JP4552673B2 - Light immobilization method for minute objects - Google Patents

Description

  The present invention relates to a technique for immobilizing a minute object on a predetermined surface, and more particularly to a technique for immobilizing a minute object on a predetermined surface with light.

  As an example of immobilizing a minute object by light irradiation, DNA and protein are arranged on a thin film surface of a polymer (azo polymer) containing a photoresponsive azo dye, and then DNA and protein are applied according to the arrangement pattern by light irradiation. Are fixed on the surface of an azo polymer (Patent Document 1). In order to fix a minute object to a carrier having a light immobilization material on its surface (hereinafter simply referred to as “carrier”), it is preferable to irradiate light with the minute object in contact with the carrier surface. In order to stably arrange the minute object on the surface of the carrier, it is disclosed that the light-immobilized material has affinity for the minute object (Patent Document 2).

In these methods, the minute object is arranged and immobilized on the surface of the carrier in a state where the minute object is dissolved or suspended in the liquid medium because the minute object can be easily developed on the surface of the carrier. It is disclosed.
JP 2003-329682 A JP 2004-251801 A

  However, according to the inventors of the present invention, it is possible to immobilize a micro object that is performing a thermal molecular motion in a solvent on the support surface by exerting a fixing force that exceeds the molecular motion for a short time after contacting the support surface. It was not easy. For this reason, the light immobilization efficiency of the minute object in the liquid medium is low, and it is difficult to immobilize the minute object at a high density. In addition, the method of irradiating light after placing a micro object on the surface of a carrier with the liquid medium removed may cause unintended physical and / or chemical stress to the micro object.

  Accordingly, an object of the present invention is to efficiently immobilize a minute object on the surface of a carrier. Another object of the present invention is to immobilize minute objects on a carrier surface at high density. Furthermore, another object of the present invention is to immobilize a minute object on the surface of a carrier under mild conditions.

  The inventors of the present invention have focused on a liquid medium for light-immobilizing a micro object contained in a liquid medium on the surface of a carrier, and variously studied the liquid medium. A medium composition that suppresses dissolution of the micro object in the liquid medium. It has been found that the use of can efficiently immobilize a micro object on the surface of the carrier. Further, it has been found that by adopting such a medium composition, it is possible to easily immobilize a minute object on the surface of a carrier with high density and to avoid or suppress stress applied to the minute object. That is, according to the present invention, the following means are provided.

According to one aspect of the present invention, a micro object-containing liquid containing the micro object is formed on the surface of a carrier having at least a part of the surface of a light immobilizing material that exhibits the ability to immobilize the micro object upon light irradiation. A light immobilization step of immobilizing the minute object on the surface of the carrier by irradiating light in a state where dissolution of the minute object-containing medium in the liquid medium is suppressed in the presence of a body. A light immobilization method is provided.

  In the light immobilization step, the liquid containing a micro object includes a component other than the micro object and a solubility suppressing component that suppresses the solubility of the micro object, and / or by changing a thermodynamic parameter. It is preferable to suppress the solubility of the minute object in the minute object-containing liquid.

  The solubility-suppressing component can be selected from components that change one or more properties selected from the pH, ionic strength, dielectric constant, and excluded volume effect of the fine object-containing liquid. Further, the solubility-suppressing component can be selected from one or more components that denature the minute object to such an extent that the minute object is not solid-phased. Furthermore, the solubility-suppressing component can be a salting-out agent that is contained to such an extent that the fine objects do not precipitate. As such a salting-out agent, the salting-out agent is one or more selected from sodium sulfate, potassium sulfate, ammonium sulfate, sodium hydrogen phosphate, sodium chloride, lithium chloride, ammonium acetate and potassium phosphate. be able to.

  The light immobilization step may be a step of changing the thermodynamic parameter by one or more operations selected from stirring, cooling, heating, pressurization, decompression, and light irradiation. . The light immobilization step may be a step of adjusting the pH of the minute object-containing liquid to the vicinity of the isoelectric point of the minute object.

  In such a light immobilization method, the minute object can be an atom, a molecule, and a particle having an arbitrary shape in which these are assembled. The minute object can be a biomolecule such as protein or nucleic acid, and is preferably a protein.

  In such a light immobilization method, it is preferable that the minute object-containing liquid contains the minute object as a solute. The minute object-containing liquid may be a solution having a saturation concentration, a saturation concentration vicinity, or a supersaturation concentration of the minute object.

  Furthermore, in any of these light immobilization methods, the fine object-containing liquid material contains two or more kinds of fine objects, and the light immobilization step has lower solubility in the liquid medium. It can be a step of selectively immobilizing a minute object on the surface of the carrier. In the light immobilization step, the micro object-containing liquid material having different solubility of the micro object in the liquid medium is present in each of the different regions of the carrier surface, and the micro object is different in each of the different regions. It can also be set as the process of fixing by density. Furthermore, the concentration of the minute objects in the two or more kinds of minute object-containing liquids present in the different regions can be made substantially the same.

  In any of these light immobilization methods, a material containing a dye structure having an azo group can be used. In addition, the minute object is an inorganic substance including at least a metal, a metal oxide, a semiconductor, ceramics, glass, an organic substance including at least a plastic and an organic compound having physiological activity, and at least a peptide, a protein, a nucleic acid, a saccharide, a lipid, and a bone. A biomolecule containing a forming substance and two or more aggregates selected from them, a biological material containing at least various biological cells and parts thereof, a tissue and a biological body, the inorganic substance, the organic substance, the biomolecule and It can be set as the composite material etc. which compounded 2 or more types selected from the said biological material.

