JP2005037331A - Substrate for detecting substance derived from organism and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a more sensitive substrate for detecting a substance derived from an organism for detecting the substance derived from the organism by fixing capture molecules of the substance derived from the organism, such as an antibody on the substrate. <P>SOLUTION: The substrate for detecting the substance derived from the organism catches and fixes the substance derived from the organism, and is used to detect the caught and fixed substance derived from the organism. At least one side of surfaces is covered with a polymer having a super-hydrophilic radical, and molecules catching specifically the substance derived from the organism or supermolecules are fixed through the polymer intruding on super-hydrophilic polymer chains and/or between the chains. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a substrate used for detecting a biological substance using a molecule that specifically captures the biological substance.

  When looking at the expression status of cell functions and responsiveness to various stresses, the purpose is achieved by looking at the expression status of genes and protein expression status related to each function or stress response. In the field of regenerative medicine, which has been actively studied in recent years, it is important to look at the expression of genes and proteins and the expression of sugar chains on the cell surface in order to see the state of cell differentiation. .

  Moreover, in the diagnosis of diseases, for example, in the diagnosis of cancer, it has been studied and actually performed to check the expression status of intracellular oncogenes and the expression status of other proteins serving as markers. Conventionally, ELISA has been used in Northern blots, Western blots, and immunoassays based on electrophoresis for the detection of biomarkers that serve as these markers. However, these methods require a lot of labor and time to obtain results from sample preparation, and the number of markers that can be detected at one time is limited.

  Therefore, in recent years, with the development of genome analysis technology, there has been a lot of movement to detect many markers quickly and with a small amount of sample, and many types of DNA are mounted in spots on one substrate. DNA microarrays have been put into practical use and are widely used in basic research fields. In addition, protein microarrays in which proteins are mounted on a substrate have been studied.

  A method for producing a microarray will be described. A widely used method is a method in which cDNA obtained by reverse transcription from synthesized oligo DNA or mRNA is spotted and fixed on a substrate. In this method, a solution in which oligo DNA or cDNA is dissolved is applied to the tip of a pin, and so-called stamping is applied to the substrate surface. In this method, it is difficult to immobilize DNA on the substrate surface simply by spotting a DNA solution on the substrate, and a process for immobilizing DNA on the substrate surface is performed in advance. For example, when immobilizing cDNA, which is a long DNA, a method of electrostatically immobilizing negatively charged DNA by coating the surface of the substrate with polylysine or polyethyleneimine and applying a positive charge is widely used. (Non-Patent Document 1).

  Since DNA is short in oligo DNA immobilization, it is difficult to immobilize DNA by electrostatic immobilization and is immobilized by covalent bond. As a method of immobilizing DNA on the substrate surface by covalent bond, a reactive active group is introduced at the end of the oligo DNA, while a reactive active group that reacts with a reactive active group introduced into the oligo DNA is introduced on the substrate surface, and then covalently bonded. Is used to immobilize oligo DNA (Non-patent Document 2, Non-patent Document 3, Non-patent Document 4). As the reactive functional group to be introduced into the oligo DNA to be fixed by forming a covalent bond, amino group, aldehyde group, SH group, biotin and the like are used. For the surface treatment of the solid phase carrier, various silane coupling agents having an amino group, an aldehyde group, an epoxy group or the like are used. By such covalent fixing, the oligo DNA can be firmly and stably fixed on the substrate surface.

In addition, protein microarrays are being studied by applying DNA microarray technology to protein detection. In the protein microarray, proteins are spotted on a substrate, and biological substances that specifically bind to the spotted proteins are detected.
As an example of the most typical protein microarray, an antibody is spotted on a substrate and used for detection of a protein derived from a living body. The measurement principle is based on the ELISA method. Instead of the enzyme-labeled antibody used for detection, the antibody is labeled, and the sandwich method is used to read the fluorescence intensity at the spot that has been spotted with a scanner. Etc. are being considered.

As a method for immobilizing the antibody or protein on the substrate, a method according to the conventional ELISA method is used. Proteins are relatively easily adsorbed on the substrate surface, and the protein can be easily immobilized on the substrate. Polystyrene is an example of a material that can easily adsorb proteins, and is widely used for ELISA plates.
In the case of stably immobilizing a protein, a method of immobilizing a protein by forming a covalent bond as in the above-described DNA microarray is used. Proteins are rich in amino groups and SH groups, and these active groups can be used to form covalent bonds and stably immobilized.

