physiological activity may be arranged.

  The cell or tissue culture method of the present invention is a method of culturing a cell or tissue using the cell culture substrate of the present invention, and the cell or tissue production method of the present invention is based on the cell or tissue culture method of the present invention. It is a method of obtaining cells or tissues by culturing. The cells to be cultured in the cell or tissue culturing / manufacturing method of the present invention are not particularly limited. According to the method for culturing / manufacturing cells or tissues of the present invention, it is possible to control and control advanced cells that induce organization, which is difficult with conventional methods, and is the minimum unit of cell arrays that forms the basis of tissue formation. Can be reproduced.

  The present invention will be further described below with reference to examples.

(Preparation of polyhedron)
A protein-immobilized carrier consisting only of a polyhedron, which is a crystalline medium formed by an insect virus-derived protein (polyhedrin), was prepared as follows. First, in order to form a cubic polyhedron of silkworm cytoplasmic polyhedrosis virus (BmCPV), a recombinant viral vector AcCP-H (Mori et al. (1993) J. Gen.) in which a polyhedrin gene is incorporated into an insect viral vector AcNPV. Virol.74, 99-102) were infected into IPLB-Sf21-AE (Sf21) cells from Spodoptera Frugiperda. Next, cubic polyhedra were collected from the infected cells on the 4th day, washed with PBS (20 mM NaH ₂ PO ₄ , 20 mM Na ₂ HPO ₄ , 150 mM NaCl, pH 7.2), and then in ice using a homogenizer. Triturated with. After washing the grinding solution with 1% Tween 20, the polyhedron is recovered by centrifugation, and further centrifuged at 50,000 × G for 45 minutes at a sucrose density gradient of 1.5M to 2.2M to separate the polyhedron. The drawing was extracted. The extracted sample was washed with PBS, and then centrifuged at 15,000 × G for 10 minutes to recover a polyhedron consisting only of crystalline polyhedrin inclusion bodies.

(Production of source substrate)
The polyhedron was dispersed in water, dropped onto a glass substrate (thickness: 100 μm) using a micropipette so as to form a droplet of about 1 mm, left at 24 ° C. for several hours, and naturally dried. Thereafter, a weak alkaline aqueous solution (pH 10.3) was dropped on the surface of the polyhedron and allowed to stand for about several minutes, the polyhedron surface was dissolved, and the alkaline aqueous solution was removed using a pipette to prepare a source substrate. .

(Polygon array)
A target substrate in which a silicon rubber sheet (thickness: 100 μm) was laminated on a glass substrate was used. On the target substrate, a spacer prepared by hollowing out a silicon rubber sheet (thickness: 100 μm) is placed, and the hollowed portion is filled with water, and further, the surface on which the polygonal body is bonded is positioned below. The source substrate was placed and sealed to produce 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 polygons and the state after the arrangement were observed with a CCD camera 26. In addition, as the laser irradiation apparatus 27, the high output femtosecond laser irradiation apparatus (120fs, 800nm, 20Hz, 10mW) was used.

  Femtosecond laser irradiation with an intensity of 150 nJ / pulse from the top of the array plate, the size of the condensing part is about 1 μm, and the light is condensed in the water layer (around 5 μm from the source substrate surface), and the polygon is placed on the target substrate Arranged. As a result, polygons could be arranged at a speed of about 1 μm / sec. The arranged polygons were not peeled off from the target substrate even after the source substrate and the aqueous layer were removed.

  An example of the result of arranging the polygons is shown in FIG. 7A and 7B are photomicrographs of a target substrate on which polygons are arranged, and FIG. 7B is an enlarged view of a portion surrounded by a square in FIG. 7A. In FIG. 7A, the portions that appear linear are polygons that are arranged, and in FIG. 7B, each that appears as a square is a polygon. As a result, according to the method of the present invention, it can be said that the line width corresponding to the size of one polygon can be arranged at an extremely high speed.

  Polyhedrons were arranged in the same manner as in Example 1 except that the intensity of the femtosecond laser was 300 nJ / pulse. The results are shown in FIGS. 8A and 8B. 8A and 8B are photomicrographs of a target substrate on which polygons are arranged, and FIG. 8B is an enlarged view of a portion surrounded by a square in FIG. 8A. The time required for arranging the three characters “JST” was about 1 minute, and the polygons could be arranged at a speed of about 10 μm / sec. The line width is almost the same as the size of animal cells. Therefore, by culturing animal cells on this polyhedron arrangement, it can be said that the polyhedron can act on specific individual cells.