  According to another aspect of the present invention, there is provided a method for manufacturing a micro object carrier comprising the steps described in any of these light immobilization methods. Furthermore, according to another embodiment of the present invention, there is provided a minute object carrier, which is obtained by a method for manufacturing the minute object carrier.

  According to another aspect of the present invention, there is provided a kit for light immobilization of a minute object, wherein the light immobilization material that exhibits the ability to immobilize a minute object upon light irradiation is at least part of the surface. And a carrier other than the minute object, which is contained in the liquid medium of the minute object-containing liquid, reduces the solubility of the minute object compared to the case where the component is not contained. A kit comprising a solubility-suppressing component or a liquid medium containing the component is provided.

The present invention relates to a light immobilization method for a minute object, a method for manufacturing a minute object carrier, a minute object carrier, a liquid medium for light immobilization of a minute object, and a kit.
In the present invention, the light immobilization is carried out by providing a micro object-containing liquid material containing the micro object on the surface of a carrier having at least a part of the surface of a light immobilization material that exhibits the ability to immobilize the micro object upon light irradiation. In the presence, light irradiation is performed in a state where dissolution of the minute object-containing liquid in the liquid medium is suppressed, and the minute object is fixed to the surface.

  According to such light immobilization in the present invention, the light immobilization is efficiently performed on the surface of the carrier by suppressing the solubility of the minute object in the liquid containing the minute object. Further, according to such light immobilization, it is possible to light immobilize minute objects on the surface of the carrier with high density. Furthermore, since the minute object can be light-immobilized on the surface of the carrier via the liquid medium, the stress applied to the minute object can be avoided or suppressed. Furthermore, according to such light immobilization, the target micro object is selectively fixed to the carrier surface by utilizing the difference in solubility (difference in solubility) of various micro objects contained in the liquid containing the micro object. can do.

  Further, according to the light immobilization in the present invention, even when a minute object is contained as a solute in the minute object-containing liquid, the light is efficiently immobilized on the surface of the carrier. Since the minute object is contained as a solute in the minute object-containing liquid, it can be light-immobilized uniformly with respect to the light-irradiated part on the surface of the carrier, and the concentration and supply part can be easily controlled. In addition, since the minute object is fixed in a solute state, the three-dimensional structure of the minute object can be controlled and light can be fixed.

  In addition, according to the light immobilization in the present invention, by changing the degree of dissolution in the liquid medium, that is, by changing the composition of the liquid medium, the fine objects are light-immobilized on the carrier surface at different densities or different amounts. Can be For this reason, the minute object can be fixed on the surface of the carrier in a wide range of density or amount, and the minute object of any concentration and amount can be easily fixed on the surface of the carrier.

Hereinafter, various embodiments of the present invention will be described in detail.
(Light immobilization method)
The light immobilization method of the present invention is a method for immobilizing a micro object on a predetermined carrier surface, and is a method for immobilizing a micro object on a surface having at least a portion of a light immobilization material that exhibits the ability to immobilize a micro object upon light irradiation. The surface is provided with a light immobilization step of immobilizing the minute object on the surface by irradiating light in the presence of the minute object-containing liquid material containing the minute object. A conceptual diagram showing an example of light immobilization of the present invention is shown in FIG.

  In the light immobilization step of the light immobilization method of the present invention, the micro object 2 is immobilized on the surface 6 of the carrier 4 by irradiating the carrier 4 with light in the presence of the micro object liquid material 10 containing the micro object 2. To do. Hereinafter, for convenience of explanation, the micro object-containing liquid 10 and the carrier 4 will be described, and then the light immobilization process will be described.

(Micro object)
The micro object 2 that is the subject of the present invention includes atoms, molecules, and particles of any shape in which these are assembled. As long as the minute object 2 is tangible, it does not ask the existence form. The size of the micro object (which means the longest size of the micro object 2) is not particularly limited, but preferably does not exceed the millimeter size, more preferably 100 μm or less, and even more preferably 50 μm or less. More preferably, it is 10 μm or less. Further, the size of the minute object may be nano-size, but is preferably 1 nm or more.

  Further, the minute object 2 does not necessarily have to have a fixed shape, and the minute object 2 may be deformable. Although it does not specifically limit as a material contained in a micro object, or comprises a micro object, For example, 1 type, or 2 or more types of materials chosen from organic substance, an inorganic substance, a metal, and 2 or more types of composites among these are included. Can be mentioned. In the present invention, the micro object 2 containing an organic substance can be preferably used, and a biomolecule can be preferably used among the organic substances.

  Here, examples of the biomolecules include polypeptides, proteins, nucleic acids, saccharides, lipids, bone-forming materials, and two or more aggregates selected from these. These biomolecules include molecules constituting the living body, modified forms thereof, and multimers thereof. The biomolecules are not limited to those collected from the living body, and may be those collected from the living body and subjected to artificial modification or artificially synthesized. These biomolecules may be bound with a label containing a dye such as a fluorescent dye. Biomolecules include peptide nucleic acids.

