JP2010077189A - Gel composition, method for producing the same, and microarray using the gel composition - Google Patents

Abstract

An object of the present invention is to provide a gel composition that has transparency in analysis and can improve the diffusibility of a sample while suppressing shrinkage of the entire gel by raising the temperature, and a microarray using the same. To do.
A base gel comprising a network structure obtained by copolymerization of an acrylamide monomer and a cross-linking agent, and a thermosensitive polymer having one end covalently bonded to the base gel. Gel composition 1. A microarray having a substrate, wherein the gel composition is disposed in a compartment formed on the substrate.
[Selection] Figure 1

Description

  The present invention relates to a gel composition, a method for producing the same, and a microarray using the gel composition.

  As a gene analysis means, a microarray that can perform DNA sequence analysis, gene expression analysis, and the like collectively using a specific probe is widely used. As a specific example of such a microarray, a microarray (Patent Document 1) in which capture probes (DNA (deoxyribonucleic acid), peptides, etc.) are sequentially synthesized on the two-dimensional surface of a substrate by photolithography is synthesized in advance. A plurality of grooves or through holes (hollow portions) are formed in a microarray (Patent Document 2) in which a capture probe placed is spotted on a two-dimensional surface of the substrate, a resin plate, and the like. A microarray in which a gel containing DNA or the like is held (Patent Document 3), a microarray in which spots of gel containing DNA or the like are arranged on a flat substrate (Patent Document 4), and the like are known. Also known is a microarray (Patent Document 5) obtained by cutting a hollow fiber array in which a gel is held in a hollow part of a hollow fiber in a direction crossing the fiber axis of the hollow fiber array. .

In the DNA microarray, the capture DNA (capture probe) fixed on the substrate is in a state of falling along the substrate, so that hybridization (double helix formation reaction) with the DNA fragment derived from the target gene is performed. Efficiency may be reduced.
However, in the microarray using the gel as in Patent Documents 3 to 5, hybridization closer to the ecological environment is possible in the three-dimensional reaction field in the gel. In addition, the amount of capture DNA that can be immobilized in one section is larger than a microarray in which capture DNA is immobilized on the two-dimensional surface of the substrate. Therefore, the hybridization efficiency between the capture DNA and the target DNA fragment is increased, and the analysis accuracy and efficiency are excellent.

  In such a microarray, a sample such as a DNA fragment to be examined (hereinafter referred to as “specimen”) is sufficiently diffused in the network structure of the gel composition, so that the captured DNA in the gel composition High hybridization efficiency is achieved. However, in the microarrays as described in Patent Documents 3 to 5, the specimen is not sufficiently diffused in the gel network structure, and the region that can be used for the inspection may be limited to the gel surface.

Thus, it is important that the gel composition used for the microarray is excellent in the diffusibility of the specimen when the capture probe reacts with the specimen. At the same time, it is also important to have excellent transparency during analysis such as fluorescence analysis.
Therefore, as shown in FIG. 3, a gel composition having characteristics that increase the effective pore size of the gel stitch structure and improve the diffusibility of the specimen is obtained by copolymerizing a monomer, a crosslinking agent, and a capture probe. A gel composition 101 including a base gel 110 made of a network structure and a thermosensitive polymer 114 that is not covalently bonded to the constituent components of the base gel 110 is shown (Patent Document 6). This gel composition 101 has a structure in which a linear temperature-sensitive polymer 114 is entangled in the network structure of the base gel 110. In addition, the temperature-sensitive polymer 114 has a property of contracting due to hydrophobic interaction when the temperature is higher than the phase transition temperature.

At a temperature lower than the phase transition temperature, the gel composition 101 is in a state in which the temperature-sensitive polymer 114 spreads as shown in FIG. 3A, and is macroscopically homogeneous and transparent. On the other hand, at a temperature higher than the phase transition temperature, as shown in FIG. 3B, the thermosensitive polymer 114 shrinks while entraining the base gel 110 due to hydrophobic interaction, and the pores 112 in the base gel 110 become smaller. Collapse to form a dense structure. At this time, since the network structure of the base gel 110 becomes a non-uniform and dense structure, the gel composition 101 becomes cloudy.
By disposing such a gel composition 101 in a section formed on the substrate, the gel composition 101 has transparency in the analysis and has a diffusibility of the specimen in the hybridization (about 40 to 60 ° C.). An improved microarray can be obtained.
US Pat. No. 5,405,783 US Pat. No. 5,601,980 JP 2000-60554 A US Pat. No. 6,682,893 JP 2000-270877 A JP 2008-13663 A

However, in the gel composition 101, the thermosensitive polymer 114 contracts while entraining the base gel 110 at a high temperature, thereby forming a dense structure. Accordingly, the base gel 110 contracts and the gel composition 101 itself contracts. Is seen. Therefore, due to the contraction of the base gel 110, the sparse part created in the base gel 110 is not sufficiently large, and the diffusibility of the specimen during hybridization may not be sufficiently improved. In some cases, the gel composition 101 may be peeled off from the compartment of the substrate due to the shrinkage.
For the reasons described above, a gel composition that is transparent during analysis and improves the diffusibility of the specimen during hybridization or the like is desired without shrinkage of the gel composition itself.

  Therefore, the present invention has an object to provide a gel composition that has transparency during analysis and can improve the diffusibility of the specimen while suppressing the shrinkage of the gel composition itself by increasing the temperature, and a method for producing the same. And Moreover, the microarray using this gel composition is provided.

The gel composition of the present invention comprises a base gel composed of a network structure obtained by copolymerization of an acrylamide monomer and a crosslinking agent, and a thermosensitive polymer having one end covalently bonded to the base gel. It is a composition.
In the gel composition of the present invention, the thermosensitive polymer may be poly (N-ethyl (meth) acrylamide), poly (Nn-propyl (meth) acrylamide), poly (N-isopropyl (meth) acrylamide). ), Poly (N-cyclopropyl (meth) acrylamide), poly (N-methyl-N-ethylacrylamide), poly (N, N-diethylacrylamide), poly (N-methyl-N-isopropylacrylamide), poly ( N-methyl-Nn-propylacrylamide), poly (N-acryloylpyrrolidone), poly (N-acryloylpiperidine), one polymer or a mixture of two or more polymers preferable.
Moreover, it is preferable that the copolymerization rate of the said thermosensitive polymer is 0.01-4 mol%.
Moreover, it is preferable that the phase transition temperature of the said thermosensitive polymer is 20-50 degreeC.
Moreover, it is preferable that the covalent bond of the thermosensitive polymer to the base gel is formed by using a thermosensitive macromonomer in which an unsaturated bond is introduced at the end of the thermosensitive polymer.
Furthermore, it is preferable that a bio-related substance covalently bonded to the base gel is included.