The cell culture substrate prepared in Example 2 was immersed in a culture solution (medium: Dulbec's modification of Eagle's medium (DMEM) containing 5% fetal bovine serum), and NIH3T3 strain was added thereto. After several minutes, the added cells were implanted on the cell culture substrate, and the cells were cultured for 24 hours in a CO ₂ incubator. The results are shown in 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, and FIG. 9C is the portion enclosed by the upper square in FIG. Is an enlarged version. In these figures, the droplet-like 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 easier for the cells to proliferate than on the cell culture substrate (target substrate). Thus, according to the present invention, it can be said that cell proliferation can be controlled.

(Preparation of polyhedron with immobilized EGFP)
A recombinant virus vector AcVP3 / EGFP capable of expressing a fusion protein of VP3 protein, which is one of the outer proteins of BmCPV, and EGFP (enhanced green fluorescein protein) protein, 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 recombinant virus vector AcCP-H.

(Preparation of source substrate)
A source substrate (see FIG. 2E) in which a polyhedron composed solely of polyhedrin and a polyhedron on which an EGFP protein was immobilized was immobilized on the same microparticle immobilization surface was prepared in the same manner as in Example 1.

(An array of two types of polygons)
In the placement apparatus of the present invention, the source substrate and the target substrate similar to those of the first embodiment are arranged as shown in the arrangement form 50 of FIG. Two types of polygons, an immobilized polygon and a polyhedrin-only polygon, were arranged on the target substrate. The result is shown in FIG. This figure is a fluorescent micrograph of the target substrate. In the figure, the part surrounded by a circle is a place where a polygon into which EGFP is introduced is arranged, and the part enclosed by a square is not introduced with EGFP. This is the place where the polygon is placed. 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 activity of the protein.

(Preparation of source substrate and target substrate)
In the same manner as in Example 4, a source substrate in which a polygon including EGFP and a polygon not including EGFP were immobilized on the same fine particle fixing surface was prepared. Further, a target substrate was prepared by coating a glass substrate with dimertylsiloxane (DMS) and then heating at 80 ° C. for 1 minute to form a polydimethylsiloxane (PDMS) coat having a thickness of about 20 μm.

(Pattern pattern of two types of polygons)
A checkerboard pattern is formed by the placement device of the present invention in the same manner as in Example 4 except that the laser frequency is 1 kHz, the intensity is 31 nJ / pulse, the light collecting portion is about 2 μm from the source substrate surface, and the placement speed is 90 μm / sec. Patterning. That is, 40 μm × 40 μm blocks were arranged by alternately using EGPF-containing polyhedra and non-EGFP-containing polyhedra. Each block was placed by laser scanning with straight lines with a spacing of 5 μm. The interval between the blocks was 5 μm. A schematic diagram of this arrangement is shown in FIG. 12A, and an example of a transmission image of a target substrate actually arranged is shown in FIG. 12B. The arrangement as shown in FIG. 12B could be performed in about 30 minutes. When this target substrate was observed with a fluorescence microscope, a checkered fluorescent image could be obtained as shown in FIG. 12C.

  As described above, the fine particle arrangement method of the present invention is an arrangement method using laser ablation. According to the present invention, for example, a carrier on which a physiologically active factor is immobilized can be provided with an alignment technique capable of maintaining physiological activity such as a protein to be arranged at high accuracy, high density, and high speed. This is useful in the field of using biochips. Among these, the present invention is useful, for example, in the field of regenerative medicine. That is, if the cell culture substrate produced by the arrangement method of the present invention is used, various immobilized cell signal transduction factors can be made to act at the single cell level, which is an advanced level that induces organization and induction. Cell arrangement and control. As described above, the present invention intends to contribute to tissue and organ regeneration technology by reproducing the minimum unit of the cell arrangement that forms the basis of tissue formation and attempting tissue induction.

