JP2010032508A - Chip manufacturing method, chip, detection method and device using chip - Google Patents

Abstract

[PROBLEMS] To immobilize a ligand on a plurality of spots including a gel while suppressing the amount used.
A gel is immobilized on a substrate to form a spot, and then a solution containing a ligand is spotted on the spot to immobilize the ligand on the spot.
[Selection] Figure 4

Description

  The present invention relates to a chip manufacturing method and a chip manufactured thereby. In particular, the present invention relates to a chip manufactured by spotting a ligand solution on a substrate on which a fine hydrogel spot is formed, a manufacturing method thereof, a detection method and a detection apparatus using the chip.

For example, functional organic molecules that have the ability to interact with a target substance are immobilized on the surface of a solid substrate for the purpose of measuring the concentration of the target substance in a specimen or for applications such as biosensors. A chip (immobilized body) has been reported (Patent Document 1).
In addition, as a functional organic molecule also referred to as “ligand”, for example, many examples using proteins have been reported. As a use of a chip on which a protein is immobilized in this manner, for example, detection of a target substance using an interaction between an antigen and an antibody, a protein and a low molecular compound, a saccharide and a lectin, etc. has been reported. An example using saccharides as a ligand has also been reported. For example, detection of viruses, bacteria, toxins, and the like has been reported as a use of chips in which saccharides are immobilized in this way.

  By the way, when immobilizing a ligand on a substrate and analyzing the interaction between the ligand and the substance to be detected, one important thing is to increase the density of the ligand that can interact with the substance to be detected and enhance the signal. To do. In general, hydrogels are often used to achieve high density of ligands. This is because the hydrogel has a fine structure and thus has a large specific surface area and can immobilize many ligands. In addition, the hydrogel is preferably used because the substance to be detected can move to the inside.

  When ligands are immobilized on a substrate and the interaction between the ligand and the substance to be detected is analyzed, many types of ligands are immobilized on one substrate, and the substance to be detected and multiple types of ligands are It is also preferable to measure these interactions simultaneously, and it is also preferable that the uniformity of the immobilized ligand is high.

JP 2007-101520 A

  One method of immobilizing ligands using hydrogels is to form a predetermined number of fine spots made of hydrogel on the substrate surface and then immobilize the ligands on each spot. . In this case, conventionally, in the step of immobilizing the ligand, the solution in which the ligand is dissolved is dropped on the substrate with a large droplet using a pipette or the like, or the substrate is immersed in it, and uniformly distributed in a plurality of spots. Ligand was immobilized. However, this tends to increase the amount of ligand to be used, which is disadvantageous economically, which may hinder research and development.

  In addition, when a plurality of hydrogel spots are formed on the substrate, it may be desirable to immobilize different ligands on each spot. As a result, it is possible to immobilize more types of ligands on the substrate (that is, the degree of integration can be improved), and it is possible to analyze more types of interactions using a single chip. Because. In this case, for the purpose of measuring a large number of interactions at the same time, it is preferable to increase the degree of integration. For this purpose, it is desirable to miniaturize the spot containing the ligand. Among these, the integration of these is particularly preferable in a chip used for inspection or diagnosis. However, in the method of immersing the substrate in the solution in which the ligand is dissolved as described above, it is difficult to produce a highly integrated chip.

  In addition, after creating a plurality of hydrogel spots on a substrate, a sheet with a plurality of grooves is pasted so that the spots are positioned in the flow path created by the substrate and the grooves, and include different ligands. There is also known a technique for immobilizing different ligands for each spot by flowing a liquid through each channel. However, this method is not a preferable method because it is necessary to prepare the sheet and the process becomes complicated. Further, it is necessary to prepare a space for the flow path on the substrate. In other words, it was difficult to increase the degree of integration and to increase the types of ligands immobilized on the substrate at the same time.

  The present invention was devised in view of the above problems, and its purpose is to manufacture a chip capable of immobilizing a ligand on a plurality of spots configured to contain a gel while suppressing the amount used, and the manufacture It is to provide a chip manufactured by the method, and a detection method and a detection apparatus using the chip.

  As a result of intensive studies in view of the above problems, the present inventors have significantly increased the amount of ligand used by spotting a solution containing the ligand on a gel spot previously formed on the substrate (that is, by overstrike). In addition to the reduction, the present inventors have found that it is possible to immobilize different ligands on a plurality of gel spots to increase the degree of spot accumulation, thereby completing the present invention. The obtained chip has sufficient sensitivity even when it is used as a sensor. In addition, a ligand is not usually present in a portion where no spot is formed, so that a high S / N ratio (Signal to Noise ratio) can be obtained. Furthermore, spotting the solution so as to overlap the spot of the gel in this way to immobilize the ligand, and immobilizing a different ligand to each spot of the gel is easy to operate, It is also preferable from the viewpoint of the process.

  Further investigations by the present inventors have revealed that, in the chip manufacturing method of the present invention, the expansion and contraction of the substrate greatly affects the quality of the product because of the micro-scale chip manufacturing process. That is, because of the very small size, the difference between the spot position when the gel is immobilized and the spot is formed and the spotting position of the ligand solution (position where the spotted ligand solution droplet adheres) It was found that the impact was very large. For example, even if the deviation is the same 100 μm, the ratio of the overlapping portion between the spot of the gel and the spotting position of the ligand solution and the portion not overlapping is greatly different between the case where the spot size is 3 mm and the case where the spot size is 300 μm.

  This deviation is presumed to be due to expansion and contraction caused by the substrate depending on the material of the substrate; the solvent used; the conditions such as temperature and humidity; or the combination and order thereof in the chip manufacturing process. In the chip manufacturing method of the present invention, since the scale is small, the deviation between the position of the gel spot and the position where the ligand solution is spotted due to the amount of expansion and contraction is largely reflected, and the sensitivity of the manufactured chip is reduced. Variations between spots will occur, which in turn affects the quality of the product.

  In particular, as a material for the substrate, a cheaper plastic is often used compared to glass or the like because of economical efficiency and ease of handling. However, on the other hand, plastics easily swell and are easily affected by external forces. For example, when an organic solvent such as ethanol is used as a solvent during the surface treatment of the substrate, the influence of swelling is particularly significant. Although the amount of expansion and contraction of the substrate due to swelling and external force can be predicted to some extent, it cannot be completely controlled. Therefore, the degree of deviation between the spot position of the gel on the substrate and the spotting position of the overlaid ligand solution cannot be determined strictly, and is particularly a problem on a microscale.

  Further, the size of the gel spot may vary due to variations in humidity, surface hydrophilicity, and the like. In this case, the ligand is fixed to the entire spot in the spot that is smaller than the design, but if the spot is larger than the design, the size of the gel spot is within the range that can spot the ligand solution. The ligand may be immobilized only on a part of the spot. In addition, when the ligand solution is changed in various ways, the size of the spot where the ligand solution can be spotted changes according to its liquidity, so if the range becomes smaller than expected, it will be part of the gel spot. However, the ligand may not be immobilized.

  Moreover, another problem resulting from the microscale has also been clarified by the study of the present inventors. At small scales, the amount of ligand solution spotted is small and the volatilization of the solvent is fast, so that the solution can easily dry faster than it penetrates the entire gel, and the ligand is only immobilized on a portion of the gel. Can happen. Some gels have properties that take time to absorb the solution. Furthermore, if the spot is misaligned as described above, the ligand solution does not soak into the part where it has not been dripped, and only a part of the ligand contained in the spotted solution is immobilized on the gel, and the data Cause the variation of. Such a problem is not conventionally known, and has been found only after the present inventors have examined the present invention.

  The inventors of the present invention have also intensively studied these new problems. As a result, (1) a wider range than the spot of the gel can be obtained for the positional deviation between the spot of the gel and the spotting position of the solution containing the ligand. Either spot the ligand solution to cover the gel spot with a spotter that can be spotted at one time, or (2) spot the ligand solution multiple times according to a pre-planned procedure. It was found that a ligand can be immobilized on At this time, it was also found that when spotting the ligand solution a plurality of times, spotting is preferably performed while shifting the position.

  With such a configuration, the ligand can be immobilized on the entire small spot of the gel, and further, the difference between each spot is reduced, and the stability of data when performing analysis or the like using the chip is improved. It becomes possible. In addition, as a method of resolving the above-mentioned spot displacement, it is also possible to perform spot recognition accurately by adjusting the spotting position of the ligand solution for each spot by performing image recognition using a computer or the like. It is thought that. However, in such a configuration, the apparatus becomes complicated and there is a problem from the viewpoint of economic rationality. According to the present invention, such a problem can also be avoided.

  That is, the gist of the present invention is a method for manufacturing a chip comprising a substrate and a plurality of spots comprising gel immobilized on the substrate, and the spots are formed by immobilizing the gel on the substrate. A chip manufacturing method comprising: forming a gel spotting step; and spotting a solution containing a ligand on the spot to immobilize the ligand on the spot (claim). 1).

At this time, in the ligand spotting step, it is preferable to fix the ligand to the spot by spotting the spot so as to cover the spot with a spotter capable of spotting a range wider than the spot at a time (claims) Item 2).
Further, in the ligand spotting step, it is also preferable to fix the ligand to the spot by shifting the spotter position and spotting a plurality of times (Claim 3). Furthermore, in the ligand spotting step, it is preferable that the solution containing the ligand is dried before penetrating the entire gel (claim 4).

  The gel is preferably formed by including a matrix having a main chain composed of a biological material and a compound capable of binding to the biological material (Claim 5). The gel is composed of a biological material that can bind to the ligand and a particulate wrinkle having a particle size of 1 μm or less, which is formed by binding a compound that can bind to the biological material. Even if it exists, it is preferable (Claim 6). At this time, it is more preferable that a space is formed between the particulate lumps (Claim 7). Moreover, it is preferable that the ratio of the weight of the biological material to the weight of the gel is 0.1 or more (claim 8).

  In the chip manufacturing method of the present invention, it is preferable that the thickness of the spot is 5 nm or more in a dry state. In addition, at least one of the compounds preferably has two or more functional groups capable of binding to the biological substance (Claim 10), preferably has no charge (Claim 11), and is miscible with water. In addition, those that are miscible with at least one organic solvent are preferred (Claim 12), those having a molecular weight of 1000 or more are preferred (Claim 13), and the diameter of the compound when mixed in a liquid is What is 1 nm or more is preferable (claim 14).

  Furthermore, the biological material is preferably a biomolecule (Claim 15), and the biomolecule is preferably a protein (Claim 16). The ligand is at least one selected from the group consisting of metal chelates, vitamins, saccharides, glutathione, boronic acids, proteins, antigens, nucleic acids, bioactive substances, lipids, hormones, environmental hormones, and chelate-forming groups. (Claim 17).

  Further, in the chip manufacturing method of the present invention, after the gel spotting step and before the ligand spotting step, a solution containing a linker capable of binding to the gel and the ligand is brought into contact with the spot, and the linker is used. It is preferable to perform a linker spotting step for immobilizing the spots (claim 18). In the chip manufacturing method of the present invention, it is also preferable to perform a linker mixing step of mixing the gel and a linker capable of binding to the ligand and a solution containing the ligand before the ligand spotting step (claim). Item 19). In this case, the linker is preferably a hydrophilic molecule (Claim 20), and more preferably, the hydrophilic molecule contains an ethylene oxide chain (Claim 21).

  Still further, after the gel spotting step and the ligand spotting step, a solution containing a compound having a functional group capable of binding to an active site of the biological substance in the gel is brought into contact with the spot to inactivate the gel. It is preferable to have a step of converting (claim 22).

  Another subject matter of the present invention resides in a chip manufactured by the manufacturing method of the present invention (claim 23). Still another subject matter of the present invention comprises a substrate and a plurality of spots comprising a gel immobilized on the substrate, wherein a ligand is immobilized on the spot, and the ligand is present between the spots. The chip is characterized in that an unfixed portion is formed (claim 24). Still another subject matter of the present invention comprises a substrate and a plurality of spots comprising a gel immobilized on the substrate, and a first ligand is immobilized on at least one of the spots. And the second ligand is immobilized on at least one other spot among the spots (Claim 25).

Still another subject matter of the present invention is a detection method characterized by detecting a nucleic acid, protein, toxin, virus, cell, saccharide, low molecular weight compound, or a conjugate thereof using the chip of the present invention. (Claim 26). The present invention also relates to a detection method characterized by detecting light having a wavelength of 580 nm or more when fluorescence detection is performed in a state where the spot is dry using the chip of the present invention (claim 27).
Still another subject matter of the present invention is a detection apparatus characterized by detecting a nucleic acid, protein, toxin, virus, cell, saccharide, low molecular weight compound, or conjugate thereof using the chip of the present invention. (Claim 28).

  According to the present invention (Claims 1 to 25), it is possible to manufacture a chip by immobilizing a ligand on a plurality of spots configured to include a gel while suppressing the amount used. In particular, according to the present invention (claims 2 to 4), a ligand can be immobilized on the entire spot. Furthermore, according to the present invention (Claim 24), it is possible to realize a chip in which the amount of ligand used is suppressed more than before while the ligand is immobilized on a plurality of spots. Further, according to the present invention (claim 25), it is possible to realize a chip in which different ligands are immobilized for each spot and the degree of integration of the spots is increased. According to the present invention (claims 25, 26, 27), various substances to be detected can be detected using the chip of the present invention.

(A) And (b) is a figure which shows typically the example of the structure of a specific gel, in order to demonstrate one Embodiment of this invention. (A) And (b) is a figure for demonstrating one Embodiment of this invention, (a) is a figure which shows a biological material and a binding compound typically, (b) is a model of a particulate lump. FIG. (A)-(c) is a figure which shows typically an example of the mode at the time of spotting a ligand solution to a spotter, in order to demonstrate one Embodiment of this invention. (A)-(c) is a figure which shows typically an example of the mode at the time of spotting a ligand solution to a spotter, in order to demonstrate one Embodiment of this invention. (A)-(d) is a figure which shows typically an example of the mode at the time of spotting a ligand solution to a spotter, in order to demonstrate one Embodiment of this invention. It is a drawing substitute photograph which shows the result of having measured the surface of the chip | tip produced in Example 4 of this invention by AFM. It is a drawing substitute photograph which shows the mode of the chip | tip at the time of the fluorescence intensity measurement image | photographed in Example 7 of this invention. It is a drawing substitute photograph which shows the mode of the chip | tip at the time of the fluorescence intensity measurement image | photographed in Example 9 and Comparative Example 1 of this invention. FIG. 9 is a drawing substitute photograph showing a part of FIG. 8 in an enlarged manner. FIG. It is a graph showing the result of the fluorescence intensity measurement performed in Example 10 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 19 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 20 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 22 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 23 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 24 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 28 of this invention. It is a graph showing the result of the fluorescence intensity measurement performed in Example 29 of this invention.

  Hereinafter, the present invention will be described in detail with reference to embodiments, examples, etc., but the present invention is not limited to the following embodiments, examples, etc., and can be arbitrarily set within the scope of the present invention. Can be changed and implemented.

[1. Chip manufacturing method]
The chip manufacturing method according to the present embodiment is a method for manufacturing a chip including a substrate and a plurality of spots including gels immobilized on the substrate. Further, in the chip manufacturing method of this embodiment, a substrate is prepared, a gel spotting step of forming a spot by immobilizing the gel on the substrate, and a solution containing a ligand on the spot (hereinafter referred to as “ligand solution” as appropriate). A ligand spotting step in which the ligand is immobilized on the spot.

[1-1. Preparation of substrate]
In the chip manufacturing method of the present embodiment, first, a substrate to be a target for immobilizing the gel is prepared. There is no particular limitation on the material of the substrate, and any material can be used as long as the gel can be immobilized. Examples include glass, quartz, silicon, metal, metal oxide, plastic and the like. These materials are usually properly used according to the purpose of the chip to be produced, the type of ligand, and the like.

For example, when the manufactured chip is used for a field-effect transistor (FET) sensor, gold, silicon, or the like is preferably used as a substrate material.
For example, when the manufactured chip is used in a QCM (Quartz Crystal Microbalance) sensor, quartz, gold, or the like is preferably used as the substrate material.
For example, when the manufactured chip is used for an SPR (Surface Plasmon Resonance) sensor, gold or the like is preferably used as the material of the substrate.
For example, when the manufactured chip is used for a sensor based on MEMS (Micro-Electro-Mechanical Systems) technology, silicon or the like is preferably used as the substrate material.
For example, if the manufactured chip is used for ELISA (enzyme-linked immunosorbent assay), plastic, glass, etc. are preferably used as the substrate material, and plastic is particularly preferred. .

  In addition, the kind of material of a board | substrate may be 1 type, and may use 2 or more types together by arbitrary combinations and a ratio. Therefore, for example, the above listed materials may be fixed to a support made of another material to form a substrate, and the gel may be fixed to the substrate. If a specific example is given, if it is a SPR sensor use, a gold thin film will be formed on the support body which consists of glass or a plastics, and this can be used as a board | substrate. For QCM sensor applications, a gold thin film can be formed on a support made of quartz and used as a substrate.

  Further, in order to stably fix the gel on the substrate surface, the substrate may be subjected to a surface treatment as necessary. In this case, surface treatment for introducing a specific functional group for bonding the gel and the substrate surface to the substrate surface is often performed. In the surface treatment, the entire surface of the substrate may be processed in advance, or the gel may be fixed only to a specific region by processing only a specific place. In particular, it is easy to perform the surface treatment on the entire surface of the substrate.

  The surface treatment as described above varies depending on the use of the chip, the type of the substrate and the gel, and the like. For example, if the manufactured chip is used for immunochromatography, it is preferable to perform a surface treatment for fixing a cellulose derivative such as cellulose or nitrocellulose on the substrate surface. For example, when plastic is used as the substrate material, the gel can be more stably immobilized by immobilizing a functional group such as an amino group on the surface of the plastic (that is, by functionalization). There is a case.

  In particular, when a hydrogel is to be immobilized on a substrate among gels, it is preferable to have a reactive functional group on the surface of the substrate. Here, the reactive functional group means a functional group that can be bonded to the hydrogel. As a result, the substrate and the hydrogel can be firmly bonded to each other and can be stably fixed. Examples of such reactive functional groups include amino groups, succinimideoxycarbonyl groups, epoxy groups, bromo groups, maleimide groups, mercapto groups, and the like. Among these, an amino group, a succinimideoxycarbonyl group, and an epoxy group are preferable, and a succinimideoxycarbonyl group and an epoxy group are particularly preferable.

  Another example of the surface treatment includes a treatment for immobilizing the protein on the substrate surface by covalent bonding or strong physical adsorption. In this case, an amino group or a hydroxycarbonyl group of the protein can be used as the reactive functional group. In this case, the hydroxycarbonyl group can be converted into a succinimideoxycarbonyl group for use. Furthermore, in any of the above cases, it is preferable to use a functional group that is not bound to the ligand as the functional group to be immobilized by the surface treatment. This is to prevent the ligand from being immobilized at an unexpected position on the substrate surface. Note that only one type of surface treatment may be performed, or two or more types of surface treatments may be arbitrarily combined.

  There is no particular limitation on the shape and size of the substrate. For example, it may have no irregularities, or may have walls or holes. A specific example of the former includes a slide glass, and a specific example of the latter includes a titer plate. Further, one or two or more flow paths may be formed on the substrate.

[1-2. Gel spotting process]
In the gel spotting step, the gel is fixed on the substrate, and spots made of the gel are formed on the surface of the substrate.

[1-2-1. gel]
The gel is not particularly limited as long as it can immobilize the ligand, and an appropriate gel may be used according to the purpose of the chip to be produced. Since the gel usually has a three-dimensional network structure, the ligand can be immobilized three-dimensionally to the inside of the gel. For this reason, compared with the case where the ligand is directly immobilized on the substrate surface, the ligand immobilization density per unit area of the substrate can be increased by immobilizing the ligand on the gel spot. In addition, when the manufactured chip is used for a sensor for detecting a substance to be detected, the substance to be detected can usually penetrate into the gel, so the substance to be detected per unit area of the substrate. Further, the signal density due to the substance to be detected can be increased.

  Of these, hydrogel is preferably used as the gel. The hydrogel is a polymer compound having a three-dimensional network structure, and the network structure is swollen by water. Since water is often used as a solvent in the medical field, agricultural field, biological field, etc., using a hydrogel as a gel is preferable when the manufactured chip is used in such a field.

  Various hydrogels are known, for example, those composed of polyacrylic acid, polyamino acids, polyacrylamide, and the like, and those using biological materials such as hyaluronic acid and collagen.

  In particular, the hydrogel is composed of a biological substance and a particulate wrinkle formed by bonding a biological substance and a compound capable of binding to the biological substance (hereinafter referred to as “binding compound” as appropriate) described below. It is preferable to use a gel (hereinafter referred to as “specific gel” where appropriate). The specific gel is preferable in terms of shortening of sensitivity and detection time as compared with a general hydrogel. As such a specific gel, for example, a biological material structure described in JP 2007-101520 A can be used.