Moreover, the microarray of the present invention has a substrate, and any one of the gel compositions is arranged in a compartment formed on the substrate.
In the microarray of the present invention, the partition is preferably formed by a groove or a through hole.

  Moreover, the manufacturing method of the gel composition of this invention is a method of manufacturing one of the said gel compositions by copolymerization reaction of an acrylamide type monomer and a thermosensitive macromonomer.

The gel composition of the present invention has transparency at the time of analysis, and by increasing the temperature, it is possible to improve the diffusibility of the specimen while suppressing the shrinkage of the gel composition itself.
Further, according to the production method of the present invention, the gel composition has transparency at the time of analysis and can improve the diffusibility of the specimen while suppressing the shrinkage of the gel composition itself by increasing the temperature. Can be obtained.
Further, the microarray of the present invention has transparency at the time of analysis, and the diffusibility of the specimen is improved by raising the temperature. Moreover, since the shrinkage | contraction of the gel itself in that case is suppressed, a gel composition does not peel from the division of a board | substrate, but can perform a stable and highly efficient analysis.

<Gel composition>
Hereinafter, an example of an embodiment of the gel composition of the present invention will be described with reference to FIGS. FIG. 1 is a conceptual diagram showing a gel composition 1 of the first embodiment. Moreover, FIG. 2 is the conceptual diagram which showed the gel composition 2 of 2nd Embodiment.

[First Embodiment]
As shown in FIG. 1, the gel composition 1 includes a base gel 10 composed of a network structure obtained by copolymerization of an acrylamide monomer and a cross-linking agent, and a temperature sensitivity in which one end is covalently bonded to the base gel 10. A composition comprising polymer 14. In the gel composition 1, the temperature-sensitive polymer 14 is disposed in the pores 12 formed by the base gel 10.

The base gel 10 is a network structure formed by copolymerization of an acrylamide monomer and a cross-linking agent, and is a gel that is the base of the gel composition 1.
Examples of acrylamide monomers include acrylamide, methacrylamide, N-methylacrylamide, N, N-dimethylacrylamide (DMAA), and N-isopropylacrylamide (NIPAM).
These acrylamide monomers may be used alone or in combination of two or more, but are preferably used alone or in combination of two. When two acrylamide monomers are used in combination, DMAA and NIPAM are more preferable.

The crosslinking agent is a polyfunctional monomer having two or more ethylenically unsaturated bonds.
Examples of the crosslinking agent include N, N′-methylenebisacrylamide, N, N′-diallyl (1,2-hydroxyethylene) -bisacrylamide, N, N′-cystamine-bisacrylamide, and N-acryloyltris (hydroxy). And methyl) aminomethane and polyethylene glycol dimethacrylate. Of these, N, N′-methylenebisacrylamide is preferable.
These crosslinking agents may be used alone or in combination of two or more.

The amount of the crosslinking agent used is such that the molar ratio between the molar amount of the monomer (acrylamide monomer and thermosensitive monomer described later) and the molar amount of the crosslinking agent is the amount of the monomer as the main agent with respect to the crosslinking agent 1. A range of 4 to 500 is preferable.
When the molar ratio is 4 or more as a main monomer with respect to the crosslinking agent 1, the network structure of the base gel 10 becomes non-uniform so that the gel composition 1 can be obtained in a low temperature environment such as during analysis (room temperature). It is easy to suppress cloudiness. Moreover, if the monomer which becomes a main ingredient with respect to the crosslinking agent 1 is 500 or less, the base gel 10 which consists of a network structure which has sufficient crosslinking degree will be easy to be obtained.

  Further, in the gel composition 1, as the acrylamide monomer forming the base gel 10, the same monomer component (for example, NIPAM or the like) as the derivative used for forming the temperature-sensitive polymer 14 described later is referred to as “monomer component (M1 However, if only such components are used as the acrylamide monomer, it is considered that the effects of shrinkage and white turbidity caused by the phase transition of the base gel 10 itself are increased. Therefore, in this invention, it is preferable that it is the base gel 10 obtained by copolymerization using a monomer component (M1) and another monomer component as an acrylamide type monomer. The copolymerization ratio of the monomer component (M1) is preferably 50 mol% or less with respect to 100 mol% of all acrylamide monomers.

  The temperature-sensitive polymer 14 is a polymer having a phase transition temperature (hereinafter referred to as “Tc”), that is, LCST (Lower Critical Solution Temperature). LCST is also called the lower critical solution temperature, and polymers having LCST cause phase separation above that temperature, become insoluble and become cloudy or precipitate. The temperature-sensitive polymer 14 has a property of transferring to hydrophobicity and contracting when the temperature exceeds the Tc.

  Examples of derivatives that form the temperature-sensitive polymer 14 include N-ethyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-isopropyl (meth) acrylamide, N-cyclopropyl (meth) acrylamide, N -Methyl-N-ethylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide, N-acryloylpyrrolidone, N-acryloylpiperidine. These derivatives may be used alone or in combination of two or more.

  The temperature-sensitive polymer 14 is poly (N-ethylacrylamide), poly (N-ethylmethacrylamide), poly (Nn-propylacrylamide), poly (Nn-propylmethacrylamide), poly (N-isopropyl). Acrylamide) (hereinafter referred to as “NIPAM polymer”), poly (N-isopropylmethacrylamide), poly (N-cyclopropylacrylamide), poly (N-cyclopropylmethacrylamide), poly (N-methyl-N-ethyl). Acrylamide), poly (N, N-diethylacrylamide), poly (N-methyl-N-isopropylacrylamide), poly (N-methyl-Nn-propylacrylamide), poly (N-acryloylpyrrolidone), poly (N -Acryloylpiperidine) It is preferably more temperature sensitive polymer species.

When the thermosensitive polymer 14 in the present invention is used, for example, in a microarray, the reaction of the biologically related substance (capture probe) and the specimen in the gel composition improves the diffusibility of the specimen, and thus reacts them. It is preferable to use a material having a Tc lower than the temperature to be used. For example, in the case of a DNA microarray, it is preferable to use a thermosensitive polymer 14 having a Tc lower than about 40 to 60 ° C. where hybridization is usually performed. On the other hand, from the viewpoint of transparency at the time of analysis by microarray (room temperature), it is preferable that the temperature-sensitive polymer 14 has a high Tc to some extent.
The temperature-sensitive polymer 14 is more preferably one having a Tc of 20 to 50 ° C. in a hybridization buffer (2 × SSC, 0.2% SDS), poly (N-isopropylacrylamide), poly (N, N— Diethylacrylamide) is particularly preferred.

