WO2006104048A1

WO2006104048A1 - Method for arranging fine particles

Info

Publication number: WO2006104048A1
Application number: PCT/JP2006/305993
Authority: WO
Inventors: Hiroshi Masuhara; Yoichiroh Hosokawa; Yuji Hiraki; Chisa Shukunami
Original assignee: Hamano Life Science Research Foundation
Priority date: 2005-03-25
Filing date: 2006-03-24
Publication date: 2006-10-05
Also published as: JPWO2006104048A1

Abstract

This invention provides a method for arranging fine particles that can arrange various types of fine particles in a liquid with high accuracy at high density and high speed. The arranging method comprises the step of providing two substrates, that is, a source substrate (1), which has a fine particle fixation face and to which the above fine particles (3) have been fixed, and a target substrate (2) having a fine particle arranging face on which the fine particles (3) are to be arranged (substrate provision step), the step of disposing the source substrate (1) and the target substrate (2) so that the fine particle fixation faces the fine particle disposition face through a liquid layer (4) (substrate disposition step), the step of separating the fine particles (3) from the fine particle fixation face (fine particle separation step), and the step of arranging the separated fine particles (3) on the fine particle disposition face by force in a direction from the source substrate to the target substrate applied to the fine particles (3) (fine particle disposition step). In the fine particle separation step, the separation of the fine particles (3) from the fixation face is carried out by physical force (6) or chemical force induced by laser irradiation (5).

Description

Specification

How to arrange fine particles

Technical field

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

[0002] In recent years, biodevices (also referred to as biodevices or biochips) that partially mimic biofunctions have been attracting attention by fusing biotechnology with technologies in other fields. 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. For example, 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. 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). Application is expected.

[0003] 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 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.

[0004] A method for arranging proteins and cells using optical lithography is a method capable of patterning proteins with a high accuracy of 1 μm (Non-patent Document 1). For example, as shown in FIG. 13A, 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. In this method, generally, 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). For example, as shown in FIG. 13B, 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. In this method, 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)

Disclosure of the invention

Problems to be solved by the invention

However, all of the above techniques have disadvantages for arranging many types of biomolecules with high accuracy, high density, and high speed. It is difficult to arrange many kinds of biomolecules by the method using optical lithography. Methods using technologies such as inkjet printing, microcontact printing, and laser direct writing can place many types of biomolecules, but a small amount of droplets 132 containing biomolecules are ejected into the air. In this case, when transferred to the substrate 133, the biomolecules contained in the droplet 132 may be deactivated by drying.

[0006] 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, 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. However, for example, when 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. Moreover, it is extremely difficult to apply the micro contact printing technique to the arrangement of fine particles because it requires a high degree of control of the adsorption characteristics between the micro stamp and the substrate and the fine particles. On the other hand, if the laser trapping technique is used, it is possible to arrange fine particles with high accuracy. However, it is disadvantageous for manufacturing highly integrated biological devices with extremely slow deployment speeds.

[0007] Accordingly, 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.

Means for solving the problem

[0008] In order to achieve the above object, a method for arranging microparticles according to the present invention 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及 Byi 匕学 specific at least one by the arrangement method of the particulates carried out of power in the irradiation. The invention's effect

[0009] 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.

[0010] According to 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.

[0011] Furthermore, according to the method for arranging microparticles of the present invention, for example, 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. In addition, 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. In addition, 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.

Brief Description of Drawings

[0012] FIGS. 1A to 1C are schematic views showing an example of a method for arranging fine particles of the present invention.

[FIG. 2] 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).

[FIG. 8] 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).

[FIG. 9] 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] 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. 12] 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. FIG. 12C is an example of a transmission image of a micrograph of a polyhedron of a kind, and 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, and FIG. 13C is a schematic diagram of laser direct writing technology.

Explanation of symbols

1 Source board

2 Target board

3 Fine particles

4 liquid layers

5 North Laser

6 Mechanical force based on laser abrasion

7 Dispersion

8 Micropipette Source board

~: LO '"Polyhedron

Weak alkaline solution

Target board

Glass substrate

Silicon rubber substrate

Spacer

Liquid layer

Arrangement of source and target substrates Upright microscope

Stage

Condenser lens

Objective lens

Light source lamp

CCD camera

Pulsed laser irradiation device

/ 2 board

Polarizer

Collimator lens

Ta

Nores laser

Placement device

, 42 Electric stage

Support for fixing source substrate

Source board

Target board

, 47 Placement area

, 49 Polyhedron 50 Arrangement of source and target substrates

51 liquid layer

60 Objective normal

100 laser

101 Light collector

103 substrate

131 inkjet

132 Droplets of protein solution

133 PCB

134 Micro Stamp

135 Protein samples

136 laser

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] In the fine particle separation step of the fine particle arrangement method of the present invention, 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.