  Protein aggregates include both active and inactive proteins, as well as aggregates having a plurality of proteins aggregated in various binding modes, such as covalent bonds, as a plurality of units. Examples of proteins and aggregates thereof include various proteins, enzymes, antigens, antibodies, and cell membrane receptors. According to the light immobilization of the present invention, the protein as the solute in the liquid medium is immobilized on the carrier surface 6, so that the protein is immobilized on the carrier surface 6 in a form that exists in the liquid. Therefore, it is possible to maintain and fix the three-dimensional structure in the liquid in both the active form and the modified form.

  Nucleic acids include artificial nucleic acids in addition to DNA and RNA having a single strand, two or more strands, or a portion of a single strand. Examples of the nucleic acid include genomic DNA and its microsatellite portion or a fragment containing at least a part of any gene, cDNA and its fragment, mRNA and its fragment, and DNA containing a mutation site such as a single nucleotide polymorphism site (cDNA And a DNA fragment containing a restriction enzyme site. The nucleic acid may be an oligonucleotide or a polynucleotide.

  Examples of the saccharide include monosaccharides, oligosaccharides, and polysaccharides. Sugars include sugar chains that are bound to proteins and lipids in vivo and some of them. Examples of the lipid include a lipid chain in lipoprotein, lipopolysaccharide or the like or a part thereof. Examples of the bone forming material include inorganic compounds such as various phosphates of calcium.

  The inorganic material that can be used for the micro object 2 in the present invention includes at least metals, metal oxides, semiconductors, ceramics, and glass, and the organic materials include at least plastics and organic compounds having physiological activity. . In addition, a living thing itself can be used for the minute object 2, and examples of the living thing include at least various kinds of living cells and parts thereof, tissues, and living organisms. Furthermore, examples of the composite material include composite materials in which two or more kinds selected from the inorganic substance, the organic substance (including the biomolecule), and the organism are combined. As these various materials, those already described in JP-A Nos. 2003-329682, 2004-93996, and 2004-251801 can also be used in the present invention.

(Optical immobilization process)
In the light immobilization step, at least in the light irradiation, the liquid body 10 containing the minute object 2 is present on the surface 6 of the carrier 4. In the minute object-containing liquid body 10, the liquid medium 12 of the minute object 2 is present. A state in which the dissolution in is suppressed. As used herein, “dissolution” includes dissolution in any form including substitutional dissolution and interstitial dissolution. In addition, the “state in which dissolution of the minute object 2 in the liquid medium 12 is suppressed” includes at least one of the aspects selected from the following aspects (1) to (4).
(1) Solvation between the micro object 2 and the liquid medium 12 is suppressed.
(2) Repulsive force acting between the minute objects 2 in the liquid medium 12 is suppressed.
(3) Aggregation between the minute objects 2 in the liquid medium 12 is promoted.
(4) The minute object 2 is destabilized in the liquid medium 12.

(Liquid material containing minute objects)
The minute object-containing liquid (hereinafter also simply referred to as a liquid) 10 may contain the minute object 2 as a solid phase, but preferably contains as a solute. More preferably, the liquid body 10 contains the micro object 2 only as a solute. By containing the minute object 2 as a solute, the minute object 2 is evenly distributed in the liquid medium 12, so that the minute object 2 is fixed to the surface 6 of the carrier 4 uniformly and easily. The liquid 10 may contain the micro object 2 as a colloidal particle instead of a solute. This is because colloidal particles are also uniformly dispersed in the liquid medium 12.

(Liquid medium)
In the present specification, the liquid medium 12 that holds the minute object 2 as solute particles or the like may be any medium that can appropriately hold the minute object 2. From the viewpoint of operation and when the minute object 2 is a biomolecule, a biological material, or the like, as the liquid medium 12, a medium mainly composed of water or only water can be used.

(Ingredients that suppress the solubility of minute objects)
The state where the dissolution of the minute object 2 is suppressed includes a case where the liquid medium 10 contains a component that suppresses the solubility of the minute object. The component that suppresses the solubility of the minute object (hereinafter referred to as “solubility suppressing component”) is a component other than the minute object 2 contained in the liquid medium 10, and the component is contained in the liquid medium 12 by being contained in the liquid medium 12. It is a component that lowers the solubility of the minute object 2 in the liquid medium 12 as compared with the case where it is not contained. In addition, even if it is the liquid body 10 containing such a solubility suppression component, it is preferable that the micro object 2 is not solid-phased (including precipitation, aggregation, etc.).

  Although it varies depending on the type of the minute object 2, generally, parameters that affect the solubility of the substance include pH, ionic strength, excluded volume effect, and dielectric constant of the liquid medium 12 surrounding the substance. Therefore, the solubility suppressing component includes a component that changes these parameters in the liquid medium 12 in the direction of decreasing the solubility of the minute object 2.

  Examples of the component that changes the pH include various acids such as organic acids and inorganic acids and salts thereof, organic and inorganic alkalis, and salts thereof. Such components include salts that impart buffering properties. For example, when the micro object 2 contains an amino acid polymer such as protein, the solubility of the micro object 2 can be lowered by setting the pH of the liquid medium 12 to be close to the isoelectric point.