[Specific gel]
As shown in FIG. 1 (a) and FIG. 1 (b), the specific gel is formed by binding together a particulate mass formed by binding a biological substance and a binding compound. In addition, this particulate lump is usually formed into a particulate shape as shown in FIG. 2B by binding a plurality of biological substances and binding compounds as shown in FIG. Although not necessarily circular, in FIGS. 1A and 1B, the particulate mass is schematically shown as a circle.

  Specifically, the specific gel, when viewed microscopically, has a matrix structure in which a binding compound binds to a biological material and has a main chain composed of these biological material and the binding compound (matrix). ing. That is, the specific gel includes a matrix containing a biological material and a binding compound, and a skeleton having a structure in which the biological material and the binding compound are bonded to each other in a chain and / or network shape. Yes. Then, by repeating the structure, a particulate lump having a chain-like and / or network-like structure inside is formed. Further, the bonds between the particulate lumps are usually formed by the bond between the biological substance contained in the particulate lumps and the binding compound.

Therefore, the specific gel usually has two or more partial structures represented by the following formula (A).
R1-R2 Formula (A)
{In the above formula (A), R1 represents a biological substance, and R2 represents a binding compound. However, when the specific gel is bonded to the substrate, R2 represents a bonded compound that is not directly bonded to the substrate. Moreover, each R1, R2 may be the same or different. }

  That is, the specific gel is a structure in which a partial structure in which a biological substance and a binding compound are bonded as in the above formula (A) is bonded in a linear and / or network form. Specifically, R1 in the above formula (A) is independently bonded to one or more other R2, and R2 is independently bonded to one or more other R1. However, the specific gel may include a partial structure in which, for example, biological substances R1 or binding compounds R2 are bonded to each other. Here, the bond between R1 and R2 indicates a bond due to physical interaction such as intermolecular attractive force, hydrophobic interaction, and electrical interaction.

  Therefore, the specific gel has a bridging structure in which a biological material exists between binding compounds and a binding compound exists between biological materials. A specific gel is constituted by both of the compounds.

  Whether or not the biological material and the binding compound have a bridging structure in the specific gel is confirmed, for example, by the fact that the specific gel is greatly disintegrated when the biological material is decomposed while preventing the binding compound from being decomposed. Can do. Furthermore, by investigating the collapsed compounds, it is possible to identify the compounds constituting the specific gel other than the biological material constituent elements. For example, when a biological substance is directly bonded to some kind of carrier, the carrier remains after decomposition. For example, the following methods can be used as the biological material decomposition method and quantitative method. However, when used for the purpose of decomposition and quantification of biological materials, the above bridge structure can be confirmed accurately for reagents that may decompose not only biological materials but also binding compounds in the examples. Use should be avoided as it may disappear.

(Degradation method of biological material)
In the case of decomposing a biological material, the biological material may be decomposed with an enzyme, a drug, or the like. Any enzyme, chemical, or the like for degrading the biological material may be appropriately used depending on the type of the biological material or binding compound used. When the specific example is given, when a biological material is a nucleic acid, nuclease, such as ribonuclease and deoxyribonuclease, etc. are mentioned, for example.

  When the biological substance is a protein, examples of the enzyme and drug include proteolytic enzymes such as microbial protease, trypsin, chymotrypsin, papain, rennet, and V8 protease, cyanogen bromide, and 2-nitro-5. -Chemical substances having protein resolution such as thiocyanic benzoic acid, hydrochloric acid, sulfuric acid, sodium hydroxide and the like. Furthermore, when the biological substance is a lipid, examples of the enzymes and chemicals include lipolytic enzymes such as lipase and phospholipase A2.

  In addition, when the biological substance is a saccharide, examples of the enzyme and the drug include sugar-degrading enzymes such as α-amylase, β-amylase, glucoamylase, pullulanase, and cellulase. In addition, said enzyme, chemical | medical agent, etc. for decomposing | disassembling a biological substance may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios.

(Quantitative method of biological material)
In order to quantify and analyze the biological material released from the substrate surface or the substrate after decomposition of the biological material, for example, liquid chromatography, gas chromatography, mass spectrometry (MS), infrared spectroscopy, nuclear magnetic resonance ( ¹ H-NMR, ¹³ C-NMR, ²⁹ Si-NMR), high performance liquid chromatography (HPLC), gel electrophoresis, capillary electrophoresis, absorbance measurement, fluorescence measurement, elemental analysis, amino acid quantification and the like. In the analysis, each measurement method may be used alone, or two or more kinds may be arbitrarily combined.

[Biological substances]
A biological material is an element constituting a specific gel. As this biological substance, any substance can be used depending on the purpose as long as the effects of the present invention are not significantly impaired. However, from the viewpoint of immobilizing the ligand on the specific gel, when the ligand is bound to the biological material, a biological material that can bind to the ligand is used.

  The binding between the biological substance and the ligand may be any binding depending on the type of chip to be manufactured and the use. Specific examples of the bond between the biological substance and the ligand include covalent bond, ionic bond, chelate bond, coordination bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic force bond. It may be another combination that does not fit into the classification.

  On the other hand, when the ligand is bound to the binding compound so as not to be bound to the biological material, any biological material can be used depending on the application. For example, when a predetermined interaction is to be caused between a ligand and some detected substance, a biological substance that does not cause a non-specific interaction with the detected substance is used. It is preferable to use it.

  Specific examples of biological materials include enzymes, antibodies, lectins, receptors, protein A, protein G, protein A / G, avidin, streptavidin, neutravidin, glutathione-S-transferase, albumin, glycoprotein and the like, Peptides, amino acids, cytokines, hormones, nucleotides, oligonucleotides, nucleic acids (DNA, RNA, PNA), sugars, oligosaccharides, polysaccharides, sialic acid derivatives, sugar chains such as sialylated sugar chains, lipids, derived from biological substances other than those mentioned above And high molecular organic substances, low molecular compounds, inorganic substances, or fusions thereof, or biomolecules such as viruses or molecules constituting cells.

  In addition, substances other than biomolecules such as cells can also be used as biological substances. Furthermore, immunoglobulins and their derivatives F (ab ′) 2, Fab ′, Fab, receptors and enzymes and their derivatives, nucleic acids, natural or artificial peptides, artificial polymers, carbohydrates, lipids, inorganic substances or organics A ligand, a virus, a cell, etc. are also mentioned as an example of a biological material.

  Further, among the examples of the biological material, the protein may be the full length of the protein or a partial peptide including a binding active site. Further, the protein may be a protein whose amino acid sequence and its function are known or unknown. These can also be used as target substances, such as synthesized peptide chains, proteins purified from living bodies, or proteins that have been translated from a cDNA library or the like using an appropriate translation system and purified. Further, the synthesized peptide chain may be a glycoprotein having a sugar chain bound thereto. Of these, a purified protein is preferable.

  By using the protein, the characteristics of the protein can be utilized in the manufactured chip. Specifically, for example, when albumin is used as a biological material, nonspecific adsorption can be suppressed when the chip is used. In another aspect, if avidin is used as a biological material, a biotinylated ligand can be immobilized easily and in large quantities. In yet another aspect, if protein A is used as the biological material, when an antibody is used as the ligand, the antibody can be immobilized easily and in large quantities.

  Examples of proteins include albumin such as BSA; avidin such as streptavidin; protein A, protein G and the like. BSA can immobilize ligands using amino groups or hydroxycarbonyl groups, Avidin can immobilize ligands with biotin, and Protein A and Protein G immobilize antibodies as ligands. Can do.

  Furthermore, among the examples of the biological material, the nucleic acid is not particularly limited, and in addition to DNA and RNA, nucleobases such as aptamers and peptide nucleic acids such as PNA can be used. The nucleic acid may be a known nucleic acid or an unknown nucleic acid. Among them, it is preferable to use a nucleic acid having a binding ability to a protein and having a known function and base sequence as a nucleic acid, or that has been cleaved and isolated from a genomic library or the like using a restriction enzyme or the like.

  Moreover, among the examples of the above-mentioned biological substances, the sugar chain may be a sugar chain having a known sugar chain or an unknown sugar chain. Preferably, a sugar chain that has already been separated and analyzed and whose sugar sequence or function is known is used. Further, among the examples of the biological material, the low molecular compound is not particularly limited as long as the requirements for the biological material are satisfied. Those having an unknown function or those having an already known ability to bind to a protein can be used.

  In addition to using the biological substance as it is, for example, a substance into which a hydroxycarbonyl group, a succinimideoxycarbonyl group, an amino group, a hydroxyl group, polyhistidine (HIS tag) or the like is introduced can be used. In addition, a biological material may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

[Binding compound]
As the binding compound, any compound can be used as long as it is a compound capable of binding to the biological substance. As the binding compound, a compound having a functional group capable of binding to the biological substance (hereinafter appropriately referred to as “binding functional group”) can be arbitrarily used. Due to the binding functional group, the binding compound binds to the biological substance, and can be further crosslinked to form a particulate mass. Here, the bond usually refers to a bond consisting of one or more of a covalent bond, ionic bond, chelate bond, coordination bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic bond, Among these, a covalent bond is preferable.

The binding functional group is not particularly limited as long as it is a functional group capable of binding to the biological material, and any functional group can be used. Usually, it is preferable to select an appropriate material according to the type of biological material, the use of the chip, and the like.
In addition, a bond functional group may be used individually by 1 type, and may be used 2 or more types by arbitrary combinations and ratios.

The binding functional group is generally classified into a group that binds to a biological material as a reactive group via a covalent bond and a group that binds to a biological material via a non-covalent bond.
When binding by a covalent bond, specific examples of the binding functional group include a succinimide group, an epoxy group, a formyl group, a maleimide group, a p-nitrophenoxy group, and the like.

  In this case, examples of the biological substance that binds to the binding functional group through a covalent bond include proteins, nucleic acids, and saccharides. When the biological material is a protein, usually, a group such as an amino group, a hydroxyl group, or a mercapto group present on the surface layer of the protein is bonded to a binding functional group of the binding compound. Examples of the bonding functional group bonded to the amino group include a succinimideoxycarbonyl group, an epoxy group, a formyl group, a maleimide group, and a p-nitrophenoxy group. Moreover, an epoxy group etc. are mentioned as a coupling | bonding functional group couple | bonded with a hydroxyl group. Furthermore, examples of the binding functional group that binds to the mercapto group include a maleimide group.

  In addition, when the biological material is a nucleic acid, a group such as an amino group, a hydroxyl group, or a mercapto group that is introduced at the end of the nucleic acid is usually bonded to a binding functional group of the binding compound. For example, succinimideoxycarbonyl group, epoxy group, formyl group, maleimide group, p-nitrophenoxy group and the like can be mentioned as the binding functional group that binds to the amino group. Moreover, an epoxy group etc. are mentioned as a coupling | bonding functional group couple | bonded with a hydroxyl group. Furthermore, examples of the binding functional group that binds to the mercapto group include a maleimide group.

  Furthermore, when the biological substance is a saccharide, usually, a group such as an amino group, a hydroxyl group, a hydroxycarbonyl group, or a mercapto group present in the side chain of the saccharide is bonded to the binding functional group of the binding compound. For example, succinimideoxycarbonyl group, epoxy group, formyl group, maleimide group, p-nitrophenoxy group and the like can be mentioned as the binding functional group that binds to the amino group. Moreover, an epoxy group etc. are mentioned as a coupling | bonding functional group couple | bonded with a hydroxyl group. Examples of the binding functional group that binds to the hydroxycarbonyl group include a hydroxyl group, an epoxy group, and an amino group. Furthermore, examples of the binding functional group that binds to the mercapto group include a maleimide group.

On the other hand, when the biological substance and the binding compound are bonded by non-covalent bonding, the binding can be performed by, for example, complex formation or interaction between biological substances.
When a biological substance and a binding compound are combined to form a complex, specific examples of the binding functional group include a boronic acid group.
Further, for example, when binding is performed by an avidin-biotin interaction among the interactions between biological substances, a specific example of the binding functional group is a biotin group.

  Further, for example, when a virus is used as a biological material, specific examples of the binding functional group include saccharides such as monosaccharides and polysaccharides. For example, when the biological substance has a hydrophobic region, it may be bonded by physical adsorption by hydrophobic interaction.

  In addition, when the binding compound has a binding functional group, the binding compound preferably contains at least one kind having a binding functional group of usually 2 or more, preferably 3 or more in one molecule. This is to facilitate the formation of a specific gel network structure. If there are two or more binding functional groups in one molecule, it is possible to easily form a particulate lump in which a biological substance and a binding compound are bonded, and further to bond these particle lumps together to form a higher order structure. A specific gel can be formed.

  However, in order to facilitate the formation of the particulate lump, it is preferable that the binding compounds do not bind to each other, and the binding between the biological substance and the binding compound is the main binding in the specific gel. This is because when the binding compounds are bonded to each other, the binding compounds are likely to aggregate with each other, the particle size of the particulate lump tends to increase, and it becomes difficult to control the particle size of the particle lump. Further, when a polymer is used as the binding compound, there is a tendency that it is difficult to immobilize the biological material if internal crosslinking of the binding compound occurs. Here, the bond between binding compounds refers to a bond excluding intermolecular attractive force, hydrophobic interaction, and electrical interaction.

  In order to form a specific gel in which the binding compounds are not bonded in this way, it is desirable to select a binding functional group that does not bond the binding compounds, and to eliminate the situation where the binding compounds are bonded. Specific examples of such a functional group include a succinimideoxycarbonyl group, an epoxy group, a formyl group, a maleimide group, a boronic acid group, and a biotin group as described above. The situation where the bonding functional groups are bonded to each other means that excessive heat is applied or that strong ultraviolet rays are irradiated.

  Moreover, as a method for examining the binding between the binding compounds contained in the specific gel, it is determined that an insoluble material is formed when the biological material in the specific gel is decomposed by the above-described method. Or it is judged by detecting the compound which suggests the coupling | bonding of coupling | bonding compounds by analyzing the specific gel after decomposition | disassembly by a pyrolytic gas chromatography. Specifically, for example, when a specific gel is formed by binding a biological substance and a binding compound having a photoreactive group by ultraviolet irradiation, binding is performed by a photoreactive group (for example, an azide group) in the binding compound. When compounds are bonded to each other, a bond involving a photoreactive group or a residual photoreactive group will be present. Therefore, by confirming the presence of these bonds or the remaining photoreactive group, bonding The binding between the compounds can be inferred (see “Affinity Chromatography” published by Tokyo Chemical Doujin, authors: Isao Matsumoto, Masatoshi Beppu, P238 et al.).

  Furthermore, in order to take advantage of the characteristics of the biological material, it is preferable to select the functional group of the biological material and the functional group of the binding compound so that the biological material is not deactivated. For example, when the biological substance is a protein and has a mercapto group in its active part, a group other than the mercapto group (for example, an amino group) may be selected as the binding functional group of the biological substance. The functional group of the corresponding binding compound is selected from a succinimideoxycarbonyl group and an epoxy group that react with an amino group.

  Examples of the bond between the ligand and the binding compound include covalent bond, ionic bond, chelate bond, coordination bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic force bond. It may be another bond that does not apply.

  The binding compound preferably has a functional group capable of binding to the ligand in addition to the binding functional group. There is no restriction | limiting in such a functional group, Arbitrary functional groups can be used and it can select according to the kind of ligand, the use of a chip | tip, etc. The functional group capable of binding to the ligand may be one type, or two or more types may be used in any combination and ratio.

  Furthermore, since the binding compound often uses water as a medium such as a solvent or a dispersion medium when producing the specific gel, it is desirable to use a binding compound that is miscible with water. When producing a specific gel, the binding compound and the biological material are mixed in the presence of water, and a process of producing a particulate lump is performed. To facilitate the reaction, the biological material and the binding compound are rapidly and uniformly mixed. It is preferable to mix. In the present invention, the mixing may be dissolved or dispersed.

  The binding compound is preferably miscible with at least one organic solvent. The choice of the solvent used for the synthesis can be increased, and the number of synthesis reaction species can be increased. Thereby, it becomes possible to increase the types of specific gels to be produced and to design the structure. In addition, a binding compound having a protected binding functional group can be prepared by a synthesis reaction in an organic solvent.

  Furthermore, it is more preferable to use a binding compound that is miscible with both water and an organic solvent. Thereby, the kind of solvent at the time of manufacturing a chip | tip can be increased, and a use can be expanded.

  The binding compound is preferably uncharged. If the binding compound has the same charge (charge having the same sign) as that of the biological material, the binding between the binding compound and the biological material may be hindered by electrostatic repulsion. On the other hand, if the binding compound has a charge opposite to that of the biological substance (reverse sign charge), the biological substance and the charged part in the binding compound are bound by electrostatic attraction, and the binding compound has This may prevent the biological substance from effectively binding to the binding functional group. In addition, when the chip of the present invention is used for separation and purification, the binding between the binding compound and the biological substance is expected to be easily broken by an additive such as pH or salt of the solution used at the time of use. Is done.

  Furthermore, when the detection target substance is detected using the chip of the present invention and the detection target substance has the same charge as the binding compound, the ligand and the detection target substance included in the specific gel If the detected substance and the binding compound have opposite charges, the detected substance and the binding compound are nonspecific due to the electric attractive force. It is assumed that adsorption occurs.

  Note that the binding compound is uncharged means that the binding compound has nonionic or zwitterionic (both positive and negative charges) at least in the structural formula, and the mutual charges substantially cancel each other. The binding compound is uncharged. However, even if the binding compound has a charge due to hydrolysis of the binding functional group or the like in the manufacturing process of the chip of the present invention, such a binding compound is preferably used as long as the effect of the present invention is not impaired. it can.

  Other examples of the binding compound include organic compounds, inorganic compounds, organic-inorganic hybrid materials, and the like. Moreover, a binding compound may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

  Furthermore, the organic compound used as the binding compound may be a low molecular compound or a high molecular compound, but is preferably a high molecular compound.

  When a polymer compound is used as the binding compound, the polymer compound may be a synthetic product or a natural product. When the polymer compound is a synthetic product, it is preferable to have a monomer that can bind to a biological substance. Moreover, in order to mix with water, it is preferable to have a hydrophilic monomer. Furthermore, a synthetic polymer compound obtained by copolymerizing a monomer capable of binding to the biological substance and a hydrophilic monomer is preferable. In addition, when a ligand couple | bonds with a binding compound, it is preferable to have the monomer which can couple | bond with a ligand in addition to said monomer.

  That is, for the synthesis of a synthetic polymer compound used as a binding compound, at least as a monomer species, a monomer capable of binding to a biological substance to form a particulate lump, and the particulate lump are bound together to form a chain and And / or a monomer having a binding functional group for constructing a network-bonded structure (that is, a structure of a specific gel), and further, a monomer having a hydrophilic or amphiphilic functional group is used. It is more preferable. In this case, the molar ratio of the monomer having a bonding functional group and the monomer having a hydrophilic or amphiphilic functional group (monomer having a bonding functional group / monomer having a hydrophilic or amphiphilic functional group) From the viewpoint of property, it is usually 5/5 or more, preferably 6/4 or more, usually 9/1 or less, preferably 8/1 or less, and 7/3 is particularly preferred. In addition, it is also preferable to include a hydrophobic monomer for the purpose of controlling the structure and spread of micelles and the like formed by the synthetic polymer compound in the solution. Furthermore, when the ligand and the binding compound are bound, it is desirable to include a monomer having a functional group capable of binding to the ligand. In addition, as for the monomer quoted here, one monomer may have 2 or more of said functions. Therefore, the synthetic polymer in this case may be a homopolymer or a copolymer.

  Specific examples of monomers constituting a synthetic polymer compound that can be used as a binding compound include monomers that can be used for radical polymerization such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene. Saturated aromatics; polymerizable unsaturated carboxylic acids such as (meth) acrylic acid, itaconic acid, maleic acid and phthalic acid; polymerizable unsaturated sulfonic acids such as styrene sulfonic acid and sodium styrene sulfonate; (meth) acrylic acid Methyl, ethyl (meth) acrylate, n-butyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, glycidyl (meth) acrylate, N- (meth) Acrylic oxysuccinimide, ethylene glycol di (meth) acrylate Polymerizable carboxylic acid esters such as (meth) acrylic acid tribromophenyl, 2- (meth) acrylic acid glycosiloxyethyl, 2-methacryloxyethyl phosphorylcholine; (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N, N-dimethylacrylamide, N-isopropyl (meth) acrylamide, N-vinylformamide, 3-acrylamidophenylboronic acid, N-acryloyl-N′-biotinyl-3,6-dioxaoctane-1,8-diamine, Unsaturated carboxylic acid amides such as butadiene, isoprene, vinyl acetate, vinyl pyridine, N-vinyl pyrrolidone, N- (meth) acryloylmorpholine, vinyl chloride, vinylidene chloride, vinyl bromide; polymerizable unsaturated nitriles; halogenated Vinyls; conjugated di Enenes; Macromonomers such as polyethylene glycol mono (meth) acrylate and polypropylene glycol mono (meth) acrylate, and the like.