[0015] In the fine particle separation step of the fine particle arrangement method of the present invention, it is preferable that the physical force induced by laser concentration is a mechanical force based on laser abrasion. By bringing the mechanical force based on the laser abrasion into contact with the fine particles, the source substrate force also separates the fine particles. The laser ablation preferably includes at least one generation of shock waves, bubbles, and convection.

[0016] In the fine particle separation step of the fine particle arrangement method of the present invention, 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. Prefer to be quality ,. Reducing the force to fix the microparticles on the source substrate by modifying the source substrate and some of the Z or microparticles by laser irradiation By eliminating, the source substrate force also separates the microparticles.

In the method for arranging microparticles of the present invention, 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.

[0018] In the fine particle arrangement step of the fine particle arrangement method of the present invention, 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.

[0019] The method for arranging fine particles of the present invention includes the substrate arranging step! In addition, it is preferable that 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. .

[0020] In the present invention, the laser light is preferably light selected from the group consisting of infrared light, visible light, and ultraviolet light. In addition, 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.

In the present invention, 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.

[0022] 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.

[0023] 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. [0024] 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. Is a culturing method comprising culturing 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.

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, microparticles can be arranged by continuously arranging 1 dot of microparticles by laser irradiation.

[0027] 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. Although not particularly limited, for example, 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.

[0028] 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 m, Preferably, lOOnm˜: LO μm, and more preferably 100 nm˜l μm. Further, the shape of the fine particles is not particularly limited.

[0029] When the microparticle is a carrier on which a biomolecule is immobilized, the carrier is not particularly limited, but a carrier that can immobilize the biomolecule and can be immobilized on a substrate is preferable. As the carrier, 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. Among these, as 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. In the present invention, a polyhedron is a polyhedral structure of 100 nm to 10 μm 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. In addition to the resulting crystal structure, 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).

In the method for arranging microparticles of the present invention, 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). 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 generally the explosive erosion phenomenon. Including dynamic force based. 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. As a result, the mechanical force due to the generation of shock waves, bubbles, convection, etc. propagates in the direction of the arrow, and the mechanical force 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 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.

[0031] 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 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, In addition, 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.

Therefore, 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. In addition, for example, a continuous wave laser or a pulse laser can be used as the laser to be used. As the pulse laser, for example, a conventionally known laser such as a nanosecond laser, a picosecond laser, or a femtosecond laser is used. it can. Specific examples include femtosecond titanium sapphire lasers, femto fiber lasers, femtoseconds, ittribium lasers, femtoseconds, excimer lasers, and picosecond YAG lasers.

[0033] 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.

[0034] (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 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. In addition, by introducing a photoreactive or photodegradable molecule into the modifying group, 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. In addition, when ablation is induced by causing the source substrate to emit laser light, as described above, a molecule having one-photon absorption may be included in the irradiated laser light.

[0035] 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. 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. When 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. It is preferable that 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. As will be described later, when a plurality of fine particles are arranged on one target substrate, the plurality of fine particles may be divided and fixed on the source substrate.

[0036] 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. In addition, the target substrate may be a laminate including two or more layers. For example, when the target substrate and the microparticles need to be fixed, the microparticle placement surface may be used to fix the microparticles. A layer suitable for the above 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. For example, when the target substrate uses a polyhedron as fine particles, 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).

[0037] (Substrate placement process)

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 100ηπι to 100 / ζ πι, more preferably 100 nm to 10 ^ m.

[0038] A liquid layer is disposed between the source substrate and the target substrate. This makes it possible to use liquid laser ablation. 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 microparticles arranged are proteins, cells, and other biomolecules, it is preferable that these biomolecules are arranged while maintaining the biofunction. However, 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. Therefore, 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.