  In DNA microarrays and protein microarrays, high detection sensitivity is desired. Even if DNA and protein can be stably immobilized on the substrate, it is difficult to obtain high detection sensitivity. In the case of an antibody, if it is firmly immobilized on the surface of the substrate, the reaction efficiency with the antigen deteriorates, causing a reduction in sensitivity. In addition, the high ability to immobilize a protein causes nonspecific adsorption of the protein, which increases the background.

As a method for suppressing nonspecific adsorption of proteins, a method of forming a superhydrophilic surface has been disclosed (Patent Documents 1 and 2). However, stable fixation of DNA and protein could not be performed on the surface of the substrate, and therefore sufficient sample detection sensitivity could not be obtained.
Schena, M. et al, Science, 270, 467-470 (1995) "Proteins, Nucleic acids, Enzymes", 43, 2004-2011 (1998); Lamture, JB et al., Nucl. Acids Res., 22, 2121-2125 (1994 Guo, Z. et al., Nucl. Acids Res., 22,5456-5465 (1994)) JP 2003-130882 A International Publication No. 00/039582

  An object of the present invention is to stably immobilize molecules or supramolecules that specifically capture biological substances such as antibodies on a substrate, and to detect the target biological substances using their specific binding properties. In other words, the present invention provides a biological substance detection substrate capable of being detected with high sensitivity.

The inventor forms a polymer layer having a superhydrophilic group having a low affinity and / or non-adsorbing property on a biological material on the surface of the substrate, and then forms biological materials such as antibodies on the polymer. By specifically immobilizing molecules to be captured in advance and contacting a polymer layer having a superhydrophilic group, it retains the action of suppressing nonspecific adsorption of the polymer having a superhydrophilic group to biological substances. And it discovered that specific capture performance, such as an antibody, was improved, and came to complete this invention.

That is, the present invention
(1) A substrate used for capturing and fixing a biological substance, and detecting the captured and fixed biological substance, wherein at least one surface is coated with a polymer having a superhydrophilic group, A biological substance-detecting substrate, wherein a molecule or a supermolecule that specifically captures a biological substance is fixed on the superhydrophilic polymer chain and / or via a polymer that enters between the chains;
(2) The molecule or supramolecule that specifically captures a biological substance is at least one selected from nucleic acids, peptides, proteins, sugar chains, lipids, and steroids, or at least one of these. (1) a substrate for detecting a biological substance, which is a complex comprising
(3) The substrate for detecting a biological substance according to (1) or (2), wherein the molecule or supramolecule that specifically captures the biological substance is an antibody,
(4) The biological substance detection substrate according to (1) or (2), wherein the molecule or supramolecule that specifically captures the biological substance is a lectin protein,
(5) The substrate for detecting a biological substance according to (1) or (2), wherein the molecule or supramolecule that specifically captures the biological substance is an aptamer,
(6) The biological substance detection substrate according to (1) or (2), wherein the molecule or supramolecule that specifically captures the biological substance is an enzyme,
(7) The biological substance-detecting substrate according to (1) or (2), wherein the molecule or supramolecule that specifically captures the biological substance is a part of a cell or a cell,
(8) The substrate for detecting a biological substance according to any one of (1) to (7), wherein the superhydrophilic group is derived from phospholipid,
(9) The biological substance-detecting substrate according to (8), wherein the polymer having a superhydrophilic group is a 2-methacryloyloxyethyl phosphorylcholine polymer and / or a copolymer of 2-methacryloyloxyethyl phosphorylcholine-containing components,
(10) The biological material is at least one selected from nucleic acids, peptides, proteins, sugar chains, lipids, and steroids, or a complex containing at least one of these (1) to ( 9) Any biological substance detection substrate,
(11) The biological substance detection substrate according to any one of (1) to (9), wherein the biological substance is a part of a cell structure or a cell.
(12) The biological substance detection substrate according to any one of (1) to (11), wherein a molecule or supramolecule that specifically captures a biological substance is fixed to the polymer by electrostatic bonding or covalent bonding,
(13) The immobilization of a molecule or supramolecule that specifically captures a biological substance is achieved by introducing a functional group having an electrostatic bond or a covalent bond into a copolymer of 2-methacryloyloxyethyl phosphorylcholine-containing components. (12) the biological substance detection substrate,
(14) The biological substance detection substrate according to (12), wherein a molecule or supramolecule that specifically captures a biological substance is immobilized on a polycationic polymer,
(15) The biological substance detection substrate according to (14), wherein the polycationic polymer is at least one selected from polyethyleneimine, polyvinylamine, polyallylamine, polyornithine, and polylysine,
(16) Immobilization of molecules or supramolecules that specifically capture biological substances is spot-shaped on the substrate and is arranged with regularity (1) to (15) Substrate for object detection,
(17) including a step of bringing a solution containing and / or dissolving a molecule that specifically captures a biological substance or a polymer immobilizing a supramolecule into contact with a substrate surface coated with a polymer having a superhydrophilic group, (1) to (16) any one of the methods for producing a biological substance-detecting substrate,
(18) (1) a step of dissolving a polymer having an active group in a solution;
(2) A step of dissolving a molecule or supramolecule that captures a biological substance in the solution of (1),
(3) a step of inactivating the active group of the polymer in the solution of (2),
And (4) the step of bringing the solution of (3) into contact with the surface of the substrate coated with the polymer having a superhydrophilic group, (1) to (16) ,
It is.