  In addition to various compounds such as various electrolytes, chelating agents and the like are also included as components that change the ionic strength. By adding salt to the liquid medium 12 to increase the ionic strength, the solubility of the micro object 2 can be lowered by the salting out action. Further, when the micro object 2 forms colloidal particles in the liquid medium 12, the micro object 2 is hydrated by addition of an electrolyte having a charge that counteracts the charge of the colloidal particles (coagulation action) or by the micro object 2. The solubility of the micro object 2 can be reduced by taking away water molecules (salting out action).

  Examples of the component that changes the dielectric constant of the liquid medium 12 include a solvent such as an organic solvent having a dielectric constant different from that of the solvent molecules constituting the liquid medium 12, and an organic compound such as a polymer. Examples of components that change the excluded volume effect include uncharged polymers.

  Such a solubility-suppressing component can be appropriately selected according to the type of the minute object 2, the type of the liquid medium 12, and the like. For example, when the micro object 2 is hydrophilic and the liquid medium 12 is aqueous, the solubility-suppressing component has a lower dielectric constant than water such as water-soluble acids and alkalis, various salts, acetone, ethanol, DMSO, and the like. An organic compound such as an organic solvent or a polymer such as uncharged or suppressed charge of polyethylene glycol can be used. Conversely, when the micro object 2 is hydrophobic and the liquid medium 12 is an organic solvent, an acid or alkali that is soluble in the organic solvent, various salts, an organic solvent having a higher dielectric constant than the organic solvent. And organic compounds such as charged polymers. Further, when the minute object 2 has a hydrophobic part and a hydrophilic part, it is appropriately selected according to the liquid medium 12.

When the minute object 2 contains a polypeptide chain such as a protein, a salting-out agent, a coagulant, a precipitating agent or the like generally used for protein aggregation or precipitation is used as such a solubility-suppressing component. it can. As the salting-out agent, a salt containing ions known as Hofmeister permutation can be used. Such permutation, the ^{_{anion, PO 4 3-> SO 4 2-}} > CH 3 COO -, Cl -> Br -> NO 3 -> ClO 4 -> I -> SCN -, also the cations ( Various ions of CH ₃ ) ₄ N ⁺ > NH ₄ ⁺ > Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ > Li ⁺ > Mg ²⁺ > Ca ²⁺ > Ba ²⁺ are included in this order. Specific examples of such salts include ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), sodium sulfate (Na ₂ SO ₄ ), potassium sulfate (K ₂ SO ₄ ), sodium hydrogen phosphate (Na ₂ HPO ₄ , NaH ₂ PO ₄ ), sodium chloride (NaCl), lithium chloride (LiCl), ammonium acetate (CH ₃ COONH ₄ ), potassium phosphate (K ₃ PO ₄ ), and the like. Of these, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) and sodium sulfate (Na ₂ SO ₄ ) can be preferably used. Examples of the salting-out agent include charged polymers such as polystyrene and polyacrylic acid.

  Examples of the precipitant include organic solvents such as acetone, ethanol, and DMSO, and uncharged polymers such as polyethylene glycol. Such a salting-out agent and a precipitating agent are preferably contained within a range in which the micro object 2 is not solidified by aggregation or precipitation.

  Moreover, when the micro object 2 contains a polypeptide chain such as a protein, a protein denaturant can generally be used as such a solubility-suppressing component. Examples of protein denaturing agents include urea, guanidine hydrochloride, guanidine thiocyanate, DTT, 2-mercaptoethanol, surfactants such as sodium lauryl sulfate, and organic solvents such as trichloroacetic acid, phenol, and methanol. For example, it is preferable to use denaturing agents such as urea, guanidine hydrochloride and guanidine thiocyanate. These denaturants are preferably contained in a range in which the micro object 2 is not solid-phased by aggregation or precipitation, or in a range in which the function of the micro object is not impaired.

  Since the solubility suppressing component is contained in the liquid medium 12, the solubility (saturation concentration) of the micro object 2 with respect to the liquid medium 12 is decreased, and as a result, the concentration of the micro object 2 can be at or near the saturation concentration. When the concentration of the minute object 2 is at or near the saturation concentration, the further dissolution of the minute object 2 in the liquid medium 12 is suppressed, so that the dissolution of the minute object 2 in the liquid medium 12 is suppressed. It can be said that Further, even when the minute object 2 is at the supersaturated concentration, it can be said that the dissolution of the minute object 2 is suppressed. The liquid material 10 in which the minute object 2 is at the saturation concentration, the vicinity thereof, and the supersaturated concentration is formed even when the minute object 2 is dissolved in the liquid medium 12 having a single composition that does not contain the solubility suppressing component. The Further, when the liquid 10 is a colloidal solution containing the minute object 2 as colloidal particles, it can be said that the dissolution of the minute object 2 is suppressed.

(Thermodynamic parameters that suppress the solubility of small objects)
Further, the state in which the dissolution of the minute object 2 in the light immobilization step of the present invention is suppressed may be formed by changing the thermodynamic parameter of the liquid 10. Such a change in thermodynamic parameters can be caused in the liquid medium 12 and / or the minute object 2 by any one or two or more kinds of operations selected from stirring, cooling, heating, pressurization, decompression, and light irradiation. . Among these operations, it is preferably selected from heating, pressurization and light irradiation, and more preferably selected from heating and pressurization. Which operation is effective or how much stirring, cooling, heating, pressurization, and light irradiation are effective in suppressing the solubility of the micro object 2, the types of the micro object 2 and the liquid medium 12, and the micro object 2 It is set appropriately according to the concentration.