Further, as a derivative of the temperature-sensitive polymer 14, an amphoteric polymer (betaine polymer) can also be used.
The zwitterionic polymer is a polymer having a quaternary ammonium base, an oxonium base, a sulfonium base or the like in the molecule, and N, N-dimethyl (acrylamidopropyl) ammonium propane sulfonate (hereinafter referred to as “DMAAPS”). It is preferable that it is a polymer using.

  The temperature-sensitive polymer having LSCT changes greatly depending on the salt concentration of the solution used. In particular, when the gel composition of the present invention is used for a microarray for nucleic acid detection or the like, it is important to select the thermosensitive polymer 14 according to the buffer composition at the time of hybridization. NIPAM polymers and the like have a low LCST in the high salt concentration region and become cloudy even at room temperature, making it difficult to apply to DNA microarrays and the like. However, it exhibits good phase transition behavior in the low salt concentration region.

On the other hand, since the polymer using DMAAPS is a zwitterionic polymer, molecules are dissolved in a solution in a high salt concentration region. Therefore, it can be set as the temperature sensitive polymer 14 according to the buffer composition to be used. For example, if the salt concentration is 2 × SSC or less (with 0.2% SDS added), the NIPAM polymer will have a salt concentration higher than 2 × SSC (with 0.2% SDS added). It is preferable that
However, in particular, when DMAAPS is used for the polymer portion, the molecule dissolves alone in the salt solution, and no phase transition occurs even when the temperature is raised. Therefore, in this case, it is preferable to use the temperature-sensitive polymer 14 obtained by copolymerizing DMAAPS and a derivative (preferably NIPAM) that forms a temperature-sensitive polymer having LCST without being dissolved in the salt solution. Thereby, the phase transition temperature can be adjusted.

When DMAAPS is used for the temperature-sensitive polymer 14, the ratio of DMAAPS is preferably 5 to 15 mol% with respect to the total of derivatives (100 mol%) used for copolymerization of the temperature-sensitive polymer 14.
When the ratio of DMAAPS is 5 mol% or more, application to high salt concentration conditions becomes easy. Moreover, if the ratio of DMAAPS is 15 mol% or less, the temperature-sensitive polymer 14 tends to cause sufficient phase transition.

The mass average molecular weight of the temperature-sensitive polymer 14 is preferably such that the total length of the temperature-sensitive polymer 14 is smaller than the size of the pores 12 (the distance between the cross-linking points), depending on the size of the pores 12. Although it is different, it is preferable that it is 2000-20000, and it is more preferable that it is 2000-10000.
If the mass average molecular weight of the temperature-sensitive polymer 14 is 2000 or more, the gel composition 1 having excellent strength can be easily obtained. Moreover, if the mass average molecular weight of the thermosensitive polymer 14 is 20000 or less, the thermosensitive polymer 14 contracts without entraining the base gel 10, and thus it is easy to suppress the gel composition 1 itself from contracting.

In the gel composition of the present invention, such a temperature-sensitive polymer 14 is covalently bonded to the base gel 10 at one end thereof.
The covalent bond of the thermosensitive polymer 14 to the base gel 10 can be formed, for example, by using a thermosensitive macromonomer in which an unsaturated bond is introduced at the end of the thermosensitive polymer described above. Specifically, for example, a vinyl group or the like is introduced at the end of the temperature-sensitive polymer to produce a temperature-sensitive macromonomer, and this is copolymerized with an acrylamide monomer and a crosslinking agent, whereby one end of the base gel 10 is added. A covalently bonded thermosensitive polymer 14 is obtained. The functional group having an unsaturated bond (unsaturated functional group) is not limited to a vinyl group, and may have an acrylate or methacrylate skeleton.

  The method for introducing an unsaturated bond at the terminal of the thermosensitive polymer is not particularly limited as long as a desired unsaturated bond can be introduced at the terminal. As a specific example, a method for obtaining a NIPAM macromonomer (temperature-sensitive macromonomer) by introducing a vinyl group at the end of a NIPAM polymer (temperature-sensitive polymer) is shown below. The synthesis scheme of NIPAM macromonomer is as follows. However, n in the synthesis scheme represents the number of repeating units.

Step (I) is a reaction utilizing the method of Kaneko et al. (Macromolecules, 28, 7712 (1995)), and using 2-aminoethanethiol (AESH) as a chain transfer agent, the end of the NIPAM polymer obtained by polymerizing NIPAM Polymer (1) can be obtained by introducing an amino group into.
As the solvent, polar solvents such as N, N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) can be used.
The reaction temperature is preferably 60 to 100 ° C.
As polymerization initiators, 2,2′-azobisisobutyronitrile (AIBN), 2,2′-azobis (2-methylbutyronitrile), 2,2′-azobis (2,4-dimethylvaleronitrile) ) Etc. can be used.

Step (II) is a method using the method of Li et al. (Small, 2, 917 (2006)), in which the polymer (1) obtained in step (I) is reacted with acryloyl chloride, and the terminal amino acid is reacted. The NIPAM macromonomer (2) is obtained by introducing a vinyl group into the base part.
As the solvent, a polar solvent such as N, N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) can be used.
The reaction temperature is preferably 30 to 50 ° C.

The total monomer concentration at the time of copolymerization in the production of the gel composition (the total of the acrylamide monomer and the thermosensitive macromonomer) is preferably 100 to 700 mol / m ³ .
If the total monomer concentration is 100 mol / m ³ or more, a gel composition with higher strength can be easily obtained. Moreover, if the total monomer concentration is 700 mol / m ³ or less, the gel composition 1 with improved diffusibility of the specimen can be obtained.

The copolymerization rate of the temperature-sensitive macromonomer is preferably 0.01 to 4 mol% with respect to the total (100 mol%) of the acrylamide monomer and the temperature-sensitive macromonomer.
If the copolymerization rate of the temperature-sensitive macromonomer is 0.01 mol% or more, the gel composition 1 having excellent strength can be easily obtained. Moreover, if the copolymerization rate of the thermosensitive macromonomer is 4 mol% or less, it is easy to obtain a gel composition 1 that has been sufficiently gelled, and it is easy to improve the diffusibility of the specimen in the gel composition 1. .

In addition, the method of manufacturing a thermosensitive macromonomer is not limited to the above-mentioned method, The method according to the unsaturated bond introduce | transduced can be used.
Further, the method of covalently bonding one end of the thermosensitive polymer 14 to the base gel 10 is not limited to the method using the thermosensitive macromonomer. For example, a reactive functional group is introduced into the polymer chain of the base gel in advance. Alternatively, a method of reacting with a temperature-sensitive polymer into which a functional group that reacts with the functional group is introduced may be used. Examples of the reaction that can be used here include a reaction that forms a Schiff base with an aldehyde group and an amino group, a reaction that uses a ring-opening reaction between ethylene oxide and an amino group, or a reaction between an epoxy and a carboxylic acid. Can be mentioned.