  According to the biological substance detection substrate of the present invention, the biological substance that is the purpose of detecting capture molecules and supramolecules can be captured efficiently and non-specific adsorption can be suppressed. The origin can be detected.

Hereinafter, the biological origin detection substrate of the present invention will be described in detail.
First, the shape of the substrate used in the present invention is a glass slide such as a DNA microarray or a protein microarray, or a so-called microfluidics for performing a reaction and detection in a flow channel by applying a fine flow channel to the substrate. A substrate, a multiwell plate having 96 or 384 wells conventionally used in ELISA, and a bead-like one may be used.

  The material of the substrate is basically not limited as long as it has impermeability and water resistance. Examples of such materials include glass, plastic, and metals.

Further, the substrate material is limited depending on what is used for detection of the captured biological substance. However, when detection is performed by fluorescence, it is necessary that fluorescence is not emitted. As such a material, glass is preferable, and a plastic that does not emit fluorescence is preferable, and examples of the plastic that does not emit fluorescence include ETFE and saturated cyclic polyolefin.
In the case of performing detection by absorbance, it is necessary to have transparency, and as such a material, glass is preferable, and plastic having transparency is preferable. Polystyrene, polyethylene, saturated cyclic polyolefin, polycarbonate and the like can be mentioned. Polystyrene is inexpensive and suitable.

  In addition, when applied to microfluidics, it is necessary that the processability of the fine flow path should be easy, so such materials include glass, plastic, and metal. The processed substrate can also be applied to a silicon wafer used for photolithography. Even in microfluidics, it is necessary to select a material depending on fluorescence and transparency by the detection method as described above.

  Next, the polymer layer having a superhydrophilic group will be described. When a polymer having a superhydrophilic group is coated on the substrate, superhydrophilicity is imparted to the substrate surface. By imparting superhydrophilicity to the substrate surface, adsorption of biological substances such as proteins, nucleic acids and sugar chains can be suppressed. As the polymer having a superhydrophilic group, a polymer containing 2-methacryloyloxyethyl phosphorylcholine polymer or 2-methacryloyloxyethyl phosphorylcholine as a component, which is also known to have antithrombogenicity, may be a nucleic acid, a protein, Glycoprotein, and further, cell adsorption and adhesion are very small, which is preferable.

  About the formation method of the polymer layer which has a super hydrophilic group, the case where the above-mentioned 2-methacryloyloxyethyl phosphorylcholine polymer is coated is described as an example. First, 2-methacryloyloxyethyl phosphorylcholine polymer is dissolved in an appropriate solution, and the substrate surface is brought into contact with the solution by immersing the substrate in this solution or spraying the solution on the substrate surface. Form. In this 2-methacryloyloxyethyl phosphorylcholine polymer, the phosphorylcholine portion is superhydrophilic, the other portion is hydrophobic, and if the substrate surface is hydrophobic, the phosphocholine portion is aligned and arranged on the substrate surface. By doing so, the substrate surface has super hydrophilicity. Thus, there is almost no adsorption | suction of biological origins, such as a protein, to the substrate surface provided with super hydrophilicity. Further, since this polymer is colorless and does not emit fluorescence, the background can be suppressed almost perfectly in color detection and detection by fluorescence.