  Moreover, as a monomer of a synthetic polymer compound that can be used as a binding compound, for example, a monomer used in addition polymerization can also be used. Specific examples of monomers used for this addition polymerization include aliphatics such as diphenylmethane diisocyanate, naphthalene diisocyanate, tolylene diisocyanate, tetramethylxylene diisocyanate, xylene diisocyanate, dicyclohexane diisocyanate, dicyclohexylmethane diisocyanate, hexamethylene diisocyanate, isophorone diisocyanate, or the like. Aromatic isocyanates, ketenes, epoxy group-containing compounds, vinyl group-containing compounds and the like can be mentioned.

  In addition, the above compound group can be reacted with a monomer having a functional group having activated hydrogen. Specific examples thereof include a compound having a hydroxyl group or an amino group, specifically, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, glycerin, trimethylolpropane, Polyols such as pentaerythritol, sorbitol, methylene glycoside, sucrose, bis (hydroxyethyl) benzene; ethylenediamine, hexamethylenediamine, N, N′-diisopropylmethylenediamine, N, N′-di-sec-butyl-p -Polyamines such as phenylenediamine and 1,3,5-triaminobenzene; oximes and the like.

  Furthermore, in the synthetic polymer compound that can be used as the binding compound, in addition to the above-described monomer, a polyfunctional compound that can serve as a crosslinking agent may coexist. Examples of the polyfunctional compound include N-methylol acrylamide, N-ethanol acrylamide, N-propanol acrylamide, N-methylol maleimide, N-ethylol maleimide, N-methylol maleamic acid, N-methylol maleamic acid ester, Examples thereof include N-alkylolamides of vinyl aromatic acids (such as N-methylol-p-vinylbenzamide) and N- (isobutoxymethyl) acrylamide.

  Furthermore, among the monomers described above, divinylbenzene, divinylnaphthalene, divinylcyclohexane, 1,3-dipropenylbenzene, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, butylene glycol, Polyfunctional monomers such as trimethylolethane tri (meth) acrylate and pentaerythritol tetra (meth) acrylate can also be used as a crosslinking agent. By using a polyfunctional compound that can be a crosslinking agent as a monomer, the spread and hardness of the binding compound in the medium can be controlled.

  Examples of the monomer having a binding functional group capable of binding to the aforementioned biological substance include monomers having a succinimide group, a succinimideoxycarbonyl group, an epoxy group, a formyl group, a maleimide group, a p-nitrophenoxy group, and the like. Examples include acrylic monomers such as N- (meth) acryloxysuccinimide; glycidyl (meth) acrylate, acrolein, maleimide acrylate, p-nitrophenyloxycarbonyl polyethylene glycol methacrylate, p-nitrophenyl methacrylate, and the like.

  Examples of the monomer having a boronic acid group as a binding functional group include 3-acrylamidophenylboronic acid. Furthermore, examples of the monomer having a biotin group as a binding functional group include N-acryloyl-N′-biotinyl-3,6-dioxaoctane-1,8-diamine. Examples of the monomer having a sugar or a polysaccharide as a binding functional group include glycosyloxyethyl 2- (meth) acrylate.

  Examples of the monomer having a functional group capable of binding to the ligand include the same monomers as those having a binding functional group capable of binding to the biological substance.

  Furthermore, specific examples of the hydrophilic monomer include (meth) acrylic acid, itaconic acid, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, maleic acid, sulfonic acid, sodium sulfonate, (Meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropylacrylamide, N-vinylformamide, (meth) acrylonitrile, N- (meth) acryloylmorpholine, N-vinylpyrrolidone, N-vinylacetamide, N- Examples include vinyl-N-acetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, 2-methacrylooxyethyl phosphorylcholine, N-acryloylmorpholine, and the like. N-acryloylmorpholine is an acrylic monomer having a morpholino group as a hydrophilic group.

  The binding compound is preferably an uncharged one as described above. Therefore, when the synthetic polymer compound used as the binding compound is uncharged, the monomer used in the uncharged synthetic polymer compound is not particularly limited as long as it is uncharged. 2-hydroxyethyl acid, 2-hydroxypropyl (meth) acrylate, (meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropylacrylamide, N-vinylformamide, (meth) acrylonitrile, N- (meta ) Acrylylmorpholine, N-vinylpyrrolidone, N-vinylacetamide, N-vinyl-N-acetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, 2-methacrylooxyethyl phosphorylcholine and the like.

  Moreover, although there is no restriction | limiting in the polymerization method of a monomer, radical polymerization is suitable. When a synthetic polymer compound is synthesized by radical polymerization of a monomer, the polymerization is usually started by mixing a radical polymerization initiator, but the radical polymerization initiator used at that time does not significantly impair the effects of the present invention. Any thing can be used. Examples of the radical polymerization initiator that can be used include 2,2′-azobisisobutyronitrile, 2,2′-azobis- (2-methylpropanenitrile), 2,2′-azobis- (2,4 -Dimethylpentanenitrile), 2,2'-azobis- (2-methylbutanenitrile), 1,1'-azobis- (cyclohexanecarbonitrile), 2,2'-azobis- (2,4-dimethyl-4- Azo (azobisnitrile) type initiators such as methoxyvaleronitrile), 2,2′-azobis- (2,4-dimethylvaleronitrile), 2,2′-azobis- (2-amidinopropane) hydrochloride, Benzoyl peroxide, cumene hydroperoxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfates (eg ammonium persulfate), peracid esters ( Example, if t- butyl peroxide octoate, etc. α- cumyl peroxypivalate and t- butyl per octoate) peroxide type initiators and the like.

  Furthermore, polymerization may be initiated by mixing a redox initiator. Any redox initiator can be used as long as the effects of the present invention are not significantly impaired. Examples thereof include ascorbic acid / iron (II) sulfate / sodium peroxydisulfate, tert-butyl hydroperoxide / dioxide. Examples include sodium sulfite, tert-butyl hydroperoxide / Na hydroxymethanesulfinic acid. In addition, each component, for example, a reducing component, may be a mixture, for example, a mixture of sodium salt of hydroxymethanesulfinic acid and sodium disulfite.

  When a synthetic polymer compound is used as the binding compound, the synthetic polymer compound may be a polymer synthesized by ring-opening polymerization or the like. Specific examples thereof include polyethylene glycol. Furthermore, the synthetic polymer compound described above may use a polymer synthesized by hydrolysis or the like. Specific examples thereof include polyvinyl alcohol synthesized by hydrolyzing polyvinyl acetate and the like. Further, the above-described synthetic polymer compound may be synthesized by modifying a functional group that binds to the aforementioned biological substance by chemical modification.

  In addition, a commercially available synthetic polymer compound can be used as the binding compound. Specific examples include SUNBRIGHT series DE-030AS, DE-030CS, DE-030GS, PTE-100GS, PTE-200GS, HGEO-100GS, and HGEO-200GS manufactured by NOF Corporation.

  On the other hand, when a natural polymer compound is used as the binding compound, specific examples thereof include polysaccharides such as dextran, carboxymethyl-dextran, starch and cellulose, proteins such as albumin, collagen and gelatin, nucleic acids such as DNA and RNA, etc. Is mentioned. These natural compounds may be used as they are, or may be used after being chemically modified.

  When a polymer compound such as a synthetic polymer compound or a natural polymer compound is used as the binding compound, the form of the polymer compound is arbitrary. For example, it may be dissolved in an aqueous solution, or may be an aggregate such as a micelle or emulsion, or a fine particle such as a polymer latex.

  Examples of the inorganic compound used as the binding compound include metal particles such as gold colloid and inorganic fine particles such as silica. Furthermore, it is good also as a binding compound which has a functional group couple | bonded with a biological substance by chemically modifying these inorganic compounds.

  In addition, organic / inorganic hybrids used as binding compounds include, for example, colloidal silica coated with a polymer, metal colloid coated with a polymer (for example, gold, silver, platinum, etc. particles coated with a protective colloid. And those obtained by adsorbing a polymer to a porous substrate such as clay. These organic-inorganic hybrids can be synthesized by known methods (see polymer-based nanocomposites, industrial research committee, Susumu Nakajo, etc.). Furthermore, these organic-inorganic hybrids can be used as a binding compound by modifying the binding functional group.

Further, the molecular weight and structure of the binding compound are not particularly limited and are arbitrary. However, when a low molecular weight compound is used, there is a possibility that a specific gel cannot be formed due to crosslinking within one biological material to be immobilized. From the viewpoint of preventing this, the molecular weight of the binding compound is preferably 1000 or more, more preferably 10,000 or more, and even more preferably 20,000 or more. Moreover, 1 million or less is preferable, Furthermore, 500,000 or less is preferable, Among these, 300,000 or less is preferable. When a synthetic or natural polymer compound is used as the binding compound, the weight average molecular weight is preferably within the above range. This is because if it falls below this range, there is a possibility that the specific gel in which the particulate lumps gather effectively cannot be formed.
Various methods can be used to measure these molecular weights. For example, GPC (gel permeation chromatography), SEC (size exclusion chromatography), static light scattering measurement, viscosity measurement, etc. should be used. Can do.

  Moreover, there is no restriction | limiting in the magnitude | size of a binding compound, and it is arbitrary unless the effect of this invention is impaired remarkably. However, in order to effectively bind the biological substance and the binding compound, the diameter of the binding compound is preferably 1 nm or more, and more preferably 2 nm when mixed in a liquid (here, medium) such as a solvent or a dispersion medium. The above is preferable, and among these, 3 nm or more is even more preferable.

  Various methods can be used to measure the size of these binding compounds. However, when metal colloids, inorganic particles, polymer fine particles, etc. dispersed in a liquid are used as binding compounds, the static light scattering measurement method is used. It can be examined by a general method such as a dynamic light scattering measurement method and a light diffraction method. In addition, a liquid obtained by removing a liquid such as a reaction medium from a dispersion liquid in which these colloidal gold, inorganic particles, and polymer fine particles are dispersed is observed with an electron microscope such as SEM (scanning electron microscope) or TEM (transmission electron microscope). Alternatively, it may be handled as the diameter of the binding compound in the liquid.

  On the other hand, when using an aggregate such as a polymer or micelle dissolved in a solution as a binding compound, investigate by a general method such as static light scattering measurement method, dynamic light scattering measurement method, or light diffraction method. be able to. In general, polymer particles or micelles dissolved in a solution have a difference in particle diameter depending on the measurement means and analysis method, but the particle diameter is evaluated based on the measured value obtained by any means or method. be able to. When measuring the size of the binding compound in the liquid by an optical method, it is effective to bind the biological substance and the binding compound so that the average particle size of the binding compound is within the above range. It is desirable to make it.

  Further, the amount of the binding functional group that the binding compound has is not particularly limited and is not generally defined by the type of the binding compound. For example, when a polymer is used as the binding compound, the mole% of the constituent unit is based on the constituent unit. It is preferable that it is 0.1% or more, preferably 0.5% or more, more preferably 1% or more, and 90% or less, preferably 80% or less, more preferably 70% or less. This is because if it is below this range, the binding compound may not be able to bind efficiently to the biological substance, and if it exceeds this range, it may not be miscible with the solvent or dispersion medium.

[Specific gel structure]
The specific gel is formed by bonding together a particulate lump formed by bonding a biological substance and a binding compound. Furthermore, in the specific gel, the particle size of the particulate lump is usually 1 μm or less.

  As schematically shown in FIGS. 2 (a) and 2 (b), the specific gel is schematically shown in FIGS. 1 (a) and 1 (b) in which a particulate mass in which a biological substance and a binding compound are bonded as a single unit. As shown specifically, the structures are connected to each other in a chain form and / or a network form.

  The specific gel thus has a structure formed by aggregation and / or bonding of the particulate mass formed by both the biological material and the binding compound. For this reason, it is possible to increase the ratio of the biological substance. Accordingly, if a biological material that does not cause the above-described non-specific interaction is used, there is an advantage that the non-specific interaction generated by the binding compound can be suppressed. That is, a binding compound that can cause non-specific interaction can be covered with a large amount of biological material. Therefore, suppression of non-specific interaction and efficient detection of the interaction between the ligand and the substance to be detected can be performed.

  In addition, since the specific gel can increase the ratio of the biological substance, the ratio of the ligand that binds to the specific gel can be increased, and a larger amount of the ligand can be retained than before. Furthermore, when a ligand can bind to a binding compound in addition to a biological substance, a larger amount of ligand can be retained.

  On the other hand, even when the ligand binds to the binding compound and does not bind to the biological material, the specific gel has a three-dimensional structure, as will be described later. It is. In addition, the above-described advantage that non-specific interaction can be suppressed is also obtained when a binding compound and a ligand bind.

In the conventional technique for fixing a biological material to a substrate, when the chip is used for detection applications, the amount of biological material immobilized is limited to a predetermined upper limit value in order to bind the biological material directly to the surface of the substrate. (Normally, protein monolayer adsorption is at most 0.3 to 1.0 μg / cm ² ), and a large amount of biological material could not be retained. Therefore, even when a ligand is bound to this biological material, the amount of the ligand is not sufficient.

  The specific gel of the present invention is configured such that the particulate lumps gather and come into contact with each other due to their attraction, or the carbon chains are entangled with each other. For example, the particulate mass is bonded only by intermolecular attractive force to form a specific gel, the particulate mass is bonded by the binding of the functional group of the binding compound and the biological material, or the specific gel is formed, or Both of the above factors combine to form a specific gel by combining particulate lumps. Here, the bond between the particulate lumps is usually a bond of at least one of covalent bond, ionic bond, chelate bond, coordination bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic bond. It means what holds, and among them, a covalent bond is preferable. In this case, it may be in a state where only a part of the particulate lumps are bonded, but it is preferable that as many particulate lumps as possible are bound, and that all the particulate lumps are bound. More preferred. In general, it is presumed that the particulate lump aggregates due to a plurality of factors such as entanglement and bonding to constitute a specific gel.

  The particle size of the particulate mass is usually 1 μm or less, preferably 0.5 μm or less, more preferably 0.1 μm or less. If the particle size of the particulate mass is large, a sufficient specific surface area cannot be obtained when used for separation and purification, and a highly accurate detection result may not be obtained when the chip of the present invention is used for detection applications, etc. There is. Furthermore, when measuring the particle size of the particulate mass individually, it is sufficient that at least a part of the particulate mass in the specific gel has a particle size in the above range, It is preferable that as many particulate lumps as possible have a particle size in the above range, and it is more preferable that all the particulate lumps have a particle size in the above range.

  Here, the particle size of the particulate mass can be measured by observing with an optical microscope, an electron microscope such as SEM or TEM, or a microscope such as AFM (atomic force microscope). In addition, when observed with a microscope, the shape of the particulate lump may be observed as a shape protruding from the specific gel into a tuft shape, but this tufted lump portion (tufted lump) Since it is inferred that the particulate lump is formed by protruding from the specific gel, the diameter (usually the short diameter) of the tuft-like lump portion may be within the above range.

  As one method for examining the presence of the particulate lump, a case of analyzing Power Spectral Density (power spectral density) from the image measured by the AFM will be described in detail. The analysis using the power spectrum is used to evaluate minute irregularities on the surface of the object, and an analysis method that decomposes changes (eg, height, depth, etc.) caused by the irregularities into waveforms and performs Fourier transform. one of.

  A commercially available AFM apparatus can be used for the AFM measurement, but preferably NanoScope IIIa manufactured by Digital Instruments is used. The probe preferably has a tip radius R of 5 nm or less, and specifically, SSS-NCH manufactured by Toyo Technica Co., Ltd. is preferable. Furthermore, it is desirable to measure in the tapping mode so as not to damage the sample surface. The measurement may be performed in the air or in a liquid, but in order to obtain a good image, the measurement is preferably performed in the air, and more preferably in a dry state.

  Further, the algorithm used for Power Spectral Density analysis is preferably in accordance with the recommendation of ASTM E42.14 STM / AFM subcommittee for analysis of the surface shape, and particularly preferably, Power Spectrum attached to NanoScope IIIa manufactured by Digital Instruments. The Density function is used (reference document: NanoScope command reference manual).

When analysis of a specific gel is performed using these devices and algorithms, for example, when AFM measurement is performed in a 1 μm × 1 μm field of view, and Power Spectral Density (PSD) analysis is performed from the obtained AFM image The ratio Ic / It of the sum of the power spectrum (Ic) and the total power spectrum (It) excluding the value of 2D isotropic PSD of 100 (nm ⁴ ) or more and a wavelength less than 100 nm is usually 30% When the content is 50% or more, preferably 60% or more, and more preferably 70% or more, and more preferably 70% or more, the presence of a submicron-order particulate lump can be confirmed. This is because the power spectrum represents irregularities on the surface of the object, and if the ratio Ic / It is in the above range, irregularities resulting from the structure of the particulate mass can be confirmed on the surface of the specific gel.

The particle size of the particulate mass can also be confirmed by spectroscopic techniques such as light scattering, X-rays, and neutron scattering. In this case, the average particle diameter to be measured may be in the above range. Even when the average particle size of the particulate mass is within the above range, the same advantages as when the particle size is within the above range can be obtained. Here, as an example, a method for confirming a particulate lump by a static light scattering measurement method will be described in more detail. For example, the particulate lump can be evaluated by obtaining the correlation length by measuring the forward light scattered light intensity of the specific gel. This method measures the scattering angle dependence of the scattering intensity of light that has passed through a specific gel, and based on the Debye-Bueche theory, the (light intensity) ^(-1/2) is the wave number. The slope plotted against the square (= {(4πn / wavelength of light) × sin (scattering angle / 2)} ² ) is obtained, and the correlation length is obtained from (slope / intercept) ² . Here, n is the refractive index of the medium. Although a commercially available light scattering apparatus can be used for the measurement, DYNA 3000 is preferable. The biomolecule complex may be swelled in a liquid or in a dry state, but measurement in a liquid is preferable.

  Moreover, the particulate lumps in the specific gel may not be in close contact with each other, and spaces (voids) may be formed between the particulate lumps. When detection is performed using the chip of the present invention, a detected substance that interacts with a ligand can enter this space, and the ligand interacts with the detected substance in this space. Is possible. A specific gel has a three-dimensional structure and can have many ligands inside (inside), but since the ligand can interact without losing activity, the specific gel Contains a larger amount of ligand than before, and can interact with a substance to be detected in a large amount.

  Although there is no restriction | limiting in the method of investigating whether the specific gel has a space between particulate masses, For example, it can confirm with microscopes, such as an optical microscope, electron microscopes, such as SEM and TEM, and AFM. In addition, it can also be confirmed by spectroscopic techniques such as light scattering, X-rays, and neutron scattering. Furthermore, when the volume in the dry state of the specific gel is compared with the volume when the liquid is included, and the volume increases, it is assumed that the volume has increased due to the liquid entering the space. The specific gel may be recognized as having a space. These volume changes may be confirmed by any method. For example, when the specific gel is formed into a film, the AFM indicates the film thickness in a dry state and the film thickness when the film is immersed in a liquid. It can be confirmed by comparing the two.

[Specific gel composition]
In the specific gel, the ratio of the contained biological material is not limited, but it is usually desirable that a larger amount of the biological material is contained. Specifically, the ratio of the weight of the biological material to the weight of the specific gel represented by “(weight of biological material) / (weight of specific gel)” is usually 0.1 or more, preferably 0.3 or more, More preferably 0.5 or more, and particularly preferably 0.7 or more. When the ratio of the biological material is below this range, the binding compound in the specific gel to be constructed cannot be sufficiently covered with the biological material, and nonspecific adsorption to the binding compound may occur. In addition, when the ligand is bound only to the biological substance, a sufficient amount of the ligand cannot be bound, and when separation and purification is performed using the chip of the present invention, the efficiency of the separation and purification is reduced. There is a possibility.

  The method for measuring the ratio of the biological material is not particularly limited. For example, the biological material contained in the specific gel is decomposed using an enzyme, a drug, or the like, and the biological material, the binding compound, and the ligand-derived material are respectively obtained. What is necessary is just to quantify with various methods. The decomposition method and the quantification method are as described above.

[1-2-2. Gel spot size, thickness, spacing, etc.]
There is no restriction | limiting in the dimension of the spot of the gel formed in a board | substrate, According to the use of a chip | tip, it can set arbitrarily. However, from the viewpoint of increasing the degree of spot accumulation, the spot diameter is usually 1 μm or more, preferably 10 μm or more, more preferably 50 μm or more, and usually 1 mm or less, preferably 500 μm or less, more preferably 300 μm or less. . Since the spot is usually formed in a circular shape, the spot diameter is also uniquely determined. However, if the spot shape is other than a circular shape, the largest one of the distances between the outer edges of the spot is the spot. Of the diameter.