[Second Embodiment]
The gel composition 2 is a composition in which a bio-related substance is further contained in the gel composition 1, and, as shown in FIG. 2, a base composed of a network structure obtained by copolymerization of an acrylamide monomer and a crosslinking agent. The composition includes a gel 10, a thermosensitive polymer 14 having one end covalently bonded to the base gel 10, and a bio-related substance 16 covalently bonded to the base gel 10. In the gel composition 2, the thermosensitive polymer 14 and the biological material 16 are disposed in the pores 12 formed by the base gel 10.
The acrylamide-based monomer, the crosslinking agent, and the temperature-sensitive polymer 14 that form the base gel 10 are the same as those in the gel composition 1 and thus will not be described.

The biological substance 16 is a capture probe that captures a substance (specimen) to be detected or measured.
Examples of the biological substance 16 include DNA, RNA (ribonucleic acid), protein, and lipid. These biological substances may be commercially available or can be obtained from living cells. For extraction of DNA from living cells, the method of Blin et al. (Nucleic Acids Res., 3, 2303 (1976)) can be used. For extraction of RNA from living cells, the method of Favaroro et al. (Methods. Enzymol., 65, 718 (1980)) can be used.

  As the DNA, a chain or circular plasmid DNA or chromosomal DNA can be used. Furthermore, a restriction enzyme or a chemically cleaved DNA fragment, DNA synthesized by an enzyme or the like in a test tube, or a chemically synthesized oligonucleotide can also be used.

The biological material 16 can be fixed to the network structure of the base gel 10 by a copolymerization reaction with an acrylamide monomer and a crosslinking agent. That is, the bio-related substance 16 is covalently bonded to the base gel 10 by introducing an unsaturated functional group capable of copolymerization reaction and copolymerizing with an acrylamide monomer and a crosslinking agent, like the thermosensitive polymer 14. be able to.
Examples of unsaturated functional groups include acrylate groups and methacrylate groups in addition to vinyl groups. The unsaturated functional group may be introduced at any site as long as the function of the biological material 16 is not impaired. For example, when the biological material 16 is a nucleic acid, the unsaturated functional group may be introduced at either the terminal or the chain of the nucleic acid. However, it is preferably introduced at the end of the nucleic acid from the viewpoint of further improving the hybridization efficiency between the biological substance 16 and the specimen.
The unsaturated functional group can be introduced by the method described in WO 02/062817 pamphlet.

The copolymerization rate of the bio-related substance 16 can be adjusted according to the purpose and application, and is 10 to 100000 μmol% with respect to the total (100 mol%) of the acrylamide monomer, the thermosensitive polymer 14 and the bio-related substance 16. It is preferable that it is 100-10000 micromol%.
If the copolymerization rate of the biological material 16 is 10 μmol% or more, it is easy to improve the efficiency and accuracy of detection and measurement when the gel composition 2 is used in a microarray or the like. Moreover, if the copolymerization rate of the bio-related substance 16 is 100000 μmol% or less, it is easy to improve the diffusibility of the specimen in the gel composition 2.

The gel composition of the present invention may contain components other than the acrylamide monomer, the crosslinking agent, the temperature sensitive polymer, and the biological substance.
Examples of other components include glycerin.

[Production method]
Hereinafter, an example of a method for producing the gel composition 1 and the gel composition 2 described above will be described as a method for producing the gel composition of the present invention.
The gel composition 1 can be obtained by copolymerizing an acrylamide monomer, a crosslinking agent, and a thermosensitive macromonomer. The gel composition 2 can be obtained by copolymerizing an acrylamide monomer, a crosslinking agent, a thermosensitive macromonomer, and a biological substance.

Examples of the copolymerization method include a method using a polymerization initiator.
The polymerization initiator is not particularly limited as long as it is a method in which the thermosensitive macromonomer and the biological substance are not decomposed during the polymerization reaction. 2,2′-azobis [2- (2-imidazolin-2-yl) propane ] Dihydrochloride, APS (ammonium persulfate), KPS (potassium persulfate), etc. are mentioned.
Moreover, when using APS and KPS as a polymerization initiator, it is preferable to use TEMED (tetramethylethylenediamine) as a polymerization accelerator.

As a solvent used for the polymerization, water, ethanol or the like can be used.
The polymerization temperature is within a range lower than the Tc of the temperature-sensitive macromonomer to be used. When an azo polymerization initiator is used, it is 45 ° C. or higher, and when APS or KPS is used alone, 50 ° C. As mentioned above, when using TEMED together with APS and KPS, it is preferable to set it within the range of 20-40 degreeC.

  In the gel composition of the present invention described above, the temperature-sensitive polymer 14 spreads in the pores 12 of the base gel 10 at a temperature lower than Tc, as shown in FIGS. 1 (A) and 2 (A). Existing. Therefore, the effective pore diameter of the pores 12 is reduced by the temperature-sensitive polymer 14, and the temperature-sensitive polymer 14 becomes an obstacle, resulting in poor specimen diffusibility. However, since the gel composition is macroscopically transparent in this state, it is suitable for analysis. Here, “transparent” in the present invention means that the light transmittance is about 80% or more at a gel thickness of 2 mm.

On the other hand, when the temperature is higher than Tc, as shown in FIGS. 1 (B) and 2 (B), the temperature-sensitive polymer 14 having one end covalently bonded to the base gel 10 contracts, thereby causing pores 12. The effective pore diameter is substantially equal to that in the case without the temperature-sensitive polymer 14. Thereby, the diffusibility of the specimen in the gel composition is improved. The temperature-sensitive polymer 14 returns to the expanded state as shown in FIGS. 1 (A) and 2 (A) by lowering the temperature, and the gel composition becomes macroscopically transparent.
As described above, when detection or measurement is performed using a gel composition having a biological substance, the reaction efficiency between the biological substance and the specimen can be improved.

  Further, in the gel composition of the present invention, the base gel 10 is involved in the contraction of the temperature-sensitive polymer 14 and contracts in order to improve the diffusibility of the specimen by such a mechanism, and the gel composition itself contracts. Is suppressed. Therefore, it is possible to suppress the effective pore diameter of the pores 12 from being reduced due to the contraction of the base gel 10, and the diffusibility of the specimen is sufficiently improved.

  As described above, the gel composition of the present invention improves the diffusibility by shrinking the temperature-sensitive macromonomer by raising the temperature above Tc during the reaction between the biological substance and the specimen. At the time of detection, the gel composition can be returned uniformly and changed to be transparent by setting the temperature lower than Tc, and excitation light at the time of fluorescence detection can be transmitted through the entire gel. Therefore, the detection sensitivity becomes extremely high.