  A method for immobilizing an antibody or DNA on a surface capable of suppressing the background without adsorbing any biological material will be described. Since the surface does not adsorb protein or nucleic acid at all, immobilization of nucleic acid or protein for capturing a biological substance cannot be performed as it is. If an active group for immobilizing nucleic acid or protein is introduced on the substrate surface and a nucleic acid or antibody solution is brought into contact with the substrate surface as in the conventional technique, the nucleic acid or antibody can be immobilized. However, the introduction of reactive groups on the surface results in loss of non-adsorbability to the nucleic acid and protein on the substrate surface to which it has been applied, and after the nucleic acid or antibody is immobilized, the active group such as blocking is lost. It is necessary to make it non-specifically adsorb proteins and nucleic acids. Furthermore, even if a blocking operation is performed after the nucleic acid or antibody is immobilized on the surface, it is difficult to completely prevent nonspecific adsorption of proteins and nucleic acids while maintaining high sensitivity.

  Accordingly, the inventor fixed the capture molecule in advance by immobilizing the capture molecule on another polymer, and entangled the polymer in a superhydrophilic polymer that has non-adsorbability with respect to a biological substance. It is possible to immobilize proteins and nucleic acids while completely retaining the non-adsorbing property of the superhydrophilic polymer surface formed on the surface, and to improve the specific capturing ability of antibodies and nucleic acids. I found it.

  The immobilization of proteins, nucleic acids and the like on the surface of a substrate on which a superhydrophilic polymer surface is formed will be described.

The polymer used for immobilizing proteins and antibodies is preferably water-soluble because the target of immobilization is derived from a living body and most of them are water-soluble and the substrate surface is superhydrophilic. It is. Examples of the water-soluble polymer include polyethylene glycol, polylysine, polyethyleneimine, and the like, and a 2-methacryloyloxyethyl phosphorylcholine polymer may be used as a base polymer. Polyethyleneimine and polylysine have a positive charge and can immobilize nucleic acids and proteins electrostatically.

An active group for immobilizing nucleic acid or protein is introduced into a polymer having no active group. Examples of the active group include those that electrostatically immobilize nucleic acids and proteins, those that form and fix a covalent bond, or those that have a specific interaction. Specifically, as the active group, Aldehyde group, amino group, epoxy group, thiol group, hydroxyl group, carboxyl group, nitro group, sulfone group, acid anhydride, carbodiimide group, vinyl group, halogenated alkyl group, halogenated allyl group, succinimide group, maleimide group, isocyanate Nalto group isocyanate group, hydrozide group, azide group, biotin derivative, avidin derivative, phosphoric acid group, azlactone group, nitrile group, amide group, imino group, nitrene group, acetyl group, etc. can be used. An aldehyde group, an amino group, an epoxy group, a thiol group, a carbodiimide group, and an acid anhydride are preferable.

Next, the actual nucleic acid and protein immobilization procedures are described.
First, a polymer having an active group is dissolved in pure water or an aqueous buffer such as PBS (−). Ethanol or the like may be added for the purpose of adjusting the viscosity or the like of the solution. The concentration of the polymer is preferably 0.01 μg / ml to 1 mg / ml, and is adjusted by the concentration of molecules and supramolecules that capture biological substances such as antibodies.

  Next, the protein or nucleic acid to be immobilized is added to the polymer solution in an amount equivalent to the polymer concentration to about 1/100, and stirred for a while. At this point, the protein or the like is immobilized on the polymer. If all the active groups on the polymer react with the protein or nucleic acid during the protein immobilization process, non-specific adsorption will not occur, but the active group may remain depending on the structure of the protein to be immobilized. If an active group remains, non-specific adsorption occurs and a back-up occurs during detection, so that the active group is inactivated.

  Inactivation is carried out in the above protein solution, and if it is a chemically reactive active group, a treatment for inactivating the chemical reactivity is performed. For example, in the case of an aldehyde group, the reactivity is deactivated by performing a reduction treatment. Further, by adding BSA or the like to the solution and stirring for a while, the active group is blocked and inactivated, and nonspecific adsorption can be suppressed.

  Next, the prepared solution is brought into contact with the surface of the substrate into which the polymer layer having a superhydrophilic group has been introduced. For contact, if the substrate has a well like a 96-well plate, the solution is dispensed into the well and allowed to stand, and then the solution is removed and washed.

  If the substrate has a glass slide shape, the solution is allowed to adhere to the substrate surface using pins or the like and washed.

  As described above, a molecule that captures a biological substance such as an antibody is immobilized on the polymer layer having a superhydrophilic group. In this way, the biological substance detection substrate to which biological substances such as antibodies are immobilized does not cause adsorption of non-specific proteins, so that there is no increase in background in detection and antigen-antibody reaction. High efficiency and high detection sensitivity can be obtained.