  In addition, the thickness of the gel spot is preferably 3 nm or more, more preferably 5 nm or more in a dry state, from the viewpoint of increasing the amount of immobilized ligand by holding the ligand three-dimensionally. Furthermore, a plurality of spots are formed on the substrate. At this time, the number of spots formed on one substrate is arbitrary as long as it is 2 or more, but a larger number is preferable.

  The distance between spots is not limited, but from the viewpoint of preventing ligands from being mixed between adjacent spots, the distance between the edges of the spots is usually 20 μm or more, preferably 100 μm or more, more preferably Is 200 μm or more, and is usually 2000 μm or less, preferably 1000 μm or less, more preferably 500 μm or less from the viewpoint of increasing the degree of spot accumulation.

[1-2-3. Gel immobilization method (spot formation method)]
There is no limitation on the method for immobilizing the gel on the substrate. For example, a solution or dispersion containing a gel in a solvent or dispersion medium may be spotted on a substrate, and the solvent or dispersion medium may be removed by drying to fix the gel. Also, for example, a solution or dispersion containing a gel raw material in a solvent or dispersion medium is spotted on the substrate, the raw material is reacted on the substrate surface to form a gel, and the solvent or dispersion medium is removed by drying to remove the gel. May be fixed.

  In particular, when a spot is formed by immobilizing a specific gel, a biological material and a binding compound are mixed in a solvent or dispersion medium (mixing step), and the resulting mixture is spotted on a substrate (spotting). Step), a method of fixing the specific gel by removing the solvent or dispersion medium from the droplets on the substrate by drying (drying step) is preferable. Hereinafter, this method will be described in detail.

[Mixing process]
In the mixing step, the biological material and the binding compound are mixed in a medium such as a solvent or a dispersion medium. When the biological material and the binding compound are mixed and coexist in the same system, the biological material and the binding compound are combined to form a particulate lump. And this specific lump aggregates and couple | bonds together and a specific gel is formed. Normally, the process in which the biological substance and the binding compound are combined to form a particulate lump and the process in which the particulate lump is aggregated to form a specific gel proceed as a series of processes. In addition, as described above, the biological substance and the binding compound do not necessarily cause a chemical reaction and bind to each other. However, in this specification, the substance that forms a field when the biological substance and the binding compound are bound is referred to as “ It shall be called broadly as “medium”.

  Any medium can be used as long as it can produce a specific gel, but it is usually desirable to use a material in which a biological substance, a binding compound, and additives (to be described later) that are appropriately used can be mixed. . At this time, the mixing state of the biological substance, the binding compound and the additive is arbitrary, and may be in a dissolved state or a dispersed state, but in order to stably bind the biological substance and the binding compound, The biological material and the binding compound are preferably present in a dissolved state in the medium.

  The medium will form a field where the biological substance and the binding compound will bind, and may affect the activity of the biological substance or binding compound, the stability of the structure, etc. It is preferable to do. Usually, water is used as the medium. Further, a liquid other than water may be used as the medium, and for example, an organic solvent can be used. Furthermore, among the organic solvents, an amphiphilic solvent, that is, an organic solvent miscible with water is preferable. Specific examples of the medium other than water include THF (tetrahydrofuran), DMF (N, N-dimethylformamide), NMP (N-methylpyrrolidone), DMSO in addition to alcohol solvents such as methanol, ethanol and 1-butanol. (Dimethyl sulfoxide), dioxane, acetonitrile, pyridine, acetone, glycerin and the like.

  Further, when liquid is used as these media, salt may be added to the media. The type of the salt is arbitrary as long as the effects of the present invention are not significantly impaired, and specific examples include NaCl, KCl, sodium phosphate, sodium acetate, calcium chloride, sodium bicarbonate, ammonium carbonate and the like. Moreover, there is no restriction | limiting in the quantity of the salt to be used, Arbitrary quantity salt can be used according to a use.

  Furthermore, when water is used as a medium, as water, an aqueous solution in which a solute other than a biological substance and a binding compound is dissolved can be used in addition to pure water. Examples thereof include various buffer solutions, and specific examples thereof include carbonate buffer, phosphate buffer, acetate buffer, HEPES buffer, TRIS buffer and the like. In addition, a medium may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

  Moreover, you may make it mix an additive in addition to a biological material and a binding compound as needed. Examples of additives include the above-mentioned salts, moisturizers such as acids, bases, buffers, and glycerin, metal ions such as zinc as a stabilizer for biological materials, antifoaming agents, denaturing agents, and the like. . In addition, an additive may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

  The mixing ratio of the biological material, the binding compound, the medium, the additive, and the like to be mixed is arbitrary as long as the specific gel can be obtained. However, the mixing ratio represented by “(weight of binding compound) / (weight of biological material)” is usually 1/100 or more, preferably 5/100 or more, more preferably 10/100 or more, and usually 10,000. / 100 or less, preferably 200/100 or less, more preferably 100/100 or less. This is because a particulate lump may not be formed outside this range.

  Further, the ratio (concentration) of the biological substance and the binding compound in the medium is arbitrary as long as the specific gel can be obtained. The total concentration of the biological substance and the binding compound is preferably 0.01% by weight or more, and 1% by weight or more is more preferable, 1% by weight or more is particularly preferable, 10% by weight or less is preferable, 5% by weight or less is more preferable, and 3% by weight or less is particularly preferable. In order to form a uniform specific gel, it is preferable to uniformly mix the biological substance and the binding compound in the medium at the stage of preparing the mixed solution. It is because it is preferable to make a compound coexist.

  In mixing, the biological material, the binding compound, the medium, the additive, and the like may be prepared in any state as long as the specific gel can be produced. However, the biological material is usually prepared as a solution or dispersion in which the biological material is dissolved or dispersed in some solvent or dispersion medium. In this case, the solvent or dispersion medium for diluting the biological material is preferably adjusted in consideration of the activity of the biological material, the stability of the structure, and the like. It can be used as a medium.

  Specific operations for mixing the prepared biological material, binding compound, medium, additive and the like are also arbitrary. For example, a solution or dispersion of a biological material and a solution or dispersion of a binding compound may be mixed, or a solution or dispersion of a biological material and a solid binding compound may be mixed. The substance and the binding compound solution or dispersion may be mixed, or the solid biological substance and the binding compound may be mixed with the solvent.

  By mixing the biological material and the binding compound, a particulate lump is formed, and the particulate lump aggregates to constitute a specific gel. The formation process of the specific gel is not clear, but can be estimated as follows. That is, when a mixed liquid in which a biological substance and a binding compound coexist is prepared, the biological substance and the binding compound are bound in the mixed liquid (see FIG. 2A), and particles as shown in FIG. A clump is generated. Confirmation of a particulate lump in the process of forming such a specific gel can be performed by dynamic light scattering measurement or the like. For example, when a protein of about 10 nm and a binding compound of about 10 nm are mixed in a solution, a submicron-order particulate mass may be confirmed. As shown in FIGS. 1 (a) and 1 (b), this particulate mass is a specific gel having a structure in which the particulate mass is assembled in a chain shape and / or a mesh shape as shown in FIGS. Is presumed to form. Further, at this time, it is presumed that the particulate mass to be bonded may be bonded to each other by a biological substance and a binding compound, and the particulate mass may be bonded to each other to form a specific gel. In this case, since the specific gel becomes more stable, it can be applied to a wide range of environments and uses, which is preferable.

[Spotting process]
The obtained mixed liquid is spotted at a desired position on the substrate. Here, spotting broadly refers to an operation of attaching a target liquid droplet to a solid-phase carrier (here, a substrate).

  There is no limitation on the device for spotting (referred to as “spotter”). For example, a pin type spotter can be used. Pin-type spotters allow liquid to adhere to the tip of a columnar pin, or after the liquid has been sucked into the interior of a cylindrical pin by surface tension, by bringing the pin into contact with or close to the substrate. The droplet is transferred to the substrate. Examples thereof include GENOMIC SOLUTIONS microarrayer OmniGrid (TM) Microarrayer Series. A spotter of a type that causes droplets to fly may be used as the spotter. Examples of spotters that cause droplets to fly include a piezo method and those using a solenoid valve. As an example, GENOMIC SOLUTIONS SynQUAD (TM) Systems can be cited.

  Further, spotting for forming one spot may be performed only once or may be performed twice or more. In particular, in the case of forming a minute spot, the medium in the liquid is quickly dried and the droplet tends to volatilize within a few minutes. Therefore, from the viewpoint of forming a thick spot, it is performed twice or more. It is preferable to perform spotting.

  The spotting position, the amount of droplets to be spotted, the number of spottings, etc. may be appropriately set according to the position, size, thickness, etc. of the spot to be formed.

[Drying process]
After the droplet of the mixed liquid is attached to the substrate by spotting, the medium in the droplet is preferably dried in order to fix the specific gel on the substrate. A spot is formed by the immobilized gel. When the amount of the medium in the mixed liquid is large, a particulate lump or a specific gel may or may not be generated in the mixed liquid. Even in these cases, by concentrating the mixed solution in the middle of drying, the particulate lump is efficiently generated, and the specific gel is efficiently generated. Further, even when the above-mentioned mixed solution contains a particulate lump or a specific gel fragment, the particulate lump or the specific gel can be further formed by concentration and drying.

  Although the method of drying a liquid mixture is arbitrary, For example, ultrafiltration, reduced pressure drying, etc. are mentioned. In addition, drying or concentration may be performed simply by evaporation under normal pressure. The temperature condition for drying the mixed solution is arbitrary, but from the viewpoint of avoiding denaturation of biological substances, it is usually 25 ° C. or less, preferably 10 ° C. or less. In addition, the pressure condition for drying the mixed solution is arbitrary, but it is usually desirable to reduce the pressure to a normal pressure or lower.

[Other processes]
In the gel spotting step, steps other than those described above may be performed as long as the gel can be immobilized on the substrate surface. For example, a mixture of a biological material, a binding compound, and a medium is centrifuged prior to spotting, a poor solvent of a specific gel is mixed into the mixture, or an additive such as ammonium sulfate is mixed. The production of particulate lumps may be promoted. This is to increase the probability that the biological substance and the binding compound are in contact with each other to facilitate the formation of particulate lumps, and to increase the probability that the particulate lumps are in contact with each other to facilitate the generation of a specific gel.

  For example, as long as a gel spot can be formed, components other than the biological material, the binding compound, and the medium may be mixed in the mixed solution. When to mix is arbitrary. Moreover, other components may be used alone or in combination of two or more in any combination and ratio.

  Furthermore, the liquid mixture in which the biological material and the medium are mixed and the liquid mixture in which the binding compound and the medium are mixed are spotted separately on the substrate, and the biological material and the binding compound are reacted in the droplets on the substrate so that the specific gel Then, the medium may be dried to form spots.

[Washing process]
You may make it wash | clean a chip | tip after the drying process of the above-mentioned gel spotting process. By washing the chip, the unreacted biological material and polymer can be removed. At this time, if an appropriate cleaning liquid is used, the gel is bonded to the substrate, so that the gel is not detached by this cleaning process, and the performance of the chip is not impaired.

  Further, after the drying step of the above-mentioned gel spotting step, a ligand spotting step described later may be performed without washing the chip. In this case, if a part of the ligand binds to the unreacted biological material used for spotting the gel and the amount of ligand immobilized on the gel spot decreases, the concentration and amount of the ligand should be adjusted appropriately. The problem is solved by choosing. In addition, according to the present method, there is a process advantage because the cleaning step after the preparation of the gel spot can be omitted.

[1-3. Ligand spotting process]
In the ligand spotting step, a ligand solution is spotted on the spot formed in the gel spotting step, and the ligand is immobilized on the spot.

[1-3-1. Ligand solution]
The ligand solution is a solution in which the ligand is dissolved in an appropriate solvent.

[Ligand]
The ligand is not limited, and any substance that can be immobilized on a gel can be used. Further, for example, a substance that can be bound and immobilized on a gel via a linker (described later) without being directly bound to the gel can also be used as a ligand.

  Examples of ligands include proteins, saccharides, low molecular compounds, physiologically active substances and the like. Examples of proteins include antibodies such as IgG, IgM, IgA, IgD, and IgE; plant-derived lectins, animal-derived lectins, lectins such as artificially produced lectins; and the ability to bind to other substances such as drugs. The protein which has is mentioned.

  “Saccharides” include monosaccharides and derivatives thereof (hereinafter collectively referred to as “monosaccharides”), and compounds and derivatives thereof (hereinafter collectively referred to as “monosaccharides”) in which two or more monosaccharides are linked by glycosidic bonds. "Polysaccharide"). In addition, polysaccharides are sometimes referred to as “sugar chains”. Saccharides can be used as ligands, both naturally occurring and synthesized molecules. In addition, a monosaccharide having a molecular structure that exists in nature or a polysaccharide composed thereof, or those obtained by adding a modification such as addition of a functional group or substitution to the polysaccharide can be used as a ligand. Among them, a monosaccharide having a molecular structure that exists in nature or a polysaccharide composed thereof is preferable.

  The type of monosaccharide is not particularly limited, and any monosaccharide can be used, and any of aldose, ketose and the like may be used. The number of carbon atoms in the main skeleton is not particularly limited, but is usually 5 or more, preferably 6 or more, and usually 9 or less, preferably 8 or less, more preferably 7 or less. That is, pentose (carbon number 5) and hexose (carbon number 6) are preferable, and hexose (carbon number 6) is particularly preferable. Specific examples of pentose include xylose, ribose, ribulose, xylulose and the like. Specific examples of hexose include glucose, mannose, fructose, galactose and the like. As other naturally-derived sugars, sialic acid (9 carbon atoms) is preferable.

  Examples of monosaccharides having a naturally occurring molecular structure include amino sugars (glycosamine) such as glucosamine, galactosamine, neuraminic acid; uronic acids such as glucuronic acid and iduronic acid; aldonic acids such as gluconic acid; Sorbitol), ribitol, xylitol and the like; deoxy sugars such as oxyribose, fucose and the like. In addition, a monosaccharide having a naturally occurring molecular structure having a substituent such as an acetyl group on a nitrogen atom or an oxygen atom of the monosaccharide described above can also be used. As examples of such derivatives of monosaccharides having an acetyl group on the nitrogen atom, N-acetylglucosamine, N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, N -Sialic acid such as glycylneuraminic acid.

  On the other hand, polysaccharides (sugar chains) are those in which two or more monosaccharides (monosaccharides and derivatives thereof) are directly connected by glycosidic bonds as described above. The number of monosaccharides constituting the polysaccharide is not particularly limited, and may be selected from a practical point of view such as ease of preparation and its function. "Sugar" may be referred to). The number of monosaccharides constituting the oligosaccharide is preferably 12 or less, more preferably 10 or less, particularly 6 or less, and especially 5 or less.

  The kind of monosaccharides constituting the polysaccharide is arbitrary, and can be arbitrarily selected from the above-mentioned monosaccharides (monosaccharides or derivatives thereof) according to the purpose. The polysaccharide may be composed of one kind of monosaccharide (homopolysaccharide, simple polysaccharide), or may be composed of two or more kinds of monosaccharide (heteropolysaccharide, complex polysaccharide). . Specific examples of the proteoglycan and glycosaminoglycan include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate.

  Examples of the ligand include metal chelates, vitamins, glutathione, boronic acids, antigens, nucleic acids, physiologically active substances, lipids, hormones, environmental hormones, and chelate-forming groups. When a metal chelate, glutathione, saccharide or the like is used as a ligand, the chip of the present invention is used when polyhistidine (His-tag), glutathione-s-transferase (GST), or maltose binding protein is used as an affinity tag. Thus, the substance to be detected can be detected effectively. If saccharide is used as the ligand, lectin, saccharide-binding protein, extracellular matrix, virus and the like can be detected using the chip of the present invention. When boronic acid is used as a ligand, a saccharide and a compound containing a sugar (for example, a protein having a sugar chain) can be effectively detected using the chip of the present invention. When an enzyme is used as a ligand, the substrate of the present invention can be interacted with, and an effective immobilized enzyme can be provided. By using lipids as ligands, lipid binding proteins and the like can be detected using the chip of the present invention. When hormones or environmental hormones are used as ligands, biological substances that bind to them can be detected using the chip of the present invention. When biotin is used as the ligand, avidin, streptavidin, avidin derivatives, or the like can be detected using the chip of the present invention. When an antibody or antigen is used as the ligand, the antigen can be detected when the ligand is an antibody, and the antibody when the ligand is an antigen, and the antibody can be detected using the chip of the present invention. If a lectin is used as a ligand, saccharides can be detected using the chip of the present invention. If a nucleic acid (for example, a nucleic acid molecule such as DNA or RNA or a peptide nucleic acid such as PNA) is used as a ligand, the chip of the present invention can be used for gene interaction analysis. When a physiologically active substance such as FK506 (Chemistry & Biology Vol. 9 691-698 (2002)) is used as a ligand, a binding protein such as FKBP12 is detected, or a protein such as a receptor contributing to a disease is detected. The chip of the present invention can be used as an action mechanism elucidation tool or screening tool of a drug candidate compound. If a chelate-forming group is used as a ligand, the chip of the present invention can be used for separation of metal ions and the like. In addition, 1 type may be used for a ligand and it may use 2 or more types together by arbitrary combinations and a ratio.

  The concentration of the ligand in the ligand solution is arbitrary as long as the ligand can be immobilized on the spot, but is usually 1 ng / ml or more, preferably 10 ng / ml or more, more preferably 100 ng / ml or more, and usually 100 mg / ml or less. , Preferably 10 mg / ml or less, more preferably 1 mg / ml or less. If the concentration is too low, the amount of immobilized ligand is small, and if the resulting chip is used in an assay, it may not be able to detect sufficient detection material, and if it is too high, it is economically disadvantageous. In addition to this, spotting may not be possible due to changes in the viscosity of the ligand solution.

〔solvent〕
The solvent contained in the ligand solution is not limited as long as it can dissolve the ligand, and both an aqueous solvent and an organic solvent can be used. Examples of the aqueous solvent include water and buffer solutions, and water is usually used from the viewpoint of suppressing the deactivation of biological substances. On the other hand, the organic solvent is, for example, methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, isobutyl alcohol, n-amyl alcohol, etc. 5 alkyl alcohols; amides such as dimethylformamide and dimethylacetamide; ketones or ketoalcohols such as acetone and diacetone alcohol; ethers such as tetrahydrofuran and dioxane; polyalkylene glycols such as polyethylene glycol and polypropylene glycol; ethylene Glycol, diethylene glycol, propylene glycol, butylene glycol, triethylene glycol, 1,2,6-hexanetriol, thiodiglycol Alkylene glycols containing 2 to 6 carbon atoms such as ethylene and hexylene glycol; ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, triethylene glycol monomethyl ether, triethylene glycol Lower alkyl ethers of polyhydric alcohols such as ethylene glycol monoethyl ether; lower dialkyl ethers of polyhydric alcohols such as triethylene glycol dimethyl ether and triethylene glycol diethyl ether; glycerin; sulfolane; N-methyl-2-pyrrolidone; 1,3-dimethyl-2-imidazolidinone; dimethyl sulfoxide (DMSO), dimethylformamide (DMF); Organic solvents. Of these, methanol, ethanol, DMSO, DMF and the like are preferable, and among them, DMSO or DMF is more preferable. Moreover, a solvent may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio. For example, when the ligand is difficult to dissolve in water, a mixed solvent in which water and a water-soluble organic solvent are mixed may be used. In this case, the ratio of the water-soluble organic solvent to the total solvent of water and the water-soluble organic solvent is arbitrary, but is usually 80% by weight or less, preferably 70% by weight or less, more preferably 50% by weight or less.

[Other ingredients]
The ligand solution may contain components other than the ligand and the solvent as long as the ligand can be immobilized on the spot. In addition, 1 type may contain these components and 2 or more types may contain them by arbitrary combinations and a ratio.

[1-3-2. Spotting method of ligand solution]
The ligand is immobilized on the spot by spotting the ligand solution on the spot of the gel formed on the substrate surface. At this time, the spotting method is not limited, and spotting can be performed using any spotter.

  As an example of the spotter, the same spotter as exemplified as a spotter for spotting a mixed solution in order to immobilize the gel may be mentioned.

  The spotting of the ligand solution may be performed only once per spot, or may be performed twice or more. In particular, in the case of a minute spot such as the spot of the present embodiment, the solvent quickly dries out from the ligand solution and volatilizes within a few minutes. Therefore, by spotting twice or more, a relatively large amount of ligand can be obtained. It can be fixed. In addition, if the amount of spotted ligand solution is increased by performing spotting twice or more, the time required for the solvent to dry from the droplet of the ligand solution can be lengthened, and the gel forming the spot is formed. This also has the effect of extending the immobilization time of the ligand.