  In the gel composition of the present invention, for example, a gel (gel composition 2) holding a biological substance (capture probe) can be used for gene analysis. For example, as described in JP-A-3-47097, by preparing a gel-holding tubular body by filling the hollow portion of the tubular body with the gel composition 2 described above, as a tool for analyzing gene mutations Can be used. The gel composition can be held in the hollow portion by the same method as that for producing a capillary column used for capillary gel electrophoresis.

<Microarray>
Hereinafter, the microarray of the present invention will be described. The microarray of the present invention has a substrate, and the gel composition described above is arranged in a section formed on the substrate. Hereinafter, an example of the embodiment will be described.

  In the microarray 20 of this embodiment, as shown in FIG. 4, a plurality of tubular bodies 24 are fixed to a substrate 22, and a hollow portion of the tubular body 24 forms a through hole 26 (partition). Further, the gel composition 2 is held in the through hole 26.

The substrate 22 is formed of an adhesive that fixes a bundle of a plurality of tubular bodies 24.
As the adhesive, a resin usually used in the production of microarrays can be used, and examples thereof include polyurethane resin adhesives such as Nipponran 4276 and Coronate 4403 (registered trademark, manufactured by Nippon Polyurethane Industry Co., Ltd.).

The shape of the substrate 22 is usually square or rectangular with regular arrangement of through holes (tubular bodies), but is not limited thereto. “Regularly” means that the number of tubular bodies included in a frame of a certain size is arranged in order so that the number is constant.
Moreover, it is preferable that the thickness of the board | substrate 22 is 50 micrometers-1 mm.

  The tubular body 24 may be in any form such as a glass tube, a stainless tube, a hollow fiber, and a pulp as long as the tubular body 24 has a hollow shape. In view of processability and ease of handling, it is preferable to use hollow fibers. In addition, although it does not specifically limit as a hollow fiber, For example, a synthetic fiber, a semi-synthetic fiber, a regenerated fiber, a natural fiber etc. are mentioned. The tubular body 24 may be either a porous tubular body or a non-porous tubular body, but suppresses diffusion of the precursor solution before polymerization of the gel composition to the outside of the hollow fibers and drying of the gel composition. From the viewpoint, a non-porous tubular body is preferable.

The material of the tubular body 24 is not particularly limited, and examples thereof include inorganic materials such as silica and glass, and organic materials shown below.
Examples of the organic material include polyamide materials such as nylon 6, nylon 66 and aromatic polyamide, polyester materials such as polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and polycarbonate, and acrylic materials such as polyacrylonitrile. , Polyolefin materials such as polyethylene and polypropylene, polymethacrylate materials such as polymethyl methacrylate, polyvinyl alcohol materials, polyvinylidene chloride materials, polyvinyl chloride materials, polyurethane materials, phenol materials, polyvinylidene fluoride, Examples include fluorine-based materials such as polytetrafluoroethylene, polyalkylene paraoxybenzoate-based materials, and the like. Moreover, black pigments, such as carbon black, may contain.

The through hole 26 (hollow part of the tubular body 24) is preferably aligned and fixed.
The inner diameter of the through hole 26 (the inner diameter of the tubular body 24) is not particularly limited, but is preferably 1 mm or less, and more preferably 10 μm to 300 μm.

The number of through holes 26 (number of sections) is determined by the number of tubular bodies 24. The number of sections of the microarray of the present invention can be the number of sections in the microarray, and is preferably 10 to 10,000 / cm ² .

[Production method]
The microarray of the present invention can be produced by the following steps (i) to (iii).
Step (i): A step of converging a plurality of tubular bodies so that their longitudinal directions coincide with each other to obtain a converged object.
Step (ii): Filling the hollow part of each tubular body of the converged product with a precursor solution (a solution before polymerization of the gel composition of the present invention) containing an acrylamide monomer, a crosslinking agent, and a thermosensitive macromonomer. And a step of carrying out a copolymerization reaction at a predetermined polymerization temperature (for example, a polymerization temperature of 40 ° C. or less) in the hollow portion.
Step (iii): A step of cutting the converging object in a direction crossing the longitudinal direction of the converging object.
In the step (ii), it is preferable to fill the hollow portion with the gel composition 2 of the present invention (a gel composition containing a biological substance).

Hereinafter, as an embodiment of a microarray manufacturing method, a method of manufacturing the microarray 20 having 100 through holes 26 and having a square substrate 22 as illustrated in FIG. 1 will be described.
In step (i), 10 tubular bodies are bundled in a row and fixed to form a single layer sheet, and then the sheet is formed into 10 layers so that the longitudinal directions of the respective tubular bodies coincide. A converging object is obtained by being fixed on the substrate. Examples of the method for immobilizing the tubular body in the step (i) include a method performed using an adhesive such as a polyurethane resin adhesive.

Subsequently, in the step (ii), the hollow portion of each tubular body of the bundle is filled with the precursor solution of the gel composition of the present invention. The method for filling the precursor solution is not particularly limited. For example, the method may be a method in which one end of the convergent product is immersed in the precursor solution and sucked from the other end side. Alternatively, the precursor solution may be pushed from one end side.
Moreover, after sealing one side of a tubular body and decompressing, the method of attracting | sucking by immersing an open end in a precursor solution and returning to a normal pressure in that state may be sufficient.

  The polymerization temperature of the copolymerization of the precursor solution in the step (ii) is preferably performed at Tc or less of the temperature-sensitive macromonomer to be used. A preferable temperature range is 40 ° C. or less. When the polymerization temperature is higher than 40 ° C., the temperature-sensitive macromonomer undergoes phase transition during the polymerization and tends to become a cloudy gel composition. Such a gel composition is not suitable for analysis because it does not become transparent even at low temperatures. As an initiator for carrying out the polymerization at the temperature as described above, a material obtained by adding TEMED to APS, a photolytic initiator, or the like can be used.

  In step (iii), the obtained converging object is cut into a thin piece by cutting in a direction crossing the longitudinal direction of the converging object. Thereby, the microarray 20 having the substrate 22 having a desired thickness in which 100 through holes 26 are formed is obtained. The cutting method is not particularly limited as long as it is a method capable of cutting the convergent material with a desired thickness, and examples thereof include a method of cutting with a microtome.

  Moreover, although the microarray 20 can be obtained by the steps (i) to (iii) described above, the order of the step (i) and the step (ii) is not particularly limited, and after the step (ii) is performed, the step ( i) may be performed. That is, after the hollow body of each tubular body is filled with a precursor solution and subjected to a polymerization reaction (step (ii)), the tubular bodies are converged to form a converging object (step (i)). A method of obtaining a microarray by slicing a product may be used (see International Publication No. 00/5376 pamphlet).