Claims (20)

A method for arranging fine particles,
Two substrates, a source substrate having a fine particle fixing surface, the source substrate having the fine particles fixed on the fine particle fixing surface, and a target substrate having a fine particle arrangement surface on which the fine particles are to be arranged A substrate preparation step to prepare
A substrate disposing step of facing the source substrate and the target substrate through a liquid layer in a state where the fine particle fixing surface and the fine particle disposition surface face each other;
A microparticle separation step of separating the microparticles from the microparticle fixing surface;
A microparticle arrangement step of arranging the microparticles separated by a force in the direction of the target substrate from the source substrate applied to the microparticles on the microparticle arrangement surface,
In the fine particle separation step, the fine particles are separated from the fixed surface by a method of arranging the fine particles by at least one of a physical force and a chemical force induced by laser irradiation.   In the fine particle separation step, the physical force or chemical force induced by laser irradiation is a physical force or chemical force induced by laser focusing by an optical system including a lens. Item 2. A method for arranging fine particles according to Item 1.   The method for arranging microparticles according to claim 2, wherein, in the microparticle separation step, the position of the condensing part of the laser is the microparticles to be separated, or the liquid layer or the source substrate in the vicinity of the microparticles to be separated.   In the fine particle separation step, a physical force induced by laser focusing is a mechanical force based on laser ablation, and the mechanical force is brought into contact with the fine particle to bring the mechanical force into contact with the fine particle. The method for arranging fine particles according to claim 2, wherein the fine particles are separated.   The method for arranging microparticles according to claim 4, wherein the laser ablation includes generation of at least one of shock waves, bubbles, and convection.   In the fine particle separation step, the chemical force induced by the laser irradiation is modification of the source substrate and / or modification of the portion of the fine particles in contact with the source substrate, and the source substrate is modified by the modification by laser irradiation. The method for arranging microparticles according to claim 2, wherein the microparticles are separated from the source substrate by weakening or eliminating the force for immobilizing the microparticles.   The arrangement of microparticles according to claim 1, wherein the microparticles to be separated in the microparticle separation step are microparticles fixed at the position of the immobilization surface opposite to a desired microparticle arrangement position of the arrangement surface. Method.   The method for arranging microparticles according to claim 1, wherein the microparticle separation step and the microparticle arrangement step are repeated to arrange the microparticles as at least one of arbitrary points and lines on the arrangement surface.   The method for arranging microparticles according to claim 1, wherein in the substrate preparing step, source substrates on which different types of microparticles are immobilized are prepared, and different types of microparticles are arranged on the same microparticle arrangement surface.   2. The method for arranging microparticles according to claim 1, wherein the force applied to the microparticles in the microparticle arranging step is a force selected from the group consisting of gravity, magnetic force, electrostatic force, light force and buoyancy.   The method for arranging fine particles according to claim 1, wherein the laser light is light selected from the group consisting of infrared light, visible light, and ultraviolet light.   The method for arranging fine particles according to claim 1, wherein the laser is a pulse laser selected from the group consisting of a nanosecond laser, a picosecond laser, and a femtosecond laser, or a continuous wave laser.   2. The method for arranging microparticles according to claim 1, wherein the microparticles are selected from the group consisting of a carrier on which biomolecules are immobilized, biomolecule crystals, biomolecule aggregates, virus particles, and cells.   14. The method for arranging microparticles according to claim 13, wherein the carrier on which the biomolecule is immobilized is a polyhedron derived from an insect virus.   The microcarrier according to claim 13, wherein the carrier on which the biomolecule is immobilized is a microparticle selected from the group consisting of a polymer, a metal, a semiconductor, an aggregate of biomolecules, a crystal thereof, and a combination thereof. Particle placement method.   A method for producing a microparticle-arranged substrate, comprising the step of arranging microparticles on a substrate by the method for arranging microparticles according to claim 1.   2. A fine particle placement apparatus for use in the fine particle placement method according to claim 1, wherein the fine particle placement device includes a source substrate placement portion, a target substrate placement portion, a laser irradiation means, and a laser focusing means. Particle placement device.   A method for culturing cells or tissues, wherein cells or tissues are cultured on a cell culture substrate produced by arranging microparticles containing biomolecules exhibiting physiological activity according to the microparticle arrangement method according to claim 1. A culture method comprising:   19. The method for culturing cells or tissues according to claim 18, comprising culturing cells on the microparticles arranged on the cell culture substrate, wherein the biomolecule exhibiting physiological activity is proliferated, differentiated, and migrated. And a culture method for transmitting a signal selected from the group consisting of death to the cells.   A method for producing a cell or tissue, comprising culturing the cell or tissue by the culture method according to claim 18.

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