  The position of spotting the ligand solution is not limited as long as the spotted ligand solution droplet is in contact with the gel spot. However, the fact that the ligand solution is widely attached to the part other than the spot on the substrate greatly deviating from the spot increases the background when the chip is used for detection, and also causes noise when using the chip. Should be avoided.

  The conditions for spotting the ligand solution are arbitrary as long as the effects of the present invention are not significantly impaired. Among them, the temperature is usually 0 ° C. or higher, preferably 4 ° C. or higher, more preferably 10 ° C. or higher, particularly preferably 20 ° C. or higher, and usually 50 ° C. or lower, preferably 40 ° C. or lower, more preferably 37 ° C. It is as follows. The pressure is preferably atmospheric pressure.

  When spotting a ligand solution to a gel spot, the ligand usually contacts and binds to the gel. As a result, the ligand is immobilized on the spot. In this case, the ligand and the gel are bound by a strong bond such as a covalent bond, an interaction between biological substances, and a strong hydrophobic bond. Specific examples include an ester bond, an amide bond, and a biotin-avidin bond. It can also be coupled by reactions such as Schiff basification reaction, reductive amination reaction, Diels-Alder reaction, diene and dienofile reaction, azide and ethynyl group reaction, HIS tag and HIS tag fixing group reaction, etc. it can. However, the binding and reaction when the ligand is bound to the gel are not limited to those exemplified.

  In addition, the spotted ligand solution penetrates into the gel, and the ligand also binds to the gel inside. For this reason, the gel can be immobilized not only on the surface portion of the spot but also inside the spot, and the ligand can be immobilized at a high density. Usually, since the solvent contained in the spotted droplets volatilizes quickly, the solvent of the ligand solution does not remain in the spot after spotting of the ligand solution.

  In this way, a chip having a small gel spot with a ligand immobilized thereon can be easily spotted by spotting the ligand solution so that it is overlaid on the gel spot previously formed on the surface of the substrate. Can be manufactured. At this time, since the ligand solution is spotted only on the spot, the ligand solution adhering to the portion between the ligands can be reduced or eliminated as in the prior art, and the amount of ligand used can be greatly reduced.

  Furthermore, although the same ligand may be immobilized on each spot, different ligand solutions can be immobilized on each spot by spotting different ligand solutions. That is, by spotting a ligand solution containing different ligands to different spots, it is possible to form a large number of minute spots on a gel in which different ligands are immobilized. By using the chip thus obtained, it is possible to simultaneously measure the interaction between one or more substances to be detected and a plurality of ligands. In addition, when analyzing a sample containing a plurality of substances to be detected using the obtained chip, it is possible to measure the interaction of each ligand corresponding to each substance to be detected. The percentage of the substance can also be determined.

  A plurality of ligand solutions containing different types of ligands may be spotted on one spot. In this case, the chip can be used to measure a synergistic effect by immobilizing multiple types of ligands in one spot.

[More preferred embodiment]
By the way, as a result of further investigation by the present inventors, since the above-described manufacturing method produces a microscale chip, the position of the gel spot formed on the substrate and spotting of the ligand solution are caused by the expansion and contraction of the substrate. It turned out that the position to do may shift. The degree of such deviation varies depending on the material of the substrate; the solvent to be used; the conditions such as temperature and humidity; or the combination and order thereof, and therefore usually differs at each spotting and is very difficult to control. is there.

  On the other hand, since the gel has a three-dimensional network structure, if the ligand solution is spotted, the ligand solution quickly penetrates into the gel, and even if the spotting position of the ligand solution is shifted as described above. It was thought that the ligand solution spread throughout the spot by permeation. However, at the microscale, the amount of spotted droplets is very small and the solvent in the droplets volatilizes quickly, so that the solvent volatilizes faster than the ligand solution penetrates the entire spot gel. The ligand may be immobilized only partially. In addition, there are cases where it takes time for the ligand solution to penetrate due to the nature of the gel itself.

  The time required for the solvent in the droplets to volatilize is not limited, but it is highly effective when it is within 30 minutes, of which within 10 minutes, among them, within 5 minutes, more particularly within 2 minutes, most Especially effective when it is within 1 minute.

  Therefore, when the position of the spotter 2 deviates from the position in front of the spot 3 as shown in FIG. 3A due to the expansion and contraction of the substrate 1, the spotted droplet of the ligand solution as shown in FIG. 4 may partially deviate from the spot 3. In this case, as described above, the ligand solution does not permeate the entire spot 3 due to volatilization of the solvent in the droplet 4 or the like, and as shown in FIG. 3a and the non-immobilized portion 3b may be formed. Such a problem occurs for the first time by the configuration of the present invention, but a solution is desired from the viewpoint of increasing the yield of manufactured chips. Therefore, it is preferable to perform the following operations in the ligand spotting step.

(1) Spotting using a large spotter;
First, as shown in FIGS. 4A to 4C, the ligand covers the spot 3 with the spotter 2 that can spot a wider area than the spot 3 at once (ie, once). It is preferred to spot the solution. That is, as shown in FIG. 4A, the ligand solution is spotted using a spotter 2 that is large enough to spot a range wider than the spot 3 at a time. As a result, as shown in FIG. 4B, the entire spot 3 can be covered with the droplet 4 of the ligand solution. Thereby, even when the solvent is volatilized before the ligand solution penetrates in the radial direction of the spot 3 (lateral direction in the figure), the ligand is immobilized on the entire spot 3 as shown in FIG. It is possible. Since the thickness of the spot 3 is usually much smaller than the size in the radial direction, the ligand solution can sufficiently penetrate in the thickness direction of the spot 3.

  In this case, the range in which the spotter 2 to be used can be spotted at a time may be set according to the degree of expansion / contraction of the substrate 1. When the substrate 1 expands / contracts, the degree of expansion / contraction is not exactly reproduced and tends to generate a certain amount of error. Therefore, in consideration of this deviation error, the entire spot 3 can be detected even when the maximum error is expected. It is preferable to use the spotter 2 capable of spotting a wide range that can be covered. The position of the deviation and the degree of error may be set according to the material of the substrate 1 and the manufacturing process. For example, when the substrate is made of resin, the diameter of the spotting spot 2 at a time is preferably 1.05 times or more, more preferably 1.5 times or more, particularly preferably 2 times the diameter of the spot 3. That's it. However, if the spotter 2 is spotted too wide, the amount of ligand solution used tends to increase.

  Further, the center position of the spotter 2 when spotting the ligand solution does not necessarily need to be at the center position of the spot 3. If the deviation of the center position is sufficiently planned, the spotted droplet of the ligand solution is used. 4 can completely cover the spot 3.

(2) Multiple spotting while shifting the position;
Second, as shown in FIGS. 5A to 5D, when spotting one spot 3, the spotter 2 is shifted and spotted a plurality of times to fix the ligand to the spot 3. Is preferred. That is, as shown in FIGS. 5A to 5C, the ligand solution is sequentially spotted while shifting the position of the spotter 2, thereby moving the spottable range, and as a result, a droplet of the ligand solution is applied to the entire spot 3. By attaching 4, the ligand can be immobilized on the entire spot 3 as shown in FIG.

  In this case, the range in which the droplet 4 of the ligand solution is attached by spotting a plurality of times may be set according to the degree of expansion / contraction of the substrate 1. As described above, when the substrate 1 expands / contracts, the degree of expansion / contraction is not strictly reproduced, and a certain amount of error tends to occur. It is preferable to perform spotting while shifting the position within a range where the droplet 4 can be attached to the entire spot 3.

  The position of the shift and the degree of error may be set according to the material of the substrate 1 and the manufacturing process. If the arrangement of the spotter 2 at the time of multiple spotting is sufficiently planned, the droplet 4 of the ligand solution can be attached to the entire spot 3 by multiple spotting.

  Further, when spotting is performed a plurality of times, spotting may be performed before the next spotted droplet is dried, or may be performed after the drying. However, the next spotting is preferably performed before drying from the viewpoint of uniformity of the amount of immobilized ligand.

  The number of spottings per spot 3 is not limited as long as it is 2 times or more, but 3 times or more is preferable and 4 times or more is more preferable from the viewpoint of surely immobilizing the ligand on the entire spot 3. However, if the number of spottings is too large, the chip manufacturing efficiency is lowered, so that it is usually 6 times or less.

  Further, although there is no limit to the extent to which the spotter 2 is shifted every time spotting is performed, the diameter of the range in which the spotter 2 is spotted at a time is usually 0.1 times or more, preferably 0.2 times or more, particularly preferably 0. It is desirable to shift the distance by 5 times or more, and it is usually desirable to shift the distance by 2 times or less, preferably 1 time or less.

  Further, there is no restriction on the direction in which the spotter 2 is displaced. As a result, the spot solution can be displaced in any direction as long as the ligand solution can be spotted on the entire spot 3. For example, it may be shifted so as to go straight in one direction, or the direction may be changed every time spotting. For example, it is preferable to shift the spotter 2 so as to draw a circle or a rectangle because the ligand solution can be spotted on the entire spot 3 more reliably.

(3) Summary of the first and second examples In general, even when the ligand is immobilized only on a very small part of the spot, the influence is within an allowable range, or the ligand is equivalent in other effective spots. There is no problem if it is partially fixed in proportion. However, when the spot is very small as in the chip of this embodiment, the effect of the deviation is large. On the other hand, as in the first and second examples, if a spotter that can spot a sufficiently wide range at a time is used, or if spotting is performed multiple times while shifting the position, the ligand is added to the entire gel spot. Can be fixed. As described above, it is preferable that the ligand is immobilized on the entire spot to increase the signal intensity when the chip is used, and to reduce the variation between the spots. Furthermore, if the operations as in the first and second examples are performed, the adhesion of the ligand to the surface portion of the substrate where no gel is present can be minimized or within a planned range.

  The operations as described in the first and second examples above are particularly effective when the size of the gel spot is different from the size of the spotting range of the ligand solution, or when the gel spot and the spot position of the ligand solution are caused by the expansion and contraction of the substrate. This is effective when deviation occurs. Further, the operations as in the first and second examples described above are performed when spotting a small spot of the ligand solution on the spot of the gel, when the ligand solution is dried before the ligand solution penetrates the entire gel, that is, This is particularly effective when the time required for the ligand solution to dry is shorter than the time required for the ligand solution to penetrate the entire gel. Therefore, the operations as in the first and second examples are particularly useful when the gel has water repellency.

[Others]
In the ligand spotting process, spotting can be performed a plurality of times without shifting the position of the spotter as described above, but in this case, the spotted droplets can be removed by continuously performing spotting. It is also possible to increase the size and cover the entire spot.

[1-4. Others]
In the chip manufacturing method of this embodiment, steps other than the above-described gel spotting step and ligand spotting step may be performed. For example, you may perform the linker spotting process, linker mixing process, washing | cleaning process, etc. which are demonstrated below.

[Linker spotting process]
In the linker spotting step, after the gel spotting step and before the ligand spotting step, a solution containing a linker capable of binding to the gel and the ligand (hereinafter sometimes referred to as “linker solution”) is brought into contact with the spot, In this step, the linker is immobilized on the spot.

  A linker is a molecule that connects a gel and a ligand. When the ligand is immobilized on the spot, the gel constituting the spot and the ligand may be directly bonded to each other via some kind of linker. Usually, by using a linker, the interaction between the ligand and the substance to be detected can be enhanced when the chip is used for detection. For this reason, it is preferable to bind a ligand and a gel indirectly using a linker.

  A linker that can bind to both the gel and the ligand is used. Therefore, for example, when a specific gel is used as the gel, one or both of the biological material and the binding compound that can bind to the linker is used. In this case, the bond between the biological substance and the binding compound and the linker may be any bond depending on the type and application of the chip to be manufactured. Examples of bonds include covalent bonds, ionic bonds, chelate bonds, coordination bonds, hydrophobic bonds, hydrogen bonds, van der Waals bonds, and electrostatic force bonds, but other bonds that do not fall into these categories. It may be. When the ligand and the specific gel are indirectly bound via a linker, the ligand, the biological material, and the binding compound may not necessarily be directly bound.

  There is no limitation on the type of linker, and it is optional as long as the effects of the present invention are not significantly impaired. Among them, it is preferable to use a hydrophilic molecule (hydrophilic molecule) as the linker. In order for the linker to be hydrophilic, in the molecule constituting the linker, for example, an amino group, —NHCO— group, hydroxycarbonyl group, hydroxyl group, sulfonic acid group, glycidyl group, nitrile group, polyethylene glycol, etc. It is desirable to include these atomic groups. Among them, since polyethylene glycol chains are known to suppress nonspecific adsorption of proteins, it is desirable to use a linker containing polyethylene glycol chains when using proteins as biological substances or target substances. In addition, a linker may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

  The solvent contained in the linker solution is not limited as long as it dissolves the linker. For example, the same solvent as the solvent of the ligand solution can be used. In addition, the solvent of a linker solution may also use 1 type and may use 2 or more types together by arbitrary combinations.

  The concentration of the linker in the linker solution is not limited, but is usually 0.01% by weight or more, preferably 0.1% by weight or more, more preferably 0.5% by weight or more, usually 10% by weight or less, preferably It is 2% by weight or less, more preferably 1% by weight or less. If the concentration is too low, the amount of linker immobilized on the gel may be insufficient, and a sufficient amount of ligand may not be immobilized on the gel. If the concentration is too high, denaturation of the gel may occur. .

  There is no limitation on the method of bringing the linker solution into contact with the spot. For example, the chip may be immersed in a linker solution, or the linker solution may be applied to the substrate. Among these, from the viewpoint of suppressing the amount of the linker solution used, it is preferable to spot the linker solution on spots on the substrate in the same manner as spotting the ligand solution in the above-described ligand spotting step. Thereby, a linker couple | bonds with the gel which comprises a spot. If the ligand spotting step is performed after the linker spotting step, the ligand is bound to the linker, and as a result, the ligand is immobilized on the spot.

[Linker mixing step]
The linker mixing step is a step in which a linker that can bind to the gel and the ligand and the ligand solution are mixed before the ligand spotting step.

  If the linker is mixed with the ligand solution in advance, the ligand and the linker are bonded in the ligand solution. Then, when a ligand spotting step is performed, the ligand binds to the gel via the linker portion, and as a result, the ligand is immobilized on the spot. This makes it possible to enhance the interaction between the ligand and the substance to be detected when the chip is used.

  The amount of linker to be mixed may be set according to the amount of ligand in the ligand solution. Usually, it is usually 0.01 mol times or more, preferably 0.1 mol times or more, more preferably 1 mol times or more, usually 1000 mol times or less, preferably 100 mol times or more with respect to the amount of the ligand in the ligand solution. The amount is not more than twice, more preferably not more than 10 mole times. If the amount is too small, the amount of linker immobilized on the gel may be insufficient, and a sufficient amount of ligand may not be immobilized on the gel. If the amount is too high, denaturation of the gel may occur. .

[Washing process]
You may make it wash | clean a chip | tip after the ligand spotting process mentioned above. By washing the chip, the ligand attached to the substrate surface can be removed. At this time, if an appropriate washing solution is used, the ligand immobilized on the spot is bonded to the gel, and therefore, this washing step does not detach from the spot and does not impair the performance of the chip.

  After the spotting step of the gel, the ligand is spotted. After that, inactivating the active sites of the biological material constituting the gel also gives favorable results to the assay results. In some cases, the solution containing the substance to be detected may contain a substance that binds to the active site of the gel without binding to the ligand. For example, if the gel contains avidin, it is clear that compounds with biotin bind strongly to the active site and may detect false signals. In order to solve this problem, it is preferable to use a substance that binds to the active site of the biological substance constituting the gel and has a structure that itself hardly causes nonspecific adsorption. To illustrate the case where the gel contains avidin, after immobilizing a ligand having a biotin group on the gel spot by overstrike, contact with a substance that has biotin in the molecule and does not cause nonspecific adsorption In addition to solving this problem, the effect of the present invention can be further enhanced.

Such a compound is not particularly limited as long as it serves its purpose. Examples of structures that hardly cause non-specific adsorption include those containing polyethylene glycol in the main chain and those containing phosphorylcholine groups in the side chain, and among them, those having polyethylene glycol in the main chain are preferred. The number of ethylene glycol units of polyethylene glycol is not limited, but is preferably 1 or more, and more preferably 2 or more. The upper limit is not particularly limited, but is preferably 10,000 or less, more preferably 1000 or less, and particularly preferably 100 or less, and particularly preferably 10 or less.
In the case where the gel contains avidin, for example, polyethylene glycol having biotin group and methoxy group at both ends can be mentioned, and the gel contains albumin, and the ligand is immobilized after NHS of carboxyl group or amino group in the molecule For example, polyethylene glycols having an amino group and a methoxy group at both ends, ethanolamine, ethanolamine methyl ether and the like can be mentioned. Moreover, although this contact method is not limited, it may be dripped so that a gel may be covered, and when the gel is installed in the flow path, you may make it contact by liquid passing. In addition, in the same manner as when the ligand is overprinted on each gel spot where the ligand is not immobilized, it may be overprinted. Furthermore, these may be combined. Further, in this step, the compound may be immobilized by bringing the solution of the compound into contact with a gel spot on which the ligand is immobilized.

[2. Chip configuration]
By the above-described chip manufacturing method of the present invention, a chip of the present invention is obtained that includes a substrate and a plurality of spots including gels fixed to the substrate, and the ligand is immobilized on the spot. In the chip of the present invention, since the ligand is immobilized on the entire spot, the sensitivity of the chip and the variation between spots do not occur. For example, the quality as a detection chip (described later) can be improved.

  In addition, since the ligand is immobilized by spotting the ligand solution on spots previously formed on the substrate during manufacturing, in the chip of the present invention, between the spots on the substrate surface. The ligand solution does not adhere to this part, and the ligand is not immobilized. That is, a portion where a ligand is not immobilized is formed between spots on the substrate surface. For this reason, a high S / N ratio (Signal to Noise ratio) is obtained.

  Furthermore, since the ligand solution is spotted for each spot with respect to spots previously formed on the substrate during manufacturing, the chip of the present invention can control the type and amount of the ligand immobilized for each spot. . Thereby, the chip of the present invention immobilizes the first ligand in at least one spot among the spots formed on the same substrate, and the second ligand (first ligand in the other at least one spot. Different types of ligands) can be immobilized. That is, it is possible to immobilize spots by immobilizing different ligands on a plurality of gel spots. For this reason, when the chip | tip of this invention is used as a chip | tip for a detection, multiple examples of interaction with a ligand and a to-be-detected substance can be measured simultaneously.

  Further, in the chip of the present invention, the gel constituting the spot and the ligand are bonded by a strong bond such as a covalent bond, an interaction between biological substances, a strong hydrophobic bond, etc. Desorption and alteration of the ligand during use of the chip under washing conditions can be suppressed.

  In the chip of the present invention, the substrate, gel and ligand are as described above. Further, the spot diameter, the thickness, and the interval between the spots are as described above. The amount of ligand immobilized on the spot can be set arbitrarily, but it is usually preferable that more ligands are immobilized. Specifically, the content ratio represented by “(ligand weight) / (whole spot weight)” is preferably 0.0001% by weight or more, more preferably 0.0003% by weight or more. The content is preferably 0.01% by weight or more. The content ratio of the ligand can be measured by, for example, elemental analysis or amino acid analysis.

  By the way, when distinguishing the chip of the present invention from other chips, for example, the structure may be analyzed by various known methods. Although there is no restriction | limiting in the analysis method in that case, For example, XPS analysis, mass spectrometry, etc. are mentioned.

When performing mass spectrometry, for example, it is possible to redissolve a ligand present on the surface or spot of the chip of the present invention in an appropriate solvent and perform analysis using the obtained solution. It is convenient to apply ions directly to the chip or spot and observe the ionized molecules and atoms. Examples of such a method include time-of-flight secondary ion mass spectrometry (ToF-SIMS). This method is, for example, a method in which accelerated Au ⁺ ions are applied to the surface and the generated secondary ions are collected, and a commercially available apparatus can be used.

  Further, for example, by labeling a substance capable of binding to a ligand and bringing it into contact, information on the presence or absence of the ligand and the state thereof can be obtained. As a labeling method, a known method can be used. For example, an isotope label, a fluorescent label, an enzyme label, a gold colloid label, and the like can be used.

[3. Detection method and apparatus]
Although there is no restriction | limiting in the use of the chip | tip of this invention mentioned above, It is preferable to use as a detection chip | tip used for the detection of a to-be-detected substance from the point which utilizes effectively the point which can integrate | stack a spot. In particular, it is preferably used in a detection apparatus that detects a biological substance as a substance to be detected (hereinafter sometimes referred to as “biosensor” as appropriate).