  By these various operations, the thermodynamic parameters of the liquid body 10, the liquid medium 12, and / or the micro object 2 are changed, so that the bond or structure of the micro object 2 is partially damaged, and the solubility is increased. Will be reduced. These changes in thermodynamic parameters all denature organic substances such as proteins and reduce solubility. For example, when the micro object 2 has a peptide chain such as a protein, these thermodynamic parameters Due to the change, hydrogen bonds and the like in the peptide chain are cut and the solubility in the liquid medium 12 is lowered. When the micro object 2 has a peptide chain, the change of the thermodynamic parameter is preferably imparted by an operation selected from heating, pressurization, and light irradiation. Even if such a thermodynamic parameter is the changing liquid material 10, it is preferable that the micro object 2 is not solid-phased.

(Supplying and irradiating liquids containing minute objects)
The minute object-containing liquid material 10 in which the dissolution of the minute object 2 in the liquid medium 12 is suppressed may be prepared in advance before being supplied to the surface 6 of the carrier 4 as shown in FIG. Alternatively, the surface 6 may be prepared in situ. Examples of the case of in-situ preparation include a case in which the solution of the micro object 2 is supplied onto the surface 6 of the carrier 4 and only the solubility-suppressing component is separately supplied as shown in FIG.

  Further, as shown in FIG. 3, various operations for changing the thermodynamic parameters may be performed on the liquid material 10 before supplying the liquid material 10 to the surface 6 of the carrier 4. 4 may be performed after supplying the liquid material 10 to the surface 6 of 4. Preferably, such an operation is not performed simultaneously with the light irradiation, but is performed in advance prior to the light irradiation. In addition, the state of reduced solubility due to such an operation is maintained during light irradiation, but it may be recovered from a change in thermodynamic parameters due to such an operation. For example, the liquid 10 containing the micro object 2 having a peptide chain is heated in advance to denature the peptide chain to reduce the solubility of the micro object 2, and then the liquid 10 is kept at an appropriate temperature of room temperature or lower. Such a liquid material 10 may be supplied to the surface 6 of the carrier 4 and irradiated with light after cooling to the temperature, or after the liquid material 10 before heating is supplied to the surface 6 of the carrier 4, the liquid material 10 is heated. Thereafter, light irradiation may be performed after cooling.

  In order to supply the liquid 10 to the surface of the carrier 4, a known method that can be used to supply the liquid 10 to the surface of the object, such as dripping, spraying, dipping, spin coating, etc. is used. it can. In addition, in order to supply the liquid material 10 to the carrier surface 6 in a predetermined pattern, a liquid supply method used in various printing methods can be employed.

(Light irradiation)
The light irradiation method is not particularly limited, and any light such as various propagation light, near-field light, or evanescent light may be irradiated so as to reach the surface 6 of the carrier 4 or the vicinity thereof. In some cases, it is preferable that the liquid 10 is stably held on the surface 6 of the carrier 4 during light irradiation. In such a case, light irradiation may be performed in a state where the carrier 4 and the liquid material 10 are held in the same chamber.

  By irradiating light to the area of the surface 6 of the carrier 4 selectively, the minute object 2 can be fixed only in the light irradiation area. Examples of such a selective light irradiation method include a lithography method using a mask corresponding to a light irradiation pattern, and a drawing method using various laser beams. A distribution can be given not only to the irradiation region but also to the intensity of light irradiation as necessary. In addition, about the light irradiation in this invention, the irradiation light already described in Unexamined-Japanese-Patent No. 2003-329682, Unexamined-Japanese-Patent No. 2004-93996, and Unexamined-Japanese-Patent No. 2004-251801 can be employ | adopted. .

(Selective light immobilization of minute objects)
Just by changing the solubility of a minute object by varying the type and amount of the solubility-suppressing component and / or the type and change amount of the thermodynamic parameter to be changed for the liquid 10 containing two or more kinds of the minute object 2. Two or more kinds of minute objects 2 from the same liquid 10 can be selectively fixed to another or the same region of the surface 6 of the carrier 4.

  For example, in the liquid 10 containing two or more kinds of minute objects 2, the first minute object 2 is irradiated with light so as to impart a solubility-suppressing component and / or change a thermodynamic parameter so that the solubility of the first minute object 2 is the lowest. Then, the first micro object 2 is fixed to a predetermined region of the surface 6 of the carrier 4, and then a solubility suppressing component is applied so that the solubility of the second micro object 2 becomes the lowest and / or the thermodynamic parameter. It is possible to fix the second minute object 2 to a predetermined region by irradiating with light while changing. It should be noted that the application amount of the solubility-suppressing component and / or the change amount of the thermodynamic parameter may be changed sequentially or continuously.

(Control of immobilization amount of minute objects)
Further, even if the concentration of the minute object 10 is not particularly changed, the kind and concentration of the solubility-suppressing component and / or the kind and amount of change of the thermodynamic parameter are made different to support the minute object 2 with different density or amount. 4 can be fixed to different areas of the surface 6. For example, when using the liquid 10 having a salting-out agent as a solubility-suppressing component, the first region of the carrier surface 6 is irradiated with light at a first salting-out agent concentration to immobilize a minute object, and the second salting-out is performed. It is possible to immobilize the minute object by irradiating the other second region of the carrier surface 6 with the agent concentration. The concentration of the minute object 2 can be made substantially the same, but may be changed as appropriate according to the amount to be immobilized on the surface 6 of the carrier 4.