Since the microarray of the present invention described above uses the gel composition of the present invention, it is transparent at the time of analysis usually performed at room temperature, and the sample is raised when the temperature such as hybridization is increased. Therefore, it is possible to analyze with high efficiency and high accuracy.
The microarray of the present invention is not limited to the microarray illustrated in FIG. For example, the section of the microarray of the present invention is not limited to the through hole as in the microarray 20, and may be a section by a groove. However, from the viewpoint of being suitable for mass production by the aforementioned steps (i) to (iii), a microarray in which compartments are formed by through holes using a tubular body is preferable.

Moreover, the microarray which arrange | positioned the gel composition 1 instead of the gel composition 2 may be sufficient.
Alternatively, a microarray in which a precursor solution containing a capture probe before polymerization or immediately after the start of polymerization is spotted on a predetermined section on a flat substrate may be used (Japanese Patent Publication No. 6-507486, US Pat. No. 5,770,721). See the description).

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description.
<Temperature sensitive macromonomer>
[Production Example 1]
A NIPAM macromonomer, which is a thermosensitive macromonomer, was synthesized using the synthesis scheme described above.
In step (I), the chain transfer agent, AESH (0.43446 g), NIPAM (11.5900 g), and the solvent, DMF, were charged into a parabolic flask with a condenser placed in an oil bath at 75 ° C. Purity nitrogen gas was purged for 2 hours. Next, AIBN (0.1774 g) of a polymerization initiator that had been dissolved in DMF in advance and purged with high-purity nitrogen gas at room temperature was added, and reacted for 15 hours in a high-purity nitrogen gas atmosphere. DMF used for the reaction was 45 mL.
After completion of the reaction, DMF was removed using a vacuum dryer to obtain a polymer (1) having an amino group at the terminal, which is a precursor of a thermosensitive macromonomer. Next, in order to purify the polymer (1), the obtained reaction product was redissolved in acetone and dropped into diethyl ether to precipitate the polymer (1). Thereafter, filtration was performed, and purification was again performed with diethyl ether, followed by drying.

  In step (II), the obtained polymer (1) (3.0 g), acryloyl chloride (20 mL), and DMF (5 mL) were put into a sample bottle, and stirred at room temperature for 30 minutes while purging high-purity nitrogen gas. did. This was transferred to a constant temperature bath at a temperature of 40 ° C. and further stirred for 2 hours in a high purity nitrogen gas atmosphere to carry out the reaction. After completion of the reaction, the resulting reaction solution was added dropwise to diethyl ether to precipitate the NIPAM macromonomer of the reaction product and filtered. Further, the same operation was performed twice to purify, and the NIPAM macromonomer recovered by filtration was vacuum-dried.

[NMR analysis]
FIG. 5 shows the result of analyzing the NIPAM macromonomer obtained in Production Example 1 by ¹ H-NMR. Heavy water (D ₂ O) was used as a measurement solvent for ¹ H-NMR.
As shown in FIGS. 5 (A) and 5 (B), peaks (g), (h), (i) peculiar to the vinyl group are observed in the range of 5.6 to 6.5 ppm of the NMR spectrum, It was confirmed that the desired NIPAM macromonomer having a vinyl group was obtained.

  Moreover, the molecular weight of the obtained NIPAM macromonomer was estimated from the peak integration ratio in the NMR spectrum. As shown in FIG. 6, from the integral ratio (1: 125.7383) between the peak of the vinyl group at the end (one peak at H) and the peak of the isopropyl group at the NIPAM polymer portion (6 peaks at H), NIPAM The number of repeating units in the polymer portion was estimated to be n = 21. Thus, the mass average molecular weight of the NIPAM macromonomer was estimated to be about 2500 g / mol.

[Measurement of phase transition temperature]
Further, the phase transition temperature (Tc) of the NIPAM macromonomer obtained in Production Example 1 was evaluated by measuring the temperature dependence of the solution permeability (wavelength 600 nm). For the measurement of transmittance, a spectrophotometer with a Peltier element temperature control function (JASCO V530 type) was used. NIPAM macromonomer was dissolved at a rate of 5 g / L in water and in a hybridization buffer (2 × SSC, 0.2% SDS), and the temperature dependence of the permeability was determined. The result is shown in FIG.

As shown in FIG. 7, in water, a significant decrease in permeability occurs at about 34 ° C., and this temperature can be regarded as Tc. The Tc of the NIPAM macromonomer obtained in Production Example 1 was a value equivalent to that of a high molecular weight NIPAM polymer (without introducing a vinyl group).
Further, in the hybridization buffer, Tc was shifted by several degrees C. on the high temperature side as compared with that in water. This is considered to be because the effect | action which raises Tc of SDS which is surfactant is larger than the effect | action which reduces Tc of SSC which is a salt.

<Gel composition>
[Example 1]
N, N-dimethylacrylamide (DMAA), which is an acrylamide monomer, N, N′-methylenebisacrylamide (MBAA), which is a cross-linking agent, and the NIPAM macromonomer are dissolved in water as a solvent to form a polymerization initiator. Using APS and TEMED, which is a polymerization accelerator, a polymerization reaction is performed at 20 ° C. lower than the Tc of the NIPAM macromonomer to obtain a gel composition having a base gel and a NIPAM polymer having one end covalently bonded to the base gel. It was. The concentration of the crosslinking agent was 4 mol / m ³ , and the concentrations of TEMED and APS were 10 mol / m ³ and 0.5 mol / m ³ , respectively. The concentration of all monomers (sum of DMAA and NIPAM macromonomer) was 400 mol / m ³ , and the copolymerization rate of NIPAM macromonomer was 5 mol%.
Total monomer concentration 400 mol / ^{m 3,} if a crosslinking agent concentration of 4 mol / ^{m 3,} the distance between crosslinking points is considered to be 100 or so DMAA units (molecular weight of about 10000), is larger than NIPAM macromonomer.

[Examples 2 to 13]
A gel composition was obtained in the same manner as in Example 1 except that the total monomer concentration and the copolymerization ratio of the NIPAM macromonomer were changed as shown in Table 1.
Table 1 shows the results of evaluating the gelation state of the gel compositions obtained in Examples 1 to 13. The gelation was evaluated based on the following criteria.
(Double-circle): It gelatinized fully.
○: Gelation was observed but soft.

As shown in Table 1, when the total monomer concentration was 400 mol / m ³ , the obtained gel composition was soft when the NIPAM macromonomer copolymerization rate was 5 mol% (Example 1). % Or less (Examples 2 to 6), a gel composition having higher strength was obtained. When the total monomer concentration was 300 mol / m ³ , the gel composition obtained was soft when the NIPAM macromonomer copolymerization rate was 3.5 mol% (Example 7), but 3 mol% or less (Example 8). And in 9), a gel composition having better strength was obtained.
When the total monomer concentration is 267 mol / m ³ , the gel is excellent in strength both when the copolymerization rate of the NIPAM macromonomer is 3 mol% (Example 10) or 1 mol% (Example 11). A composition was obtained, and it was found that a gel composition excellent in strength was easily obtained as compared with the case where the total monomer concentration was 200 mol / m ³ (Examples 12 and 13).