  At the time of detection, the measurement may be performed in a state where the gel is immersed in a liquid. The measurement method in this case is not particularly limited, but is particularly effective when measuring and detecting fluorescence. In the dry state, the gel may have autofluorescence emission due to its constituent components and fine structure, which can be reduced. That is, scattering occurs due to a difference in refractive index between the air in the components of the gel and a substance such as a biomolecule or a polymer constituting the gel, and this may be observed as autofluorescence. When immersed in a liquid, the contained air is replaced by the liquid, and the refractive index of the protein, polymer, or the like is close, so that the scattering becomes very small. Liquids that can be used for this purpose include oils, water, buffer solutions such as phosphate buffer solutions and carbonate buffer solutions. In addition, for example, an organic solvent, a surfactant, a protein, a salt, or the like may be dissolved in the liquid.

  The organic solvent is not limited, and examples thereof include those that can be dissolved in water, such as DMSO and isopropanol. The surfactant is not limited, and examples include tween 20 and triton X. The protein is not limited, and examples thereof include BSA. Although salts are not limited, For example, sodium chloride, sodium azide, etc. are mentioned. Further, for the purpose of preventing stray light from the outside, a coloring agent such as a dye or a pigment may be mixed in the liquid. In this case, the colorant is preferably black.

  There is no limitation on the method of immersing the gel in a liquid, but examples thereof include a method of dropping a liquid so as to cover the entire gel produced on a substrate, and a method of placing and passing the gel in a flow path. When a gel is installed in the flow path, it is possible to perform detection by continuously circulating several kinds of liquids. As such a method, the substance to be detected is first immobilized on the gel on which the ligand is immobilized. A solution containing a fluorescent-labeled antibody that is bound to the substance to be detected, followed by a solution containing a fluorescent-labeled antibody to immobilize the antibody, and finally a washing solution. Then, the labeled antibody not bound can be removed, and the fluorescence can be measured. A cleaning step by passing a cleaning solution may be inserted between each step.

The thickness of the liquid for immersing the gel is not particularly limited, as long as the liquid is immersed in the entire gel, but preferably twice or more of the maximum thickness of the gel, and more preferably 10 or more times. Of these, 100 times or more is preferable, and 500 times or more is particularly preferable. In addition, the depth of the liquid is preferably 50 μm or more, more preferably 100 μm or more, and particularly preferably 500 μm or more. The upper limit of the depth of the liquid is not particularly limited, but is preferably 1 cm or less from the viewpoint of handling, more preferably 5 mm or less, particularly preferably 2 mm or less, and particularly preferably 1 mm or less.
In addition, performing measurement while immersed in a solution has an advantage of increasing throughput because a drying step can be omitted after the final step of the assay.
That is, when measuring fluorescence emission, the effect of this invention can be further strengthened by measuring in the state immersed in the solution.

  When the chip of the present invention is used in a biosensor, a biosensor having the chip of the present invention can be obtained by using the sensor surface of the biosensor as a substrate surface to form a chip by immobilizing a ligand according to the procedure described above.

[3-1. Sensor type]
Examples of the biosensor include a sensor using fluorescent light emission, FET, ELISA, chemiluminescence, SPR, QCM, cantilever, magnetic material, optical waveguide, surface acoustic wave, and the like.

  For example, when configured as a fluorescence sensor, a spot on which a ligand is immobilized is formed on a glass substrate or plastic substrate serving as a sensor surface, and cleaning is performed after contacting the ligand and a fluorescently labeled target substance. Thus, the interaction between the ligand and the substance to be detected can be detected as fluorescence emission. In addition, even if the substance to be detected is not fluorescently labeled, washing is performed after bringing the ligand into contact with the substance to be detected, and further, a substance that interacts with the substance to be detected and is labeled (for example, an antibody) By washing after contacting, the substance to be detected can be detected as fluorescence. The wavelength of the fluorescence emission of the dye of the fluorescent label is preferably matched with the measurement wavelength.

When measuring and detecting fluorescence emission, it is easy to handle the chip in a dry state at the time of measurement because of easy handling. However, in the dry state, as described above, the gel spot itself may fluoresce. If the fluorescence emission is measured in such a state, there is a possibility that the measurement light is mixed with the autofluorescence emission of the gel and the fluorescence emission for detecting the substance to be detected. In particular, when detecting a low-concentration target substance, the intensity of the target fluorescence emission becomes small, so that the self-fluorescence emission of the gel becomes relatively strong and reliable measurement cannot be performed. This means that the lower detection limit concentration cannot be lowered. Therefore, in addition, it is preferable to perform fluorescence measurement at a wavelength where the self-fluorescence emission of the gel is weak.
For this purpose, the wavelength of the detection light may be selected according to the convenience of the substance to be detected and the device, but is preferably 580 nm or more, more preferably 610 nm or more, and particularly preferably 630 nm or more, particularly 650 nm or more. preferable. Although there is no particular upper limit, it is preferably 2500 nm or less, preferably 1700 nm or less, more preferably 1000 nm or less, particularly preferably 800 nm or less, and particularly preferably 800 nm or less. Is preferred. The dye molecule having an emission wavelength at such a wavelength is not particularly limited, but as commercially available products, Cy5 (TM), Alexa Floor 633 (TM), Alexa Floor 635 (TM), Alexa Floor 647 (TM), Examples include BODIPY 650/665 (TM), DiD (TM), TOTO-3 (TM), DDAO phosphate (TM), among which Cy5 (TM), Alexa Floor 633 (TM), Alexa Floor 635 (TM), Alexa Floor 647. (TM) is preferred, and among them, Cy5 (TM) and Alexa Floor 647 (TM) are preferred, and Cy5 (TM) is most preferred. In addition, a reagent for binding a dye molecule to a protein or antibody is commercially available, and it can also be used.
That is, when the fluorescence measurement is performed in a dry state, the effect of the present invention can be further enhanced by limiting the measurement wavelength.

  For example, when configured as an FET sensor, the interaction between the ligand and the substance to be detected is detected as a change in potential by forming a spot on which the ligand is immobilized on or near the gate electrode serving as the sensor surface. be able to.

  For example, when configured as an SPR sensor, by forming a spot on which a ligand is immobilized on a gold surface serving as a sensor surface, the interaction between the ligand and the substance to be detected is caused by a change in reflection angle due to surface plasmon resonance, or It can be detected as a reflection intensity at a specific reflection angle.

  For example, when configured as a QCM sensor, a spot in which a ligand is immobilized is formed on the surface of the crystal oscillator serving as a sensor surface, or a gold thin film is formed on the surface of the crystal oscillator and then the ligand is formed on the gold thin film. To form a fixed spot. As a result, the interaction between the ligand and the substance to be detected can be observed as a change in frequency.

  For example, when configured as a cantilever sensor, the surface of the cantilever serving as a sensor surface is formed of a material such as silicon, gold, or silicon oxide, and a spot on which a ligand is immobilized is formed on the surface. Thereby, the interaction between the ligand and the substance to be detected can be observed as a shape change such as a cantilever warp or a change in resonance frequency.

  Further, for example, when configured as a magnetic sensor, by forming a spot with a ligand immobilized on the sensor surface, the interaction between the ligand and a substance to be detected has a superconducting quantum interference device (superconducting quantum interference device). (Hereinafter abbreviated as “SQUID”) and a device using a Hall element.

  For example, when configured as an optical waveguide sensor, the optical path serving as the sensor surface is divided into two, and a spot on which a ligand is immobilized is formed on one part of the surface. When the interaction between the ligand and the substance to be detected occurs, a change in the speed or phase of light occurs, and this can be detected as interference with the other light.

[3-2. Sensor configuration]
There is no restriction | limiting in the specific structure of the said detection apparatus, What is necessary is just to select an appropriate thing according to the detection method to be used, the kind of specimen, etc. As a typical apparatus configuration, the above-described chip of the present invention, a sample supply unit for supplying the sample to bring the sample into contact with the chip, and the interaction between the ligand and the substance to be detected generated in the chip are shown. The structure which has a detection part for detecting is mentioned.

  When the chip of the present invention is used in a biosensor, the chip is usually configured to be detachable from the apparatus body. When the chip is used, the chip is attached to the chip mounting portion provided in the apparatus main body, and the detection target substance is detected. At this time, it is preferable to provide a flow path through which the specimen can be circulated in the apparatus main body, and to provide a chip mounting portion in the flow path. Thereby, a chip | tip can be attached in a flow path and the detection procedure can be implemented continuously.

[3-3. Detected substance]
There is no limitation on the target substance to be detected using the chip of the present invention. For example, when the chip of the present invention is used in a biosensor, examples of the target substance include nucleic acids, proteins, toxins, viruses. , Cells, saccharides, low-molecular compounds, or conjugates thereof.

  These to-be-detected substances interact with the ligand. The specific type differs depending on the type of ligand of the detection chip, and a substance that can interact with the ligand is detected as a substance to be detected, and other substances do not cause an interaction. Therefore, if the interaction is detected, it can be determined whether or not the substance to be detected is contained in the specimen. If the magnitude of the interaction can be measured, the amount or concentration of the substance to be detected can be measured from the magnitude of the interaction.

  Usually, the specimen is prepared in a liquid form such as liquid or slurry, and this specimen is brought into contact with a spot on the chip surface to detect a substance to be detected in the specimen. At this time, the substance to be detected is prepared in a dissolved or dispersed state in a solvent such as water. In addition, there is no restriction | limiting in the kind, quantity, density | concentration, etc. of the solvent in a test substance, It is arbitrary according to a to-be-detected substance.

  One of the substances contained in one specimen may be detected as a substance to be detected, but two or more kinds may be detected as a substance to be detected. As described above, since the chip of the present invention can adjust the ligand to be immobilized for each spot formed on the substrate surface, for example, for each spot or each group of spot groups having different ligands, the type, amount, etc. The ligand immobilization conditions can be set. Therefore, detection conditions can be set for each spot or for each group of spot groups having different ligands, and detection can be performed under a large number of conditions simultaneously as a so-called combinatorial assay. In this case, since many substances to be detected can be detected with one chip, it is efficient. In particular, the chip of the present invention is advantageous for this purpose because of its small spot scale.

[3-4. Other uses]
The chip of the present invention can also be used for applications other than the sensor described above. For example, the chip of the present invention can also be used to concentrate a substance to be concentrated. As an example, the target substance can be eluted by bringing the target target substance into contact with the chip, capturing the target substance on the ligand, and then dissociating it with an appropriate drug or condition. It is. Although there is no restriction | limiting in the substance to concentrate, Usually, the thing couple | bonded with a ligand is preferable. Further, it is possible to separate the separation target substance from the composition and purify the separation target substance by the same principle.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples, and can be arbitrarily modified without departing from the gist of the present invention.

[Reagent used]
Commercially available products were used as reagents and solvents used in the reaction.
The biotin hyaluronate used was Sigma-Aldrich. A hyaluronic acid binding protein manufactured by Seikagaku Corporation was used. Hereinafter referred to as “HABP”. Moreover, what labeled this with Cy3 is described as "Cy3-HABP."
As the Japanese elder collectin, a product manufactured by J-Oil Mills was used. Hereinafter referred to as “SSA”. Moreover, what labeled this with Cy3 is described as "Cy3-SSA."
For biotinylation of the amino group, EZ-NHS-Biotin and EZ-Link Sulfo-NHS-LC-Biotin manufactured by Pierce were used.
A lectin manufactured by Seikagaku Corporation was used. Hereinafter, wheat germ lectin, those labeled with Cy5, and those labeled with Cy3 are referred to as “WGA”, “Cy5-WGA”, and “Cy3-WGA”, respectively, and concabanaline A, and those labeled with Cy5, These are described as “ConA” and “Cy5-ConA”, respectively, and the castor bean lectin and the Cy5-labeled product thereof are described as “RCA120” and “Cy5-RCA120”, respectively.
A product manufactured by GE Healthcare was used for fluorescent labeling. Cy5 Mono-Reactive Dye Pack (for 1 mg) was used for Cy5 labeling, and Cy3 Mono-Reactive Dye Pack (for 1 mg) was used for Cy3 labeling.
Streptavidin manufactured by Wako Pure Chemical Industries, Ltd. was used. BSA used was made by Sigma-Aldrich.
The pure water was produced using Milli-Q Gradient A10 manufactured by MILLIPORE.
PBS is a phosphate buffer solution used by Sigma-Aldrich.
As the carbonate buffer (pH = 9.4), sodium hydrogen carbonate and sodium carbonate manufactured by Junsei Kagaku Co., Ltd. were used. After preparing respective 100 mM aqueous solutions, the two solutions were mixed and adjusted to pH = 9.4.
The acryloxy succinimide used the product made from ACROS ORGANICS.
N-acryloyl morpholine manufactured by KOHJIN was used.
16-mercaptohexadecanoic acid was manufactured by Aldrich, and ethanol was ethanol for electronics industry manufactured by Kishida Chemical.
Methanol used was made by Pure Chemical.
N-hydroxysuccinimide (hereinafter abbreviated as “NHS”) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as “EDC”) manufactured by Tokyo Chemical Industry Co., Ltd. are used. did.
Ethanolamine hydrochloride manufactured by Tokyo Chemical Industry Co., Ltd. was used.
As p-nitrophenyl tetra-N-acetyl-β-chitotetraoside and p-nitrophenyl β-D-mannopyranoside, p-nitrophenyl α-D-galactopyranoside manufactured by Seikagaku Biobusiness was used.
As the fluorescence detector, GenePix 4000A manufactured by AXON and INNOSCAN manufactured by INNOPSYS were used.

[Example 1: Preparation of a solution of Cy3-HABP]
Carbonate buffer (pH = 9.4, 1.0 ml) was added to Cy3 labeling kit and dissolved. 0.1 ml of this solution was added to HABP (0.1 mg) and dissolved. The reaction was performed at room temperature and stirred using a micropipette (registered trademark Pipetteman) every 10 minutes. After 30 minutes, the reaction solution was diluted to 2.5 ml with PBS (pH = 7.4), and subjected to gel column chromatography (GE Healthcare PD-10 Desaling Column, gel volume: 8.3 ml, eluent: PBS, pH = 7.4). The fraction collected was an elution volume of 2.5 to 5.5 ml, and the concentration of Cy3-HABP was 33.3 μg / ml.

[Example 2: Preparation of a solution of Cy3-SSA]
Carbonic acid buffer (pH = 9.4, 1.3 ml) was added to SSA (1.3 mg) and dissolved. The solution was added to the Cy3 labeling kit. Separately, a carbonate buffer (pH = 9.4, 1.0 ml) of Cy3 labeling kit was prepared, and 0.3 ml was extracted and added. Stir at room temperature using a micropipette every 10 minutes. After 45 minutes, the reaction solution was diluted to 2.5 ml with PBS (pH = 7.4), and subjected to gel column chromatography (GE Healthcare PD-10 Desaling Column, gel volume: 8.3 ml, eluent: PBS, pH = 7.4). The fraction collected was an elution volume of 2.5 to 5.5 ml, and the concentration of Cy3-SSA was 433.3 μg / ml.

[Example 3: Production of polymer]
A polymer was prepared from acryloxysuccinimide and N-acryloylmorpholine according to the description in Example 1 of JP-A-2007-101520.

[Example 4: Production of micro-spots of hydrogel]
[Preparation of substrate]
Chrome and gold were vapor-deposited on a plastic plate (2.5 × 7.5 cm, thickness 1.1 mm) to produce a gold chip. An ethanol solution (5 mM) of 16-mercaptohexadecanoic acid was prepared and filtered with a filter having a pore size of 0.25 μm. A gold chip was immersed in this solution, and a self-assembled film (hereinafter abbreviated as “SAM”) was formed on the gold surface over 16 hours at room temperature. The surface was washed with ethanol, ethanol remaining on the surface was removed by a nitrogen stream, and the surface was sufficiently dried. After drying, 2 ml of a mixture of pure water (9 ml), NHS aqueous solution (0.1 M, 0.5 ml) and EDC aqueous solution (0.4 M, 0.5 ml) was dropped on the entire surface of the SAM chip. After leaving still for 15 minutes at room temperature in a wet box, the surface was thoroughly washed with pure water, and water was removed by a nitrogen stream.

[Gel spotting process]
Subsequently, 0.5 ml each of an aqueous solution (10 mg / ml) of the polymer prepared in Example 3 and a carbonate buffer solution of streptavidin (1%, pH = 9.4) were prepared. 10 μl and 100 μl of each were extracted and mixed to prepare 110 μl of a solution having a polymer: streptavidin ratio of 1:10. This is called a mixed solution for gel. Using a spotter equipped with a spot pin (Model No. SMP7B) manufactured by ArrayIt, 144 points (12 rows × 12 columns) of the gel mixed solution were spotted on the NHS gold chip. Spotting was performed on the determined coordinates on the chip, and the center-to-center distance in the vertical and horizontal directions was set to 800 μm. The size of the formed spot was about 300 μm. After spotting, it was allowed to stand at room temperature for 1 hour and dried. As a result, a spot containing streptavidin as a biological material and a hydrogel containing the polymer produced in Example 3 as a binding compound was formed on the gold chip.

[Blocking process]
Ethanolamine hydrochloride (19.5 g) and BSA (6.0 g) are dissolved in water, 1N NaOH aqueous solution is added to adjust to pH = 8.5, and water is added so that the solution volume becomes 200 ml. 1M ethanolamine aqueous solution containing 3% BSA was prepared. After filtering with a filter having a pore size of 0.25 μm, this solution was gently dropped with a pipette onto the spot portion of the gold chip. As a result, the NHS group remaining in the polymer in the spot hydrogel and the NHS group on the gold chip surface caused an exchange reaction with ethanolamine or the amino group of BSA, and were inactivated and blocked by BSA. . After allowing to stand at room temperature for 30 minutes, the surface was sufficiently washed with pure water, water remaining on the surface was removed with nitrogen, and a gold chip on which a spot containing hydrogel was formed was produced. The result of measuring the AFM of the manufactured chip is shown in FIG. It can be seen that the formed spot has a hydrogel composed of particulate soot having a size of 100 nm or more.

[Example 5: Binding of biotinylated protein to hydrogel]
To the chip prepared in Example 4, 500 μl of Cy3-HRP-biotin PBS (pH = 7.4) prepared to 10 μg / ml was gently dropped with a pipette and allowed to stand at room temperature for 30 minutes in a wet box. . Thereafter, the surface was sufficiently washed with pure water, and water remaining on the surface was removed by nitrogen. When measured with a fluorescence scanner, fluorescence was strongly observed at all spots, and it was found that avidin in the hydrogel spots had the ability to bind to biotin.

[Example 6: Immobilization of biotin hyaluronate (ligand spotting step)]
PBS of biotin hyaluronate (pH = 7.4) was prepared to be 100 μg / ml, and this was spotted using a spotter on the chip on which the spot produced in Example 4 was formed. At this time, using exactly the same coordinate setting as that used when the hydrogel was immobilized in Example 4, PBS of biotin hyaluronate was spotted on a spot using a spotter. Then, it left still at room temperature for 1 hour, it was made to dry, the surface was wash | cleaned with the pure water, and the water remaining on the surface was removed with nitrogen. As a result, biotin hyaluronate was immobilized as a ligand on the spot spotted with PBS of biotin hyaluronate.

[Example 7: Detection of binding between hyaluronic acid and Cy3-HABP]
PBS of 3.0 w / v% BSA (pH = 7.4) was gently dropped onto the entire chip prepared in Example 6 with a pipette and allowed to stand at room temperature for 30 minutes. The surface was washed with pure water. Cy3-HABP PBS (200 μl) prepared in Example 1 was gently dropped onto the entire chip with a pipette and allowed to stand at room temperature for 30 minutes. After washing the surface with pure water, water remaining on the surface was removed with nitrogen. The gold chip was scanned (excitation wavelength: 532 nm, fluorescence wavelength: 570 nm) with a fluorescence detector (GenPix 4000A manufactured by AXON), and the fluorescence intensity was measured. The results are shown in Table 7. FIG. 7 shows a drawing substitute photograph taken at the time of measurement. In addition, the spot of the center row | line of FIG. 7 is a spot which fix | immobilized the hyaluronic acid biotin.

  As shown in Table 1, binding between hyaluronic acid and Cy3-HABP was detected. However, as shown in FIG. 7, it was found that the portion where biotin hyaluronate was immobilized was smaller than the hydrogel spot. This means that the spotted PBS of biotin hyaluronate had been volatilized before it soaked into the spot and spread sufficiently. In addition, the fact that the shape of the fluorescent portion observed in FIG. 7 is not a circle but a square that matches the shape of the pin tip confirms this. That is, the spot formed by this hydrogel is expected to absorb the ligand aqueous solution and the whole spot of the hydrogel emits fluorescence, but in fact, a fluorescent spot in the shape of the tip of the spotter pin appears. ing. This means that the ligand is present only in the portion pressed at the tip of the spot pin in the spot. For this reason, if the PBS of biotin hyaluronate is strongly pressed with a spot pin, this solution will quickly soak into the hydrogel, but it will take time to soak if it is simply in contact with it. Also means that volatilization occurs first. Since no lateral spread is observed, this phenomenon is considered to occur in the same manner regardless of how the ligand solution is spotted on the hydrogel.