  In such selective immobilization of minute objects and control of the amount of immobilization of minute objects, the amount of change in solubility-inhibiting components and / or thermodynamic parameters may be changed in stages, but continuously You may let them. In addition, liquid bodies 10 having different solubility-inhibiting components and / or thermodynamic parameter variations are simultaneously present in different regions of the carrier surface 6 in advance, and collectively irradiated with different light to different regions. It is also possible to fix the same minute object 2 to different regions at different concentrations or amounts at once.

  As described above, according to the light immobilization method of the present invention, since the dissolution of the minute object 2 in the liquid medium 10 in the liquid medium 12 is limited, the light immobilization is efficiently performed on the carrier surface 6. Thus, the micro object 2 can be optically immobilized on the carrier surface 6 with high density. Furthermore, since the minute object can be optically immobilized on the surface of the carrier via the liquid medium 12, the stress applied to the minute object 2 can be avoided or suppressed. Furthermore, according to the light immobilization method of the present invention, when the micro object 2 is contained in the liquid 10 as a solute, it is efficiently light immobilized on the carrier surface 6 and the light irradiation of the carrier surface 6 is performed. Light fixation can be achieved by uniformly controlling the concentration and the supply site uniformly with respect to the site. Further, since the minute object 2 is fixed in a solute state, the three-dimensional structure of the minute object 2 and the like can be controlled to be light-immobilized. Therefore, the photo-immobilization method of the present invention is a tertiary structure in which useful structures such as biomolecules including nucleic acids such as proteins and DNA and biological materials such as cells are susceptible to damage depending on conventional immobilization methods such as precipitation and drying. The original structure is preferred for immobilization on useful materials.

  Furthermore, according to the light immobilization method of the present invention, the target micro object 2 is selectively transferred to the surface of the carrier by utilizing the solubility difference (solubility difference) of various micro objects 2 contained in the liquid 10. 6, and by changing the solubility of the minute object 2 in the liquid medium 12, it is possible to vary the amount of immobilization of the minute object 2 without greatly changing the concentration of the minute object 2. Therefore, even various types of minute objects 2 can be easily fixed to different regions of the carrier surface 6 with an arbitrary amount of fixation. Therefore, it becomes possible to easily manufacture the carrier 4 in which two or more kinds of the minute objects 2 are immobilized at different concentrations, and the degree of freedom and accuracy of the immobilization pattern and the immobilization concentration of the minute objects 2 on the carrier surface 6. And accuracy can be improved.

(Manufacturing method of micro object carrier)
In this way, the micro object 2 can be optically immobilized on the surface 6 of the carrier 4, but the carrier 4 carrying the micro object 2 thus obtained can be used as the micro object carrier 20 as it is, or as required. The liquid product 10 is removed from the surface 6 of the carrier 4, and the final product form or a form close to it is obtained by appropriately washing, drying, and supplying or enclosing an appropriate medium or combining two or more kinds. be able to. Further, the micro object 2 once fixed can be further modified.

(Micro object carrier)
Since the minute object carrier 20 obtained in this way has the minute object 2 fixed at a higher concentration than in the prior art, for example, a highly sensitive analysis chip or an analysis object in which many kinds of minute objects 2 are highly integrated. In addition, various reaction chips can be easily obtained. In addition, since the minute object 2 is fixed to the carrier surface 6 in the liquid medium 12, chemical and / or physical stress on the minute object 2 is avoided or suppressed, so that the minute object is formed on the carrier surface 6. The original function of 2 is effectively exhibited. Therefore, the micro object carrying body 20 of the present invention is preferable as the carrying body 20 carrying the micro object 2 containing biomolecules and biological materials such as biologically active peptides and proteins, and is preferably used as a protein function analysis chip. .

(Light immobilization kit for minute objects)
According to the present invention, a kit for light immobilization of a minute object is also provided. This kit for light immobilization is a combination of drugs used for light immobilization of the micro object 2, and can hold the micro object 2 to be optically immobilized as solute particles and / or colloid particles. One or two or more solubility-suppressing components used by adding to a liquid medium, and a carrier 4 having at least a part of the surface 6 with a light-immobilizing material that exhibits the ability to immobilize the micro object 2 upon light irradiation; It is equipped with. Such a solubility-suppressing component and the carrier 4 are as already described. The solubility-suppressing component may be preliminarily dissolved in an appropriate solvent according to the type of the micro object 2 to be light-immobilized and supplied as the liquid medium 12 and its stock solution (diluted and used as the liquid medium 12). According to such a kit for light immobilization, the solubility of the minute object 2 in the liquid medium 12 is lowered by any of the solubility-suppressing components included in the kit, and the object 2 is light-immobilized on the surface 6 of the carrier 4. It becomes possible. In order to prevent the liquid 10 on the carrier 4 from evaporating, the kit can include a container-like chamber or lid capable of sealing the liquid 10 on the carrier 4.