[Examples 14 to 17]
In a space formed by sandwiching a spacer with a thickness of 3 mm between two glass plates with a total monomer concentration of 400 mol / m ³ , a copolymerization rate of NIPAM macromonomer of 0.5, 1, 2, 3 mol%, And TEMED were used to conduct a polymerization reaction at 20 ° C. to produce a plate-like gel having a thickness of 3 mm. The concentration of the crosslinking agent was 4 mol / m ³ , and the concentrations of TEMED and APS were 10 mol / m ³ and 0.5 mol / m ³ , respectively. Other than that was performed similarly to Examples 1-13.

[Evaluation of clouding]
Using a quartz cell with an optical path length of 10 mm, the plate-like gel is cut and inserted so as to enter the quartz cell, and a spectrophotometer with a Peltier element temperature control function ( Installed in JASCO V530), the permeability in water was measured. The measurement results are shown in FIG.
Further, the permeability in a hybridization buffer (2 × SSC, 0.2% SDS) was measured in the same manner. The measurement results are shown in FIG.

As shown in FIG. 8A, in the case of water, the gel (plate-like gel) became cloudy when the temperature exceeded 34 ° C., which is the Tc of the NIPAM macromonomer. Further, the degree of white turbidity increased as the copolymerization rate of the NIPAM macromonomer increased. However, when the copolymerization rate was 3 mol%, the degree of cloudiness was reduced. This is considered to be because the degree of gelation is reduced by increasing the proportion of NIPAM macromonomer during copolymerization.
Further, in the hybridization buffer, as shown in FIG. 8 (B), when the copolymerization rate of the NIPAM macromonomer was 2 mol%, a slight turbidity was observed at 70 ° C. In the case of, no clouding was observed.

[Example 18]
Cylindrical gels were prepared in the same manner as in Examples 14 to 17 except that the NIPAM macromonomer copolymerization rate was 1 mol% and synthesis was performed in a glass tube having an inner diameter of 3.4 mm.

[Reference Example 1]
A columnar gel was prepared in the same manner as in Example 18 except that the NIPAM macromonomer was not used.

[Temperature dependence of swelling degree]
For the columnar gel obtained in Example 18 and Reference Example 1, the swelling diameter of the columnar gel was read at each temperature in water and in a hybridization buffer (2 × SSC, 0.2% SDS) using a microscope. The temperature dependence was evaluated. The results in water are shown in FIG. 9A, and the results in hybridization buffer are shown in FIG. 9B. The broken lines in FIGS. 9A and 9B indicate the inner diameter (3.4 mm) of the glass tube used for the gel synthesis.

As shown in FIG. 9A, the swelling diameter of the columnar gel of Example 18 was somewhat contracted in water as compared with Reference Example 1 containing only the base gel (DMAA), but its temperature dependence. Was small. In the hybridization buffer, as shown in FIG. 9B, the swelling diameter was almost the same as in Reference Example 1, and the temperature dependency was very small.
The reason why the temperature dependence in the hybridization buffer is smaller than in water is considered to be because the shrinkage of the NIPAM polymer is reduced to some extent by SDS as a surfactant.

  Next, in order to evaluate the improvement in the diffusibility of the specimen due to the shrinkage of the NIPAM polymer (temperature-sensitive polymer) having one end copolymerized with the base gel, rhodamine B, which is easy to measure the concentration, is used as a model substance. The temperature dependence of the diffusion coefficient was measured. Hereinafter, measurement of the temperature dependence of the diffusion coefficient will be described.

[Example 19]
With the total monomer concentration being 400 mol / m ³ and the copolymerization rate of NIPAM macromonomer being 1 mol%, a polymerization reaction was carried out in a glass gel holder 30 shown in FIG. The concentration of the crosslinking agent was 4 mol / m ³ , and the concentrations of TEMED and APS were 10 mol / m ³ and 0.5 mol / m ³ , respectively.
The glass gel holder 30 has a configuration in which a glass plate 32 having an opening for introducing a precursor solution of the gel composition is sandwiched between two glass plates 34 and further sandwiched by a glass plate 36 having a wire mesh 38. The gel film can be manufactured in a space surrounded by the glass plates 32 and 34 and the wire mesh 38. A solution that can pass through the gel film 1A can pass from one side to the other side through the wire mesh 38, the gel film, and the wire mesh 38 in order.
The opening formed in the upper part of the glass plate 32 was sealed with an adhesive 40.

  Next, a diffusion coefficient measuring apparatus that has two glass cells 52 (capacity 500 mL), each glass cell 52 is filled with 300 mL water, and both glass cells 52A and 52B are both placed in a constant temperature bath 54. Between the two glass cells 52A and 52B of 50 (FIG. 11), the glass gel holder 30 which has the gel film 1A was installed. Thereafter, the temperature of the water in the gel film 1A and the glass cells 52A and 52B is raised to a predetermined temperature, and 100 mL of the rhodamine B aqueous solution (rhodamine B concentration: 0.2 g / L) having the predetermined temperature is injected into the water of the glass cell 52A. 100 ml of water was poured into the glass cell 52B. Thereafter, the concentration of rhodamine B diffused to the glass cell 52B side was measured over time, and the temperature dependence of the diffusion coefficient was evaluated. The concentration of rhodamine B in the glass cell 52B was measured using a spectrophotometer (JASCO V530 type) at a wavelength of 533 nm.

[Comparative Example 1]
The concentration of rhodamine B in the glass cell 52B was measured in the same manner as in Example 19 except that the gel film 1B of only DMAA not using the NIPAM macromonomer was formed, and the temperature dependence of the diffusion coefficient was evaluated.

ここで、式中のＣ（ｔ）、Ｖ、Ｘは以下の意味を示す。
Ｃ（ｔ）：時間ｔにおけるガラスセル５２Ｂ側のローダミンＢの濃度
Ｖ：ガラスセル５２Ｂ内の溶液の容積
Ｘ：ゲル膜の面積 [Measurement and evaluation of diffusion coefficient]
The diffusion coefficient can be calculated by the following procedure.
First, the amount Q per unit area of rhodamine B that has passed through the gel membrane by time t is represented by the following formula.