[0039] (Microparticle separation step)

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 particle fixing surface force and fine particles by a physical force induced by laser irradiation. It is done. As described above, 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. . However, it is preferable not to bring the light condensing part into contact with the fine particles from the viewpoint of preventing the fine particles from deteriorating. That is, 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. In addition, 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. For example, 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 μm, 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. There is a step of separating the fine particles from the fine particle fixing surface by modifying the portion of the fine particles in contact therewith. 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. For example, the laser condensing part is directly focused on the microparticle. Alternatively, it may be a source substrate in the vicinity of the fine particles to be separated. From the viewpoint of preventing deterioration of the microparticles, the condensing part is in contact with the microparticles. Preferably not.

[0041] 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.

[0042] 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. In other words, 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.

[0043] 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 μm, preferably more than 0 and not more than 10 μm, and more preferably more than 0 and not more than 1 μm.

[0044] In the fine particle separation step, 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 μm.

[0045] In the first aspect of the fine particle separation step, 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 μm is shown. Since 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. When the pulse width of the pulse laser is Δt, the relationship between the pulse laser intensity (I) and the light density (D) of the pulse laser is expressed by the following equation.

I = D X At

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. When using a general-purpose femtosecond laser (Δ t = lOOfs) Examples of these are shown in Table 1 below.

[0047] [Table 1]

(Table 1) Intensity of pulse laser (J / pu l se)

Distance General strength Preferred strength More preferred strength

l Within OOO ym 2x 10— ⁵ ~ 4x10 '2x10— ⁵ ~ 8x10— ² 2x 10 " ⁵ ~ 2x10" ³ 50; um or less 5x 10— ⁸ ~ 10x10—' 5χ10 " ^ε ~ 2χ10" ⁴ 5x 10 " ^s ~ 2x10 " ⁶

Within 10 m 2x10 " ⁹ to 4x10" ³ 2x10— ⁹ to 8x10— ⁶ 2x 10 to 2x10 "'_

[0048] Te second aspect Nio, of the fine particle separation step, 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). When using a continuous wave laser, 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).

[0049] 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, the wavelength of the pulse laser 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 400ηπ! More preferably, l lOOnm is more preferable, and 600 l: L lOOnm.

[0050] 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. 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. In addition, 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, 1Ηζ to 20〜ζ.

[0051] (Microparticle placement process)

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.

[0052] 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. First, as shown in the schematic diagram of FIG. 1A, a source substrate 1 on which fine particles 3 are fixed is placed on a target substrate 2 via a liquid layer 4. Next, as shown in the schematic diagram of Fig. 1B, 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. Then, as shown in the schematic diagram of FIG. 1C, the separated fine particles 3 fall and are arranged on the target substrate 2. As described above, according to the arrangement method of the present invention, 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.

[0053] Further, as another aspect of the force for moving the microparticles, for example, 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.

[0054] 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. In addition, when using a PDMS-coated target substrate and fixing a polygon to this, 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.

[0055] In the fine particle arrangement method of the present invention described above, by repeating the fine particle separation step and the fine particle arrangement step, 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.

[0056] Next, 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. Here, 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. (See, for example, Patent Document 1, Japanese Patent Application Laid-Open No. 2003-319778, and WO 2002Z036785 pamphlet). However, the fine particles of the present invention are not limited to the polygon.

[0057] (Preparation of source substrate)

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. 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 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. Thus, 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. 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 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. Finally, 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. Examples of the weakly alkaline aqueous solution include ρΗΙΟ.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. 2E, 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.

(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. FIG. 3 shows a schematic diagram of an example of the arrangement form. As shown in the figure, 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. In this configuration, 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. As will be described later, 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. 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 100 m, for example. The thickness of the silicon rubber sheet 14 is not particularly limited, and is 100 m, for example. [0059] (Polygon separation and arrangement process)

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. 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. On the stage 22, 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 λλ2 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. 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 focused on the image plane of the microscope. 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 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! /

[0060] Separation and arrangement of polygons using the apparatus 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 shown 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 the polygon to be separated. Then, the stage 22 is adjusted so that the focused position of the pulse laser becomes an appropriate position. Then 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. 1B described above. As shown in the figure, 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. When this occurs, 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. Then, the dropped microparticles (polyhedrons) 3 land on the target substrate 2 and are arranged as shown in FIG. 1C. Here, as described above, when 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.