  As the carrier 4 used in the various embodiments of the present invention described above, any carrier can be used as long as it has a light-immobilizing material that expresses the ability to immobilize the minute object 2 at the time of light irradiation. The shape may be a flat shape such as a substrate, or may be various particle shapes such as a spherical shape, an indeterminate shape, a needle shape, a rod shape, and a flake shape. The carrier 4 may be a dense body or a porous body. Furthermore, the surface of the carrier 4 may be not only the outer surface but also the inner surface. Further, the entire surface of the carrier 6 or a part of it may have the light fixing material. When the light fixing material is provided on a part of the carrier surface 6, it may have a light fixing material region in a desired two-dimensional or three-dimensional design. The constituent material of the carrier 4 itself is not particularly limited, but is preferably a light transmissive material.

  The carrier 4 has a light fixing material on at least a part of its surface. The light immobilization material is not particularly limited as long as it is a material that exhibits the ability to immobilize the minute object 2 when irradiated with light, but may be any material that develops deformation (light deformation) induced by light.

  The light-fixing material is not particularly limited, but the matrix contains components (photoreactive components) that cause ablation, photochromism, photo-induced orientation of molecules, etc. when light reaches, resulting in the volume and density of the material. , A material in which a free volume or the like changes to generate optical deformation.

  Although the kind of photoreactive component is not limited, For example, the photoisomerization component and photopolymerizable component which are components which can raise | generate anisotropic photoreaction accompanying the shape change of material are mentioned. When the micro object 2 to be immobilized is a biological substance whose activity is impaired by a chemical reaction with the photofixation material, a photoisomerization component is preferable as the photoreactive component. As the photoisomerization component, for example, a component that causes trans-cis photoisomerization, particularly typically a dye structure having an azo group (—N═N—), in particular, a chemical structure of azobenzene or a derivative thereof. Ingredients can be preferably exemplified.

  The photoreactive component may be dispersed in the matrix, or may be provided as a monomer or as a side chain in the polymer material constituting the matrix. As the matrix material of the light fixing material, an organic material such as a normal polymer material or an inorganic material such as glass can be used. In consideration of the uniform dispersibility or binding property of the photoreactive component to the matrix, an organic material, particularly an organic polymer material is preferable.

  The type of the polymer material is not limited, but a polymer structural unit containing a urethane group, urea group, or amide group, and a ring structure such as a phenylene group in the polymer main chain. It is preferable in terms of heat resistance.

  In addition, as the light immobilization material, inorganic materials such as those generally called chalcogenite glass including a structure in which any of sulfur, selenium, and tellurium and any of germanium, arsenic, and antimony are combined are also used. it can.

In view of the above, the polymer materials containing the photoreactive component are preferably exemplified by the following “Chemical Formula 1” to “Chemical Formula 6” in addition to those described in the Examples.

  Other examples of the carrier 4 and the light immobilization material that can be used in the present invention include the carriers and the light immobilization materials described in JP-A Nos. 2003-329682, 2004-93996, and 2004-251801. Is mentioned. Further, in this specification, all matters described in Japanese Patent Application Laid-Open Nos. 2003-329682, 2004-93996, and 2004-251801 are incorporated by reference.

  Hereinafter, the present invention will be described with reference to specific examples. However, the present invention is not limited to the following examples.

In Examples, Cy5-labeled IgG (CHEMICON, International Inc., Anti-igG (Fc), Mouse, Goat-Polv. Cy5) as a micro object, PBS (0.15M NaCl, phosphate buffer, pH 7.4) as a liquid medium. ), Ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) as a salting-out agent was used as a solubility-suppressing component, the Cy5-labeled IgG concentration was 1 μg / ml, and the ammonium sulfate concentration was varied from 0 to 1.6 M in total 31 types A liquid containing a fine object was prepared. These fine substance-containing liquids were centrifuged at 6000 rpm, and 1 μl of the supernatant was placed on an azopolymer film of a slide glass coated with an azopolymer film (film thickness: about 40 nm) having the structure shown in the following formula (Formula 7). The slide glass was sealed in a reaction chamber so that the droplet did not dry, and then irradiated with light (maximum wavelength 470 nm, 20 mW / cm ² ) for 30 minutes. Thereafter, the slide glass was immersed in a phosphate buffer solution (PBS) containing 0.01% Tween 20, and stirred and washed for 5 minutes. After this washing operation was repeated three times, the fluorescence on the slide glass was measured with a commercially available array scanner. The results are shown in FIG.

  As shown in FIG. 4, the fluorescence intensity increased in proportion to the ammonium sulfate concentration until the ammonium sulfate concentration reached 1.2M, and decreased when the concentration exceeded 1.2M. The fluorescence intensity was 318 when the ammonium sulfate concentration was 0M, whereas it was 6848 at 1.2M. This means that the amount of Cy5-labeled IgG on the slide glass is 20 times the amount. It was thought that the fluorescence intensity decreased at an ammonium sulfate concentration of 1.2 M or more was that aggregated Cy5-labeled IgG was removed from the supernatant by centrifugation.

  From the above, it has been found that according to the present invention, by irradiating light in a state where the dissolution of the minute object in the liquid medium is suppressed, the minute object can be optically immobilized on the surface of the carrier with different immobilization densities. It was also found that the smaller the degree of dissolution suppression, in other words, the lower the solubility, the more efficiently and densely fix the micro object on the carrier surface.