  Furthermore, most of the deviation between the position of the hydrogel spot and the position of the portion where biotin hyaluronate is immobilized occurs in the major axis direction of the chip, and the deviation in the minor axis direction is small. From this, it is clear that this deviation is caused by the expansion and contraction of the chip. In FIG. 7, the horizontal direction is the minor axis direction, and the vertical direction is the major axis direction. This is due to the fact that the gold chip swelled due to the ethanol used at the time of SAM production, and the chip size slightly increased. Moreover, since the change is proportional to the length, it is remarkable in the major axis direction. The steps after the SAM production include a gel spotting step, a ligand spotting step, and washing and drying necessary for each. During that time, ethanol gradually evaporates from the plastic plate as a substrate, However, the size becomes smaller and tries to return to the original size. Therefore, it is considered that the size of the chip is different between the gel spotting step after the SAM formation and the subsequent linker spotting step.

[Example 8: Immobilization of hyaluronic acid (ligand spotting step)]
A biotin hyaluronate PBS (pH = 7.4) was spotted in the same manner as in Example 6 except that the spot program of the spotter was changed as follows. If the coordinates (x, y) when spotting the mixed solution for gel in the gel spotting step is (0, 0), the program is (−0.2, 0), (−0.2, 0). .2) Spot four spots of biotin hyaluronate PBS for each spot on the hydrogel so that it becomes (0,0) and (0,0.2) to cover the spots of the hydrogel completely I made it. The unit of the numerical value of the coordinates is mm. In addition, the coordinate axes in FIG. 7 are the x-axis in the horizontal direction and positive from left to right. Also, the vertical direction was the y-axis, and positive from top to bottom.

[Example 9: Detection of binding between hyaluronic acid and Cy3-HABP]
For the chip produced in Example 8, Cy3-HABP was used in the same manner as in Example 7, and the fluorescence intensity was measured. The results are shown in Table 2. FIG. 8 shows a drawing substitute photograph taken at the time of measurement. In FIG. 8, the left three rows that appear white correspond to the ninth embodiment, and the right five rows that appear white correspond to Comparative Example 1 (described later). FIG. 9 shows a drawing-substituting photograph in which two upper left points in FIG. 8 are enlarged.

  In Example 7, only one spot of PBS of biotin hyaluronate was spotted per spot, and biotin hyaluronate was immobilized only on a part of the hydrogel spot. In this example, Since the entire hydrogel spot emits fluorescence, it can be seen that biotin hyaluronate, which is a ligand, is immobilized on the entire hydrogel spot. In addition, the fluorescence of biotin hyaluronate and the portion where no gel was spotted was very low, indicating that the ligand, ie, biotin hyaluronate was not present. In Table 2, entry3 has a slightly higher value than Entry2, but is clearly within the measurement error range.

[Comparative Example 1; Preparation of gold chip with hyaluronic acid immobilized by doweling]
200 μl of biotin hyaluronate PBS (250 μg / ml, pH = 7.4) was applied so as to cover the right half spot (see FIG. 8) of the chip prepared in Example 8 together. It was allowed to stand at room temperature for 1 hour and dried. The surface was washed with pure water, and water remaining on the surface was removed with nitrogen. PBS (pH = 7.4) of 3.0 w / v% BSA was gently dropped on the entire chip with a pipette and allowed to stand at room temperature for 30 minutes. The surface was washed with pure water.

In addition, the chip | tip of the comparative example 1 uses the same board | substrate as the chip | tip produced in Example 8, and used Cy3-HABP in the same way as Example 9 simultaneously with Example 9, and measured the fluorescence intensity. . The results are shown in Table 3. FIG. 8 shows a drawing substitute photograph taken at the time of measurement. In FIG. 8, the right five columns correspond to Comparative Example 1.

  As shown in Entry 2, the fluorescence of Cy3-HABP was significantly detected even in the portion without the gel. Comparing the result of Comparative Example 1 and Example 9, it is clear that hyaluronic acid is also present in the portion where the gel spot is not formed. The reason for the presence of biotin hyaluronate in the absence of gel is not clear, but it may be due to nonspecific adsorption.

[Example 10: Elution of captured HABP]
In the same manner as in Example 8 and Example 9, a spot comprising hydrogel was formed on the substrate surface, and biotinylated hyaluronic acid was immobilized as a ligand to the spot, and Cy3-HABP was immobilized on this biotinylated hyaluronic acid. Was prepared, and the fluorescence intensity was measured. The concentration of biotinylated hyaluronic acid in PBS used for immobilizing biotinylated hyaluronic acid was 0 μg / ml, 100 μg / ml, 300 μg / ml, and 1000 μg / ml, respectively. Further, after the first fluorescence measurement, the surface of the chip was washed with 0.1N hydrochloric acid, and the fluorescence measurement was performed again. The results are shown in Table 4 and FIG.

  As a result, it was found that the fluorescence intensity decreased by about 40 to 50% by washing with HCl, and the captured Cy3-HABP was eluted.

[Example 11: Synthesis of sialyl sugar ligand]
A sialyl sugar ligand was prepared as described in JP-A-2007-298334.
Step 1 (reductive amination)

  Trisaccharide (compound (1)) 86.8 mg (0.129 mmol) and monoamine (compound (2)) 25 mg (0.115 mmol) were placed in a 10 ml eggplant flask and purged with nitrogen, and then a mixed solvent of methanol and acetic acid (10 1) 1 ml was added and stirred at room temperature for 45 hours. 393 mg (1.80 mmol) of monoamine (compound (2)) was added, and the mixture was heated at 40 ° C. for 2.5 hours and at 50 ° C. for 2.5 hours. 2-Picoline borane (45.9 mg, 0.429 mmol) was added and the mixture was heated at 50 ° C. for 6 hours, and then the solvent was distilled off. Gel filtration (Amersham LH-20, methanol) was performed. After adding 29.3 g (31.6 mmol) of benzaldehyde polystyrene and purging with nitrogen, 28.0 ml of methylene chloride was added. The mixture was stirred at 40 ° C. for 15 hours and filtered. The resin was washed with methylene chloride (30 ml × 5) and methanol (30 ml × 5), and the filtrate was evaporated to isolate a sugar compound (compound (3), yield 40 mg, yield 68%). . The obtained compound was identified by NMR and ESI-MS. The results are shown below.

¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 4.45 (m, 1H), 4.17 to 4.09 (m, 2H), 3.87 to 3.77 (m, 8H), 3 .74 to 3.60 (m, 24H), 3.53 to 3.46 (m, 3H), 3.39 (t, J = 4.9 Hz, 2H), 3.16 (q, J = 7. 3 Hz, 1 H), 2.82 (brd, J = 10.4 Hz, 1 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 1.63 (t, J = 10.5 Hz, 1H)

¹³ C NMR (100 MHz, CDCl ₃ ) δ 175.39, 172.69, 165.72, 105.24, 98.50, 82.60, 74.49, 73.14, 71.62, 71.54, 71.48, 71.37, 71.07, 67.45, 64.73, 51.77, 47.66, 22.66

Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50-1500 m / e, 4.0 sec / scan
Applied voltage: 3.8 kV, Cone voltage: +/− 50, −100V
Solvent: methanol m / z 877 (M + H) was observed by the ESI (+) method. Thereafter, m / z 899 (M + Na) and m / z 921 (M−H + 2Na) were observed. Further, m / z 875 (M−H) was observed by the ESI (−) method. The mass number of the target substance was MW876, which coincided with the mass number corresponding to the synthesis target product.

・ Process 2 (reduction)

  In a 100 ml four-necked round bottom flask, 21.4 mg (0.0244 mmol) of a sugar compound (compound (3)) was added, and 5 ml of methanol was added. 6.4 mg (0.005 mmol) of 10% palladium carbon (Pd / C) was added, and 1.3 ml of 1N hydrochloric acid was added in an ice bath. The reaction vessel was returned to room temperature and purged with hydrogen, followed by stirring for 12 hours. After purging with nitrogen, the mixture was filtered and the filtrate was evaporated. Gel filtration (gel filtration (Amersham LH-20, methanol) was performed, and the solvent was distilled off. Dissolve in methanol (4.3 ml), slowly add methanol solution of 0.01M sodium acetate in an ice bath. After the solvent was distilled off, the reduced product (compound (4), yield 19.9 mg, yield 96%) was obtained by gel filtration (Amersham LH-20, methanol). The obtained compound was identified by NMR and ESI-MS, and the results are shown below.

¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 4.53 to 4.46 (m, 1H), 4.17 to 4.09 (m, 2H), 3.87 to 3.77 (m, 8H), 3.74 to 3.60 (m, 24H), 3.53 to 3.46 (m, 3H), 3.22 to 3.16 (m, 3H), 2.82 (brd, J = 10.1 Hz, 1 H), 2.02 (s, 6 H), 1.60 (m, 1 H), 1.14 (brs, 2 H)

Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50 to 2000 m / e, 4.0 sec / scan
Applied voltage: 3.8 kV, Cone voltage: 30, 80 V
Solvent: methanol m / z 849 (M−H) was observed by ESI (−) method. The mass number of the target substance was MW850, which coincided with the mass number corresponding to the synthesis target product.

-Step 3 (biotinylation)

  In a 5 ml eggplant flask, 2.0 mg (2.3 μmol) of the saccharide compound (compound (4)) obtained in the previous step and 1.3 mg (2.3 μmol) of a commercially available biotinylating agent (compound (5)) are placed. Under a nitrogen atmosphere, DMF (0.25 ml) and triethylamine (0.020 ml) were added. After stirring at room temperature for 4.5 hours, the solvent was distilled off. Purification by gel filtration (Amersham LH-20, methanol) was performed to obtain a biotinylated compound (compound (6), yield 0.9 mg, yield 32%). The obtained compound was identified by ESI-MS. The results are shown below.

Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 200 to 1800 m / e, 4.0 sec / scan
Applied voltage: +/- 3.8kV, Cone voltage: +/- 30, +/- 80V
Solvent: methanol m / z 1188 (M−H) was observed by ESI (−) method. The mass number of the target substance was MW1189, which coincided with the mass number corresponding to the synthesis target product.

[Example 12: Production of gold chip on which hyaluronic acid and sialyl sugar are immobilized]
After preparing biotin hyaluronate PBS (250 μg / ml, pH = 7.4) and an aqueous solution of sialyl sugar ligand (100 μmol / l), a spotter was used for the gel on the gold chip prepared in Example 4, Biotin hyaluronate and sialyl sugar ligands were spotted multiple times on different hydrogel spots using the same program as in Example 8. After allowing to stand at room temperature for 1 hour and drying, the surface was washed with pure water, and water remaining on the surface was removed with nitrogen.

[Example 13: Measurement of fluorescence intensity of gold chip on which hyaluronic acid and sialyl sugar are immobilized (1)]
BSA PBS (3.0 w / v%, pH = 7.4) was added dropwise to the entire gel spot on the gold chip produced in Example 12, and allowed to stand at room temperature for 30 minutes. The surface was washed with pure water, and water remaining on the surface was removed with nitrogen. Thereafter, 200 μl of Cy3-HABP PBS prepared in Example 1 was gently added dropwise with a pipette and allowed to stand at room temperature for 30 minutes. The surface was washed with pure water, and water remaining on the surface was removed with nitrogen. The gold chip was scanned with a fluorescence detector (GenePix 4000A, excitation wavelength 532 nm, fluorescence wavelength 570 nm), and the fluorescence intensity was measured. The results are shown in Table 5.

  As can be seen from Table 5, fluorescence derived from Cy3-HABP was detected only in spots where biotin hyaluronate was immobilized, and almost no fluorescence was observed in spots where sialyl sugar ligands were immobilized.

[Example 13: Measurement of fluorescence intensity of gold chip on which hyaluronic acid and sialyl sugar are immobilized (2)]
The fluorescence intensity was measured in the same manner as in Example 12 except that the fluorescent protein was replaced with PBS of Cy3-SSA prepared in Example 2 (pH = 7.4, 30 μg / ml, 200 μl). The results are shown in Table 6.

  As can be seen from Table 6, fluorescence derived from Cy3-SSA was observed in the spots where the sialyl sugar ligands were immobilized, but no fluorescence was observed in the spots where biotin hyaluronate was immobilized.

[Example 14: Synthesis of N-acetylglucosamine ligand]
The nitro group of p-nitrophenyltetra-N-acetyl-β-chitotetraoside was reduced and converted to an amino group, and then an N-acetylglucosamine ligand having biotin at the end was synthesized using a biotinating agent. This ligand has a structure in which four molecules of N-acetylglucosamine are bound, and it is known that wheat germ lectin (WGA) binds specifically.

・ Process 1 (reduction reaction)
The nitro group was converted to an amino group by catalytic reduction.
A nitro compound (1-nitrophenyl-N-acetyl-β-chitotetraoside, 21.0 mg, 22.1 μmol) was placed in a 100 ml eggplant flask, and 980 μl of methanol and 670 μl of water were added and dissolved. Under a nitrogen atmosphere, 3.5 mg (32.9 μmol) of 10% palladium carbon (Pd / C) was added, and after purging with hydrogen, the mixture was stirred for 4 hours. After purging with nitrogen, Pd / C was separated with celite. The filtrate was evaporated to give an amino compound (yield 21.4 mg, yield 100%). The obtained compound was identified by ¹ H, ¹³ C NMR and ESI-MS. The results are shown below.

¹ HNMR (D ₂ O, 400 MHz, J in Hz) δ 6.76 (d, 8.8, 2H), 6.64 (d, 8.8, 2H), 4.83 (d, 8.3) 1H), 4.76 (s, 1H), 4.71 (s, 1H), 4.59 (s, 11H), 4.43 (t, 7.5, 3H), 3.84-3.31 (M, 28H), 3.19 (s, 2H), 2.09 (s3H), 1.91 (s3H), 1.90 (s, 3H), 1.88 (s, 3H).

¹³ C NMR (D ₂ O, 100 MHz): δ 175.2 (1C), 175.0 (3C), 150.6 (1C), 142.3 (1C), 118.6 (2C), 117.9 (2C) ), 101.9 (1C), 101.7 (1C), 101.6 (1C), 101.0 (1C), 79.5, 79.3, 79.3, 76.3, 75.1, 74.9, 73.8, 72.6, 72.5, 72.5, 70.1, 60.9 (1C), 60.3 (3C), 56.0 (1C), 55.4 (3C) ), 22.5 (4C).

Mass spectrometry: Multi-mode ESI + APCI method (Agilent technology 6130 Quadrupole LC / MS)
Mass scanning conditions: 500 to 1800 m / e, 2.9 sec / scan
Applied voltage: 3.8 kV, Cone voltage: +/− 50, −100V
M / z 922 (M + H) and m / z 944 (M + Na) were observed by the ESI (+) method. The mass number of the target substance was MW 921.4, which coincided with the mass number corresponding to the synthesis target product.

Step 2 (Biotinylation reaction)

An amino compound (10.8 mg, 11.7 μmol) is placed in a 1.5 ml Eppendorf tube, and 400 μl of an aqueous solution of a biotinylation reagent (EZ-Link Sulfo-NHS-LC-Biotin) (4.9 mg, 8 for biotinylation reagent) .8 μmol) was added. Further, 400 μl of water was added and stirred for 3 hours. The reaction solution was diluted to 1.5 ml, and solid phase extraction was performed using oasis HLB (3 cc, 60 mg) manufactured by Waters. As an eluent, 50 ml of a mixed solvent of water and methanol (50:50) was used. After the solvent was distilled off, the target N-acetylglucosamine ligand (biotin compound, yield 3.6 mg, yield 23%) was obtained. The obtained compound was identified by ¹ H NMR and ESI-MS. The results are shown below.

¹ HNMR (D ₂ O, 400 MHz, J in Hz) δ 7.36 (d, 8.8, 2H), 7.07 (d, 8.8, 2H), 5.13 (d, 8.3, 1H), 4.90 (s, 1H), 4.85 (t, 0.8, 1H), 4.80 (s, 10H), 4.75 (s, 1H), 4.59 (t, 8 .1, 4H), 4.55 (dd, 4.8 & 8.1, 1H), 4.31 (dd, 4.3 & 8.1, 1H), 4.01 (dd, 8.3 & 10.1, 1H) , 3.94-3.47 (m, 28H), 3.24-3.17 (m, 3H), 2.94 (dd, 4.8 & 13.0, 1H), 2.73 (d, 13.H). 0, 1H), 2.44-2.41 (m, 2H), 2.23-2.21 (m, 2H), 2.08 (s, 3H), 2.074 (s, 3H), 2 .071 (s, 3H), 2. 04 (s, 3H), 1.75-1.30 (m, 12H).

Mass spectrometry: ESI-MS method (Micromass type-ZMD)
Mass scanning conditions: 50-1500 m / e, 4.0 sec / scan
Applied voltage: 3.8 kV, Cone voltage: +/− 50, −100V
Solvent: methanol By the ESI (+) method, m / z1283 (M-H + Na) and m / z1284 (M + Na) were observed. The mass number of the target substance was MW1260.5, which coincided with the mass number corresponding to the synthesis target product.

Example 15: Preparation of Cy5-WGA
A carbonate buffer solution of WGA (1.4 ml, 1.0 mg / ml) was added to a vial of Cy5 labeling kit from GE Healthcare. The reaction was performed at room temperature and stirred with a pipette every 10 minutes. After 50 minutes, the reaction solution was purified by gel column chromatography (PD-10 Desalting Column, gel volume: 8.3 ml, manufactured by GE Healthcare). PBS (pH = 7.4 was used as the eluent. The fraction collected was an elution volume of 2.0 to 4.5 ml, the concentration of Cy5-WGA was 501 μg / ml, and the Cy5 conversion rate per molecule of WGA. Was 2.3.

[Example 16: Preparation of a solution of Cy3-WGA]
It was prepared in the same manner as in Example 15 except that the Cy3 labeling kit was used. The concentration of Cy3-WGA obtained was 702 μg / ml, and the Cy3 conversion rate per WGA molecule was 2.3.

[Example 17: Production of spot on gold chip of glass substrate]
Example 4 except that gold vapor-deposited (adhesive layer is chrome) was used instead of gold-deposited plastic slide, glass glass Nexter Glass B (2.5 × 7.6 cm, thickness 1 mm) manufactured by SCHOTT. In the same manner as above, SAM formation and NHS formation were performed. A gold chip on which hydrogel was spotted was obtained in the same manner as in Example 4 except that the gel spot was changed to 60 points (5 columns × 12 rows) at room temperature and under a humidity of 75%. Five chips were produced.

[Example 18: Immobilization of N-acetylglucosamine ligand]
[Ligand spotting process]
An aqueous solution of N-acetylglucosamine ligand was prepared to be 100 μmol / L. This was spotted on the chip on which the spot produced in Example 17 was formed to the spot (12 points) in the middle row of 5 rows using a spotter. If the coordinates (x, y) when spotting the mixed solution for gel in the gel spotting step is (0, 0), the program is (−0.1, −0.1), (−0. 1, 0.1), (0.1, 0.1), and (0.1, -0.1), spotting an aqueous solution of N-acetylglucosamine ligand for each spot of the hydrogel. The hydrogel spots were completely covered. Since the tip is made of glass and is not affected by swelling due to ethanol, the center coordinates of the four-point spot are preferably the same as the coordinates of the hydrogel spot. After allowing to stand at room temperature for 1 hour and drying, the surface was washed with pure water, and water remaining on the surface was removed with nitrogen. As a result, the N-acetylglucosamine ligand was immobilized on the hydrogel spot.

[Example 19: Detection of binding between N-acetylglucosamine ligand and Cy5-WGA]
All the chips prepared in Example 18 were gently dropped with a pipette of a 3.0 w / v% BSA PBS solution (pH = 7.4) over the entire surface, and allowed to stand at room temperature for 30 minutes in a wet box. . Thereafter, the surface was washed with pure water. The PBS of Cy5-WGA prepared in Example 15 was diluted with PBS to prepare solutions of 1 mg / ml, 100 ng / ml, 10 ng / ml, and 1 ng / ml. Each of the prepared chips was gently dropped with a pipette so as to cover the spot group. Leave in a humid box for 30 minutes at room temperature. After washing the surface with pure water, water remaining on the surface was removed with nitrogen. The gold chip was scanned with a fluorescence detector (excitation wavelength: 635 nm, fluorescence detection wavelength: 655 to 700 nm), and the fluorescence intensity was measured. The results are shown in Table 7 and FIG.
The ligand (+) is the fluorescence intensity of the spot where the N-acetylglucosamine ligand is immobilized, and the ligand (−) is the fluorescence intensity of the spot where the N-acetylglucosamine ligand is not immobilized. The outer circumference of the spot group (5 × 12) was excluded from the measurement target. Moreover, since the intensity of the fluorescent light emission of the spot differs depending on the WGA concentration, the measurement was performed with the optimum fluorescence intensity by adjusting the gain of the detection device. The results are shown in the table. S / N is ligand (+) average value ÷ ligand (−) average value. σ is {ligand (+) average value−ligand (−) average value} ÷ {ligand (+) SD + ligand (−) SD}.