[0061] By using the placement device 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 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 μm to 50 cm, preferably in the range of 1 μm to lcm. More preferably, it can be in the range of 10 / ζ πι to 1πιπι. 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 μmZsec to: LmmZsec, and more preferably in the range of 10 mZsec lOO / z mZsec. It can be a range. 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. 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.

[0062] As described above, 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. In the figure, 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. Note that 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. In 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. 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 irradiation position of the laser, 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 on which the polygon 48 is placed in the region 46 are moved onto the broken line 60 of the laser irradiation position, respectively, and the laser irradiation is performed as shown in FIG. 6B. As a result, the 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 on the electric stage 42 (see FIG. 5), and the polygon 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 part always covered with the liquid layer 51.

[0064] In the source substrate 44 of FIG. 6, if 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. In addition, the electric stage can be controlled with an accuracy of a few zm. As described above, according to the polygon arrangement method of the present invention, 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.

[0065] The force described above as an example of the polygon arrangement method of the present invention The arrangement form, arrangement apparatus, and arrangement method of the source substrate and target substrate of the present invention in this example are not limited to polygons, It can also be used in the method for arranging microparticles of the present invention for microparticles.

[0066] Next, the fine particle arrangement substrate of the present invention will be described. 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.

[0067] 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 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. Has been manufactured. Examples of 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. 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 the growth and distribution of each cell. Specific examples of 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.

[0068] 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. In this method, 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.

[0069] The present invention will be further described below with reference to examples.

Example 1

[0070] (Preparation of polyhedron)

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.

2 4 2 4

, 150 mM NaCl, pH 7.2), and then ground in ice using a homogenizer. After washing the homogenate with 1% T _W een20, by centrifugation to collect the polyhedra, further 1. 5M~2. Centrifuged for 45 minutes at a sucrose density gradient at 50, 000 XG of 2M, polyhedra Fractions were extracted. The extracted sample was washed with PBS, and then centrifuged at 15,000 XG for 10 minutes to recover a polyhedron having only crystalline polyhedrin inclusion bodies.

[0071] (Production of source substrate)

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 (ρΗΙΟ.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.

[0072] (polygon array)

A silicon rubber sheet (thickness 100 μm) 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.

[0073] From the upper part of the array plate, femtosecond laser irradiation with an intensity of 150 njZpulse was performed, and the size of the condensing part was about 1 μm, and the light was condensed in the water layer (around 5 μm from the source substrate surface). The horns were arranged on the target substrate. As a result, polygons could be arranged at a speed of about 1 μmZsec. The arranged polygons did not peel off the force on the target substrate even when the source substrate and the water layer were removed.

[0074] 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, and FIG. 7B is an enlarged view of the portion surrounded by the square in FIG. 7A. In FIG. 7A, the portions that appear to be linear are polygons 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 a line width corresponding to the size of one polygon can be arranged at an extremely high speed.

Example 2

[0075] Polyhedrons were arranged in the same manner as in Example 1 except that the intensity of the femtosecond laser was 300 njZpulse. The results are shown in FIGS. 8A and B. 8A and B are photomicrographs of the target substrate on which polygons are arranged, and 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.

Example 3

[0076] 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

2

Cells were cultured in one for 24 hours. 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 enlargement of In these figures, 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). Thus, according to the present invention, it can be said that cell proliferation can be controlled.

Example 4

[0077] (Preparation of polyhedron with immobilized EGFP)

A recombinant viral vector that can express a fusion protein of VP3 protein, one of the outer proteins of BmCPV, and EGFP (enhanced green fl uorescent protein) protein. 1 AcVP3ZEGFP 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.

[0078] (Preparation of source substrate)

Prepare a source substrate (see Fig. 2E) in which the polyhedrin-only polyhedron and the EGFP protein-immobilized polyhedron are immobilized on the same microparticle immobilization surface as in Example 1. did.

[0079] (Array of two types of polygons)

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. Two types of polygons, an EGFP-immobilized polyhedron and a polyhedrin-only polyhedron, were placed on the target substrate. The results are shown in Fig. 10. This figure is a fluorescence micrograph of the target substrate. In the figure, 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.

Example 5

[0080] (Preparation of source substrate and target substrate)

In the same manner as in Example 4, 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. Also, 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 μm.

[0081] (Two kinds of polygonal pattern patterning)

Except for the laser frequency of lkHz, the intensity of 31rjZpulse, the condensing part of about 2 μm from the source substrate surface, and the arrangement speed of mZsec, 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. 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.