In this example, 1 μg / ml of Cy5-labeled IgG (CHEMICON, International Inc., Anti-igG (Fc), Mouse, Goat-Polv. Cy5) as a micro object, water as a liquid medium, and 0.1 M as a solubility suppressing component Using sodium citrate (pH 5.0 to 5.8) or 0.1 M cacodylic acid (pH 6.0 to 6.8), a total of 10 kinds of fine substance-containing liquids were prepared. These 10 kinds of fine object-containing liquids are dropped on an azo polymer film having a structure represented by the following formula (Chemical Formula 7), and the slide glass is sealed in a reaction chamber so that the liquid droplets are not dried. Light irradiation (maximum wavelength: 470 nm, 20 mW / cm ² ) was performed for minutes. Thereafter, the slide glass was immersed in a phosphate buffer solution (PBS) containing 0.01% Tween 20, and stirred and washed for 5 minutes. After this washing operation was repeated three times, the fluorescence on the slide glass was measured with a commercially available array scanner. As a control, 1 μg / ml Cy5-labeled IgG and PBS (pH 7.4) as a liquid medium were used, but a control liquid without a solubility-suppressing component was prepared, and this was similarly irradiated with light and washed. And fluorescence was measured. These results are shown in Table 1.

  As shown in Table 1, in the range of pH 5.0 to pH 6.8, the immobilization efficiency of Cy5-labeled IgG was higher than that of the control liquid using PBS (7.4) alone.

  From the above, according to the present invention, the dissolution of minute objects in the liquid medium is suppressed by increasing the ionic strength and adjusting the pH of the liquid medium near the isoelectric point of the protein, and light irradiation is performed under these conditions. As a result, it was found that the micro object can be fixed to the support surface efficiently and with high density.

The conceptual diagram which shows an example of light fixation of a micro object. Process drawing which shows an example of the optical fixation process of a micro object. Process drawing which shows another example of the optical fixation process of a micro object. The figure which shows the relationship between the ammonium sulfate density | concentration in Cy5-labeled IgG solution, and the fluorescence intensity on a slide glass.

Explanation of symbols

  2 micro object, 4 carrier, 6 carrier surface, 10 micro object-containing liquid, 12 liquid medium, 20 micro object carrier.

Claims (16)

In the presence of the liquid containing the micro object on the surface of the carrier having at least a part of the surface of the optical immobilization material that expresses the immobilization ability of the micro object when irradiated with light, the micro object includes the liquid object. A light immobilization method of a minute object, comprising: a light immobilization step of immobilizing the minute object on the surface of the carrier by irradiating light in a state where dissolution of the minute object-containing medium in the liquid medium is suppressed.   In the light immobilization step, the liquid containing a micro object includes a component other than the micro object and a solubility suppressing component that suppresses the solubility of the micro object, and / or by changing a thermodynamic parameter. The light immobilization method according to claim 1, wherein the solubility of the minute object in the minute object-containing liquid is suppressed.   The said solubility suppression component is selected from the component which changes the 1 type (s) or 2 or more types of property selected from pH, ionic strength, a dielectric constant, and the excluded volume effect of the said fine body containing liquid. Light immobilization method.   The light immobilization method according to claim 2 or 3, wherein the solubility-suppressing component is selected from one or more components that denature the minute object so that the minute object is not solid-phased.   The light immobilization method according to any one of claims 2 to 4, wherein the solubility-suppressing component is a salting-out agent contained to such an extent that the fine objects do not precipitate.   The said salting-out agent is 1 type (s) or 2 or more types selected from sodium sulfate, potassium sulfate, ammonium sulfate, sodium hydrogenphosphate, sodium chloride, lithium chloride, ammonium acetate, and potassium phosphate. Light immobilization method.   The photofixation step is a step of changing the thermodynamic parameter by one or more operations selected from stirring, cooling, heating, pressurization, reduced pressure, and light irradiation. 7. The light immobilization method according to any one of 6 above.   The light immobilization method according to any one of claims 1 to 7, wherein the light immobilization step is a step of adjusting the pH of the fine object-containing liquid material in the vicinity of an isoelectric point of the fine object.   The optical immobilization method according to claim 1, wherein the micro object is an atom, a molecule, or a particle having an arbitrary shape in which these are aggregated.   The optical immobilization method according to claim 1, wherein the micro object is a biomolecule such as a protein or a nucleic acid.   The optical immobilization method according to claim 10, wherein the micro object is a protein. The minute object-containing liquid contains two or more kinds of minute objects,
The light immobilization method according to any one of claims 1 to 11, wherein the light immobilization step is a step of selectively immobilizing a micro object having a lower solubility in the liquid medium on the surface of the carrier.   In the light immobilization step, a micro object-containing liquid material having different solubility in the liquid medium of the micro object is present in each of different regions on the surface of the carrier, and the micro object is provided in each of the different regions with different densities. The light immobilization method according to claim 1, which is a step of immobilization.   The optical immobilization method according to claim 13, wherein the concentration of the minute objects in the two or more kinds of minute object-containing liquids present in the different regions is substantially the same. Each step according to claim 1 is provided.
A manufacturing method of a micro object carrier. A kit for light immobilization of minute objects,
A carrier having at least a part of a surface with a light-immobilizing material that expresses the ability to immobilize a micro object upon light irradiation;
A solubility-suppressing component that is a component other than the micro-object and suppresses the solubility of the micro-object in the micro-object-containing liquid, or a liquid medium containing the solubility-suppressing component;
A kit comprising:

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