By linear approximation, the qualitative limit value (σ = 3.29) was calculated to be 3.1 ng / ml, and the quantitative limit value (σ = 10.0) was calculated to be 7.5 ng / ml.

[Example 20: Detection of binding between N-acetylglucosamine ligand and Cy3-WGA]
Experiments were performed in the same manner as in Examples 17, 18, and 19 except that Cy3-WGA was used instead of Cy5-WGA, the scan wavelength was 532 nm, and the fluorescence detection wavelength was 555-606 nm. The results are shown in Table 8 and FIG.

By linear approximation, the qualitative limit value (σ = 3.29) was calculated to be 50.8 ng / ml, and the quantification limit value (σ = 10.0) was calculated to be 100 ng / ml or more.

In Example 19, since high sensitivity was provided as compared with Example 20, the superiority of performing detection at a long emission wavelength (in this case, Cy5 dye) was shown. In the following, in order to further increase the sensitivity, in Examples 22 and 23, a low molecular compound (biotin-PEG ₂ -OMe) that does not bind to the substance to be detected (WGA) is immobilized on the spot where the sugar chain is not immobilized. I tried to make it. For this purpose, a compound in which the end of diethylene glycol was biotin and the other end was a methoxy group was synthesized. The methoxy group and diethylene glycol are predicted to have high hydrophilicity and hardly cause nonspecific adsorption.

[Example 21: Synthesis of biotin masking agent (biotin-PEG ₂ -OMe)]
Process 1 (Tosylation)
Commercial diethylene glycol methyl ether was tosylated.
A THF solution (8.0 ml) of diethylene glycol methyl ether (4.03 g, 33.6 mmol) was placed in a four-necked flask and placed in an ice bath. Aqueous NaOH (7.5 M, 8.0 ml) was added slowly and stirred. A THF solution of TsCl (7.08 g, 37.1 mmol) was added dropwise over 45 minutes and stirred for 17 hours. 10% HCl aqueous solution was added to adjust the pH to 4, followed by extraction with ethyl acetate (100 ml × 3). The organic phase was collected and washed with water (100 ml × 2), 0.5N NaHCO ₃ aqueous solution (100 ml) and water (100 ml) in this order. The organic phase was dehydrated with MgSO ₄ and filtered, and then the solvent was distilled off. The tosylated product (9.08 g, 99%) was isolated by silica gel column chromatography (Biotage column 25 + M, hexane / ethyl acetate).

¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 7.80 (d, 8.3, 2H), 7.33 (d, 7.8 Hz, 2H), 4.17 (t, 4.8 Hz, 2H) ), 3.69 (t, 4.8, 2H), 3.58 (m, 2H), 3.48 (m, 2H), 3.35 (s, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 144.8, 133.0, 129.8 (2C), 128.0 (2C), 71.8, 70.7, 69.2, 68.7, 59.0 , 21.6.

Process 2 (azidation process)
The tosylated product thus obtained was azided.

A tosylated product (7.80 g, 28.4 mmol) was placed in a four-necked flask, and the atmosphere in the flask was replaced with nitrogen. Then, dehydrated DMF (28 ml) was added. NaN ₃ (2.23 g, 34.3 mmol) was added slowly, heated to 50 ° C. and stirred for 5 hours. After cooling to room temperature, ethyl acetate (80 ml) and water (100 ml) were added to extract the organic phase. Further, the aqueous phase was extracted with ethyl acetate (80 ml × 2). The organic phase was collected, washed with H ₂ O (100 ml), dehydrated with Na ₂ SO ₄ and filtered, and then the solvent was distilled off. The azide compound (3.33 g, 81%) was isolated by performing silica gel column chromatography (Biotage column 25 + M, hexane / ethyl acetate).

¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 3.68 (t, 4.8, 4H), 3.67 (m, 2H), 3.57 (m, 2H), 3.40 (t, 4.8, 4H), 3.39 (s, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 72.0, 70.6, 70.0, 59.1, 50.7.

Process 3 (Reduction process)
An azide (1.34 g, 9.23 mmol) and 150 ml of methanol were added to a 300 ml four-necked round bottom flask, and the mixture was purged with nitrogen and stirred. After adding 10% Pd / C (201 mg) and replacing with nitrogen again, 14 ml of 1N hydrochloric acid was added in an ice bath. After returning to room temperature, the reaction system was purged with hydrogen and stirred for 22 hours. After celite filtration, the filtrate was distilled off, and the oily liquid was purified by gel filtration (Amersham LH-20, methanol). Thereafter, the oil was dissolved in 5 ml of methanol, and a methanol solution of 1N-NaOAc (9.3 ml) was added in an ice bath. Stir at room temperature for 30 minutes. After the solvent was distilled off, the residue was purified by silica gel column chromatography (spherical silica gel, 15 g, MeOH / ethyl acetate) to obtain an amino compound (1.044 g, 88%).

¹ H NMR (400 MHz, CDCl ₃ δ 3.68 (m, 4H), 3.55 (m, 2H), 3.38 (s, 3H), 3.05 (t, 2H, 5.2)
¹³ C NMR (100 MHz, CDCl ₃ ) δ 71.7, 70.2, 68.8, 58.9, 39.7.

Process 4 (Biotinylation process)
An amino compound (119 mg, 1.0 mmol) was placed in a one-necked eggplant flask (25 ml), and DMF (3.3 ml) was added. A biotinylation reagent (EZ-Biotin, (341 mg, 1.0 mmol) was added. After stirring at room temperature for 6 hours, the solvent was distilled off by silica gel column chromatography (spherical silica gel, MeOH: CH ₂ Cl ₂ ). A biotinylated product (35 mg, 10%) was isolated.

¹ H NMR (400 MHz, CDCl ₃ , J in Hz) δ 6.83 (t, 5.3, 1H), 6.72 (s, 1H), 5.75 (s, 1H), 4.50 (dd, 4.8 & 7.8, 1H), 4.32 (dd, 5.0 & 7.0, 1H), 4.32 (dd, 5.0 & 7.8, 1H), 3.67-3.53 (m, 6H) ), 3.44 (t, 5.4, 2H), 3.38 (s, 1H), 3.14 (dt, 4.6 & 7.3, 2H), 2.90 (dd, 4.9 & 12.8) , 1H), 3.14 (dt, 4.5 & 7.3, 1H), 2.74 (d, 12.6, 1H), 2.23 (t, 7.6, 2H), 1.75-1 .62 (m, 4H), 1.49-1.40 (m, 2H)

¹³ C NMR (100 MHz, CDCl ₃ ) δ 173.4, 164.0, 71.6, 70.0, 69.9, 61.7, 60.2, 58.9, 55.7, 40.5, 39 1, 36.0, 28.2, 28.1, 25.6.

[Example 22: Detection of Cy5-WGA binding (masking with biotin-PEG ₂ -OMe)]
The N-acetylglucosamine ligand was immobilized in the same manner as in Example 18 except that the ligand was spotted and an aqueous solution of biotin masking agent (1 mM, 1 ml) was dropped between the drying step and the subsequent water washing step, and allowed to stand for 30 minutes. The assay was performed in the same manner as in Example 19. The results are shown in Table 9 and FIG.

By linear approximation, the qualitative limit value (σ = 3.29) was calculated to be 0.1 ng / ml, and the quantitative limit value (σ = 10.0) was calculated to be 0.6 ng / ml.

[Example 23: Detection of Cy3-WGA binding (masking using biotin-PEG ₂ -OMe)]
The experiment was performed in the same manner as in Example 22 except that Cy3-WGA was used instead of Cy5-WGA. The results are shown in Table 10 and FIG.

By linear approximation, the qualitative limit value (σ = 3.29) was calculated to be 0.5 ng / ml, and the quantification limit value (σ = 10.0) was calculated to be 34.7 ng / ml.
The result of Example 22 gave higher sensitivity than that of Example 23, and showed the superiority of performing detection at the emission wavelength of Cy5 dye, which is a long wavelength.

[Example 24: Detection of Cy5-WGA binding (examination of assay time)]
Except that six chips were prepared, the concentration of Cy5WGA was 10 (ng / ml, and the assay time was 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes, respectively, as in Example 22. The results are shown in Table 11 and Fig. 15. However, the values of S / N and σ for the assay time of 0 minutes in the tables and figures are not experimental values, but are defined for easy understanding. The more obvious one was used.

It can be seen that the signal is saturated in about 30 minutes after the assay. According to the present invention, the assay time was found to be very short. The cause of the decrease in S / N and σ values after 30 minutes is unknown, but it is presumed that the cause is mainly nonspecific adsorption to the ligand (−).

  A combination of a sugar chain and a lectin that specifically binds is determined. WGA corresponds to N-acetylglucosamine, ConA corresponds to mannose, and RCA120 corresponds to galactose. In the following, cross-assay was attempted by immobilizing different sugar chain ligands on a plurality of adjacent microspots using this.

Example 25: Preparation of Cy5-WGA and Cy5-RCA120
Except that ConA was used instead of WGA, the same procedure as in Example 15 was performed to obtain Cy5-converted ConA. The Cy5 conversion rate per molecule (104000 Da) of ConA was 2.7. Similarly, RCA120 was similarly used, and the Cy5 conversion rate per molecule (120,000 Da) was 1.1.

[Example 26: Synthesis of mannose ligand and galactose ligand]
The corresponding ligand was synthesized in the same manner as in Example 14 except that commercially available p-nitrophenyl β-D-mannopyranoside and commercially available p-nitrophenyl α-D-galactopyranoside were used as starting materials.

The resulting mannose ligand was identified by ESI-MS. The results are shown below.
Mass spectrometry: Multi-mode ESI + APCI method (Agilent technology 6130 Quadrupole LC / MS)
Mass scanning condition: 200 to 2000 m / e, 2.76 sec / scan
Capillary voltage: 3.8 kV, fragment voltage: 100 V
m / z 633 (M + Na) was observed. The mass number of the target substance is 610.27, which corresponds to M + Na.

Similarly, the galactose ligand was identified by ESI-MS. The results are shown below.
Mass spectrometry: Multi-mode ESI + APCI method (Agilent technology 6130 Quadrupole LC / MS)
Mass spectrometry: MM-ES + APCI method, mass range: 200 to 2000 m / e, 2.76 sec / scan
Capillary voltage: 2.0 kV, fragment voltage: 100 V
m / z 633 (M + Na) was observed. The mass number of the target substance is 610.27, which corresponds to M + Na.

[Example 27: Cross-assay between sugar chain and lectin]
Three chips were produced in the same manner as in Example 17 except that 96 gel spots (12 × 8) were produced at a humidity of 50%. The N-acetylglucosamine ligand synthesized in Example 14 in the third column from the left of the eight columns, the mannose ligand synthesized in Example 26 in the fifth column, and the galactose synthesized in Example 26 in the seventh column are the same. The ligand was immobilized in the same manner as in Example 18 except that 12 points of the ligand were overprinted.
In each chip, Cy5-WGA solution (PBS, pH = 7.4, 10 ng / ml), Cy5ConA (PBS, pH = 7.4, 100 ng / ml), Cy5-RCA120 (PBS, pH = 7. 4 and 100 ng / ml) were added dropwise, and the assay was performed in the same manner as in Example 22. The results are shown in Table 12.

It was clarified that, by overstrike, different ligands could be immobilized on each of the dense spots, and each of them showed high specificity with the corresponding lectin.

[Example 28: Production of protein A / G chip and fluorescence detection]
[Preparation of protein A / G chip]
100 μL of 1% Protein A / G aqueous solution (100 mM, carbonate buffer, pH 9.4) and 10 μL of 1% aqueous polymer solution were mixed. A gel spot was prepared in the same manner as in Example 4 except that 48 spots (6 columns × 8 rows) of this solution were spotted. Separately, 100 μL of a 1% Protein A / G aqueous solution (100 mM, carbonate buffer, pH 9.4) not mixed with an aqueous polymer solution was prepared, and a similarly spotted substrate was prepared. Hereinafter, a spot containing the polymer is referred to as 3D, and a spot not including the polymer is referred to as 2D.
After standing at a humidity of 65% and 25 ° C. for 10 hours, as in Example 4, 1 mL of a solution containing 3% BSA and 1M ethanolamine was dropped on the entire surface of the chip and allowed to stand at room temperature for 30 minutes. Then, it wash | cleaned with MilliQ water and the water droplet on a chip | tip was removed using compressed air.
Next, anti-Biotin antibody (manufactured by SIGMA) was dissolved in 100 mM PBS (pH 7.4), and the concentrations were 0 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL. Prepared. One concentration of liquid was overprinted on 8 gel spots. The liquid volume is 3 nl each. It carried out similarly about all the liquids of a six step density | concentration. Overstrike was performed only once at the same coordinates as the gel spot. As a result, all spots were overstruck. After standing at room temperature and humidity of 80% for 1 hour, it was washed with PBS containing 0.05% Tween 20, and moisture on the chip was removed using compressed air.

Cy3-HRP-biotin was detected using the produced chip. 500 μL of 50 μg / mL Cy3-HRP-biotin aqueous solution (PBS, pH 7.4) was added dropwise so that all spots on the chip were covered. It was allowed to stand for 1 hour in a humid atmosphere at room temperature. Subsequently, the substrate was washed with PBS containing 0.05% Tween 20, and moisture on the substrate was removed using compressed air. Thereafter, the fluorescence was measured with a microarray fluorescence detector Genepix (manufactured by Axon Instruments).
The results are shown in FIG. 3D increased fluorescence intensity (Intensity) as the concentration of anti-Biotin antibody increased.

[Example 29: Immobilization of ligand overlaid on a spot overlaid with gel itself]
In the same manner as in Example 4, the functional group at the bottom of a 96-well multi-well plate C type (manufactured by Sumitomo Bakery Co., Ltd., surface is carboxyl group) was changed to NHS. 100 μL of 1% Streptavidin aqueous solution (100 mM carbonate buffer, pH 9.4) and 10 μL of 1% polymer aqueous solution were mixed and diluted with carbonate buffer (100 mM, pH 9.4) to prepare a 0.125% solution. Separately, a 0.125% Streptavidin aqueous solution (100 mM carbonate buffer, pH 9.4) was prepared.
These prepared solutions were spotted by dropping 0.2 μL of each of the prepared solutions into 9 wells of a multi-well plate using a micropipette. Hereinafter, a spot containing the polymer is 3D, and a spot not containing the polymer is 2D. The solution was allowed to stand at room temperature and the solution was visually confirmed to be dry. The same amount of the same solution was dropped at the same position as the spot at the bottom of 6 wells out of 9 spots, and spotted twice. After drying, the spot was spotted four times by performing the third and subsequent fourth drops on three of the six wells in the same manner. By performing a series of operations, for each of 3D and 2D, a spot spotted only once, a spot spotted twice at the same position, and a spot spot spotted four times were also prepared in three wells.
100 μL of an aqueous solution containing 3% BSA and 1M ethanolamine was added dropwise to each well in which the spots were formed, and left at room temperature for 30 minutes. Then, it wash | cleaned with MilliQ water and the water droplet of each well was removed using compressed air.
0.2 μL of Cy3-HRP-biotin aqueous solution (50 μg / mL, PBS, pH 7.4) was dropped at the same position on all spots in each well. It left still at room temperature for 1 hour. Subsequently, the substrate was washed with PBS containing 0.05% Tween 20, and moisture on the substrate was removed using compressed air. Thereafter, fluorescence measurement was performed with a fluorescence detector BioFX (manufactured by BioRad).
The results are shown in FIG. As the number of gel spots increased, the fluorescence intensity (Intensity) improved in 3D.

  Although the present invention can be used in any industrial field, it can be suitably applied to an analysis chip relating to biological, chemical, medical, etc.

DESCRIPTION OF SYMBOLS 1 Substrate 2 Spotter 3 Spot 3a Spot 3 ligand-immobilized part 3b Spot 3 ligand-immobilized part 4 Ligand solution droplet

Claims (28)

A method of manufacturing a chip comprising a substrate and a plurality of spots comprising gel immobilized on the substrate,
A gel spotting step in which the gel is immobilized on a substrate to form spots;
A chip manufacturing method comprising spotting a solution containing a ligand on the spot and immobilizing the ligand on the spot. In the ligand spotting step, spotting is performed so as to cover the spot by a spotter capable of spotting a range larger than the spot at a time, and the ligand is immobilized on the spot. 2. A method for manufacturing the chip according to 1. 2. The chip manufacturing method according to claim 1, wherein, in the ligand spotting step, spotting is performed a plurality of times by shifting the position of the spotter to immobilize the ligand on the spot. The chip manufacturing method according to claim 1, wherein in the ligand spotting step, the solution containing the ligand is dried before penetrating the entire gel. 5. The gel according to claim 1, wherein the gel is formed including a matrix having a main chain composed of a biological substance and a compound capable of binding to the biological substance. 6. Chip manufacturing method. The gel is composed of a biological material that can bind to the ligand and a compound having a particle size of 1 μm or less formed by binding a compound that can bind to the biological material, to each other. The manufacturing method of the chip | tip as described in any one of Claims 1-4. The chip manufacturing method according to claim 6, wherein a space is formed between the particulate lumps. The method of manufacturing a chip according to claim 5, wherein a ratio of the weight of the biological material to the weight of the gel is 0.1 or more. The method of manufacturing a chip according to any one of claims 5 to 8, wherein the spot has a thickness of 5 nm or more in a dry state. The method for producing a chip according to any one of claims 5 to 9, wherein at least one of the compounds has two or more functional groups capable of binding to the biological substance. The method for producing a chip according to claim 5, wherein the compound is uncharged. The method for producing a chip according to claim 5, wherein the compound is miscible with water and miscible with at least one organic solvent. The method for producing a chip according to any one of claims 5 to 12, wherein the molecular weight of the compound is 1000 or more. The method for producing a chip according to any one of claims 5 to 13, wherein the diameter of the compound in a state of being mixed in a liquid is 1 nm or more. The method for manufacturing a chip according to claim 5, wherein the biological material is a biomolecule. The chip manufacturing method according to claim 15, wherein the biomolecule is a protein. The ligand is at least one selected from the group consisting of metal chelates, vitamins, sugars, glutathione, boronic acids, proteins, antigens, nucleic acids, bioactive substances, lipids, hormones, environmental hormones, and chelating groups. The method for manufacturing a chip according to any one of claims 1 to 16. After the gel spotting step, prior to the ligand spotting step, a linker spotting step is performed in which a solution containing a linker capable of binding to the gel and the ligand is brought into contact with the spot to immobilize the linker on the spot. The method for manufacturing a chip according to claim 1, wherein: The linker mixing step of mixing the gel and a linker capable of binding to the ligand and a solution containing the ligand before the ligand spotting step is provided. A method for manufacturing the chip according to claim 1. The chip manufacturing method according to claim 18, wherein the linker is a hydrophilic molecule. The chip manufacturing method according to claim 20, wherein the hydrophilic molecule contains an ethylene oxide chain. After the gel spotting step and the ligand spotting step, a solution containing a compound having a functional group capable of binding to an active site of the biological substance in the gel is brought into contact with the spot to inactivate the gel. The method for manufacturing a chip according to any one of claims 5 to 21, wherein: A chip manufactured by the manufacturing method according to claim 1. Comprising a substrate and a plurality of spots comprising gel immobilized on the substrate,
24. The chip according to claim 23, wherein a ligand is immobilized on the spot, and a portion where the ligand is not immobilized is formed between the spots. Comprising a substrate and a plurality of spots comprising gel immobilized on the substrate,
A first ligand is immobilized on at least one of the spots;
24. The chip according to claim 23, wherein a second ligand is immobilized on at least one other spot among the spots. A detection method comprising detecting a nucleic acid, protein, toxin, virus, cell, saccharide, low molecular weight compound, or a conjugate thereof using the chip according to any one of claims 23 to 25. . A detection method characterized by detecting light having a wavelength of 580 nm or more when fluorescence detection is performed in a state where the spot is dry using the chip according to any one of claims 23 to 25. 26. A detection apparatus for detecting a nucleic acid, protein, toxin, virus, cell, saccharide, low molecular weight compound, or a conjugate thereof using the chip according to any one of claims 23 to 25. .