Industrial applicability

[0082] As described above, the method for arranging fine particles of the present invention is an arrangement method using laser abrasion. According to the present invention, for example, it is possible to provide 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. Among these, 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. Thus, 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.

Claims

The scope of the claims

[1] A method for arranging fine particles,

Two substrates, a source substrate having a microparticle fixing surface, the source substrate having the microparticles immobilized on the microparticle fixing surface, and a target substrate having a microparticle arrangement surface on which the microparticles are to be arranged A substrate preparation process 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 disposing surface face each other;

A microparticle separation step for separating the microparticles from the microparticle fixing surface, and a source substrate force acting on the microparticles. Microparticles that dispose the microparticles separated by a force toward the target substrate on the microparticle arrangement surface. Including an arrangement step,

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 an ionic force induced by laser irradiation.

[2] In the fine particle separation step, the physical force or chemical force induced by laser irradiation is the physical force induced by the focusing of the laser by the optical system including the lens. 2. The method for arranging fine particles according to claim 1, wherein the method is a chemical force.

[3] In the fine particle separation step, the position of the condensing part of the laser is 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 arrangement method of the microparticles described.

[4] In the fine particle separation step, the physical force induced by laser focusing is a mechanical force based on laser abrasion, and the mechanical force is brought into contact with the fine particles. The method for arranging microparticles according to claim 2, wherein the microparticles are separated from the source substrate.

5. The method for arranging microparticles according to claim 4, wherein the laser ablation includes at least one generation of shock waves, bubbles, and convection.

[6] In the fine particle separation step, the chemical force induced by laser irradiation is modification of the source substrate and modification of the portion of the fine particles in contact with Z or the source substrate, and the modification by laser irradiation. Weakens or eliminates the force of fixing fine particles on the source substrate 3. The method for arranging microparticles according to claim 2, wherein the source substrate force is used to separate the microparticles.

[7] In the fine particle separation step, the fine particles to be separated are fine particles fixed at a position on the fixed surface opposite to a desired fine particle arrangement position on the arrangement surface. The method for arranging fine particles according to claim 1.

8. The method for arranging microparticles according to claim 1, wherein the microparticle separation step and the microparticle arrangement step are repeated, and the microparticles are arranged on the arrangement surface as at least one of arbitrary points and lines.

[9] The arrangement of microparticles according to claim 1, wherein in the substrate preparation 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. Method.

[10] The method for arranging microparticles according to [1], wherein in the microparticle arranging step, the force exerted on the microparticles is a force selected from the group consisting of gravity, magnetic force, electrostatic force, light force and buoyancy. .

11. The method for arranging fine particles according to claim 1, wherein the laser beam is a light selected from the group consisting of infrared light, visible light, and ultraviolet light.

12. The method for arranging fine particles according to claim 1, wherein the laser is a pulsed laser selected from a group power consisting of a nanosecond laser, a picosecond laser, and a femtosecond laser, or a continuous wave laser.

13. 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, a crystal of biomolecules, an aggregate of biomolecules, 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.

[15] The carrier of the carrier on which the biomolecule is immobilized is a polymer, a metal, a semiconductor, an aggregate of biomolecules, a crystal thereof, and a group force consisting of a combination force thereof. 4. A method for arranging fine particles according to 1.

[16] A method for producing a microparticle arrangement substrate according to claim 1, which is a method for producing a microparticle arrangement substrate. The manufacturing method of the microparticle arrangement | positioning board | substrate including the process of arrange | positioning a microparticle on a board | substrate.

[17] A device for arranging microparticles used in the method for arranging microparticles according to claim 1, wherein the installation portion of the source substrate, the installation portion of the target substrate, the laser irradiation means, and the laser A device for arranging microparticles including a light collecting means.

[18] A method for culturing a cell or tissue, wherein a cell or a tissue is produced 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 culturing a tissue.

[19] The method for culturing a cell or tissue according to claim 18, comprising culturing cells on the microparticles arranged on the cell culture substrate, wherein the biomolecules exhibit proliferation of the physiological activity. A culture method for transmitting a signal selected from the group consisting of differentiation, migration and death to the cells.

[20] A method for producing a cell or tissue, comprising culturing the cell or tissue by the culture method according to claim 18.