Here, C (t), V, and X in the formula have the following meanings.
C (t): concentration of rhodamine B on the glass cell 52B side at time t V: volume of the solution in the glass cell 52B X: area of the gel film

ここで、式中、Ｄおよびｄは以下の意味を示す。
Ｄ：ゲル膜中の拡散係数
ｄ：ゲル膜の厚さ
これにより、拡散係数Ｄは次式のように表されるので、ガラスセル５２Ｂ側のローダミンＢの濃度の経時変化を測定することにより拡散係数を算出することができる。

In the above formulas, C (t) is very small compared to the initial concentration C ₀ of Rhodamine B in the glass cell 52A side, the permeation rate of the gel film of Rhodamine B is assumed to be constant, the equation of Fick on Diffusion When applied, the following equation is derived.

Here, in the formula, D and d have the following meanings.
D: Diffusion coefficient in the gel film d: Gel film thickness As a result, the diffusion coefficient D is expressed by the following equation. Therefore, diffusion is measured by measuring the change over time in the concentration of rhodamine B on the glass cell 52B side. A coefficient can be calculated.

Regarding the measurement, an interaction was observed between the buffer and rhodamine B in the hybridization buffer.
The measurement results of the temperature dependence of the diffusion coefficient in Example 19 and Comparative Example 1 are shown in FIG. Further, an Arrhenius plot of the diffusion coefficient in Example 19 is shown in FIG.

As shown in FIG. 12, in Example 19, which is a gel film having a NIPAM polymer copolymerized with a NIPAM macromonomer, the diffusion coefficient increases greatly with increasing temperature, and the diffusion coefficient at 70 ° C. is higher than that at 25 ° C. It was about 4 times. Moreover, the increasing tendency of the diffusion coefficient was different around 34 ° C., which is the Tc of the NIPAM macromonomer (NIPAM polymer) in water. On the other hand, in Comparative Example 1 in which only DMAA without NIPAM macromonomer was used, the diffusion coefficient increased with increasing temperature, but the diffusion coefficient was larger than that in Example 19 in the low temperature region.
Thus, Example 19 and Comparative Example 1 showed a difference in the diffusion coefficient in the low temperature region, but the difference decreased as the temperature increased, and the diffusion coefficient became almost the same in the high temperature region. .

This result is considered as follows. Since the gel film 1A of Example 19 and the gel film 1B of Comparative Example 1 both have a total monomer amount of 400 mol / m ³ , the gel film 1A of Example 19 having a long-chain NIPAM polymer having a higher molecular weight. Is dense. Therefore, at a temperature lower than Tc, the NIPAM polymer is present in an expanded structure, so that the effective pore diameter of the gel membrane 1A is small, and the diffusion of rhodamine B is further suppressed compared to the gel membrane 1B. ing. On the other hand, at a temperature higher than Tc, the effective pore diameter of the gel membrane 1A becomes almost equal to the effective pore size of the gel membrane 1B due to the shrinkage of the NIPAM polymer, so the influence of the NIPAM polymer on the temperature dependence of the diffusion coefficient Is very small, and the diffusion coefficient is considered to be almost the same.

  Further, in the gel film 1A of Example 19, as shown in FIG. 13, the gradient of the Arrhenius plot (−Ea / RT, Ea: activation energy, R: gas constant) is greatly changed near Tc. The activation energy was lower at higher temperatures compared to lower temperatures. This result also seems to indicate that the diffusibility of rhodamine B is improved by shrinkage of the NIPAM polymer when Tc is exceeded.

  The gel composition of the present invention has transparency in the analysis, and can improve the diffusibility of the specimen by increasing the temperature. Therefore, it can be suitably used as a microarray that can perform detection or measurement with high efficiency and high accuracy.

It is the conceptual diagram which showed one example of embodiment of the gel composition of this invention. It is the conceptual diagram which showed other embodiment examples of the gel composition of this invention. It is the conceptual diagram which showed an example of embodiment of the conventional gel composition. It is the perspective view which showed one example of embodiment of the microarray of this invention. It is the figure which showed the NMR spectrum of the NIPAM macromonomer in a present Example. It is the figure which showed the NMR spectrum of the NIPAM macromonomer in a present Example. It is the figure which showed the measurement result of the transmittance | permeability of the NIPAM macromonomer in a present Example. It is the figure which showed the measurement result of the transmittance | permeability of the NIPAM macromonomer of Examples 14-17. (A) in water, (B) in hybridization buffer. It is the figure which showed the measurement result of the swelling diameter of Example 18 and Reference Example 1. (A) in water, (B) in hybridization buffer. It is the figure which showed the glass-made gel holder used in the present Example. (A) The front view of the glass plate lamination | stacking part of the center part of a glass-made gel holder, (B) Longitudinal exploded sectional drawing. It is the schematic diagram which showed the diffusion coefficient measuring apparatus used in the present Example. It is the figure which showed the temperature dependence of the diffusion coefficient of Example 19 and Comparative Example 1. FIG. 20 shows an Arrhenius plot of the diffusion coefficient of Example 19.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 Gel composition 2 Gel composition 10 Base gel 12 Pore 14 Thermosensitive polymer 16 Bio-related substance 20 Microarray 22 Substrate 24 Tubular body 26 Through-hole

Claims (9)

  A gel composition comprising: a base gel composed of a network structure obtained by copolymerization of an acrylamide monomer and a crosslinking agent; and a thermosensitive polymer having one end covalently bonded to the base gel.   The temperature-sensitive polymer is poly (N-ethyl (meth) acrylamide), poly (Nn-propyl (meth) acrylamide), poly (N-isopropyl (meth) acrylamide), poly (N-cyclopropyl (meta) ) Acrylamide), poly (N-methyl-N-ethylacrylamide), poly (N, N-diethylacrylamide), poly (N-methyl-N-isopropylacrylamide), poly (N-methyl-Nn-propylacrylamide) ), Poly (N-acryloylpyrrolidone), poly (N-acryloylpiperidine), one polymer selected from the group consisting of two or more polymers, or a mixture of two or more polymers.   The gel composition according to claim 1 or 2, wherein a copolymerization ratio of the temperature-sensitive polymer is 0.01 to 4 mol%.   The gel composition in any one of Claims 1-3 whose phase transition temperature of the said thermosensitive polymer is 20-50 degreeC.   The covalent bond of the thermosensitive polymer to the base gel is formed by using a thermosensitive macromonomer in which an unsaturated bond is introduced at the end of the thermosensitive polymer. The gel composition according to any one of the above.   Furthermore, the gel composition of Claims 1-5 containing the bio-related substance covalently bonded to the said base gel.   The microarray which has a board | substrate and arrange | positions the gel composition in any one of Claims 1-6 in the division formed on this board | substrate.   The microarray according to claim 4, wherein the partition is formed by a groove or a through hole.   The manufacturing method of a gel composition which manufactures the gel composition in any one of Claims 1-6 by copolymerization reaction of an acrylamide type monomer and a thermosensitive macromonomer.

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