  Example 1 A glass substrate having a superhydrophilic surface was obtained by coating a glass slide surface with a 2-methacryloyloxyethyl phosphorylcholine polymer. On the other hand, polylysine was dissolved in PBS (−) at a concentration of 10 μg / ml, followed by dissolving anti-rat albumin antibody at a concentration of 1 μg / ml, stirring for 30 minutes, and then adding BSA at a concentration of 10 μg / ml. The mixture was stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with a pin having a pin diameter of 500 μm was spotted on a glass substrate having a superhydrophilic surface, and 25 spots in an area of 2 cm × 2 cm. I wore it. After leaving it for 30 minutes, it was immersed in pure water and washed, and was used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Example 2)
The glass slide surface was coated with 2-methacryloyloxyethyl phosphorylcholine polymer to obtain a glass substrate having a superhydrophilic surface. On the other hand, polylysine was dissolved in PBS (−) at a concentration of 10 μg / ml, then anti-rat albumin antibody was dissolved at a concentration of 1 μg / ml, stirred for 30 minutes, and then 1 μg / ml of glutaraldehyde was added. After stirring for a minute, BSA was added at a concentration of 10 μg / ml and stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a glass substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving it for 30 minutes, it was immersed in pure water and washed, and was used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Example 3)
A 2-methacryloyloxyethyl phosphorylcholine polymer was coated on the surface of a saturated cyclic polyolefin slide molded product to obtain a saturated cyclic polyolefin substrate having a superhydrophilic surface. On the other hand, polylysine was dissolved in PBS (−) at a concentration of 10 μg / ml, followed by dissolving anti-rat albumin antibody at a concentration of 1 μg / ml, stirring for 30 minutes, and then adding BSA at a concentration of 10 μg / ml. The mixture was stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a saturated cyclic polyolefin substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving it for 30 minutes, it was immersed in pure water and washed, and was used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Example 4)
A 2-methacryloyloxyethyl phosphorylcholine polymer was coated on the surface of a saturated cyclic polyolefin slide molded product to obtain a saturated cyclic polyolefin substrate having a superhydrophilic surface. On the other hand, polylysine was dissolved in PBS (−) at a concentration of 10 μg / ml, then anti-rat albumin antibody was dissolved at a concentration of 1 μg / ml, stirred for 30 minutes, and then 1 μg / ml of glutaraldehyde was added. After stirring for a minute, BSA was added at a concentration of 10 μg / ml and stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a saturated cyclic polyolefin substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving it for 30 minutes, it was immersed in pure water and washed, and was used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Comparative Example 1)
The glass slide surface was coated with 2-methacryloyloxyethyl phosphorylcholine polymer to obtain a glass substrate having a superhydrophilic surface. On the other hand, an anti-rat albumin antibody was dissolved in PBS (−) at a concentration of 1 μg / ml and stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a glass substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving it for 30 minutes, it was immersed in pure water and washed, and was used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Comparative Example 2)
A 2-methacryloyloxyethyl phosphorylcholine polymer was coated on the surface of a saturated cyclic polyolefin slide molded product to obtain a saturated cyclic polyolefin substrate having a superhydrophilic surface. On the other hand, an anti-rat albumin antibody was dissolved in PBS (−) at a concentration of 1 μg / ml and stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a glass substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving it for 30 minutes, it was immersed in pure water and washed, and was used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Comparative Example 3)
A glass slide surface was coated with polylysine to obtain an antibody immobilization substrate. On the other hand, an anti-rat albumin antibody was dissolved in PBS (−) at a concentration of 1 μg / ml and stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a glass substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving for 30 minutes, it was immersed in pure water for cleaning. Subsequently, after leaving to stand at room temperature for 1 hour in a blocking solution in which BSA is dissolved in PBS (−) at a concentration of 1 μg / ml, it is immersed in pure water and washed to detect rat albumin. It used for the sensitivity evaluation test as an object detection board | substrate.

(Comparative Example 4)
A glass slide surface is coated with polylysine, and then immersed in a solution of 1 μg / ml glutaraldehyde in PBS (−) for 30 minutes, washed and dried to obtain a substrate for immobilizing an antibody. It was. On the other hand, an anti-rat albumin antibody was dissolved in PBS (−) at a concentration of 1 μg / ml and stirred for 30 minutes to prepare an antibody solution. Subsequently, an antibody solution prepared by a pin-type spotter equipped with pins having a pin diameter of 500 μm was spotted on a glass substrate having a superhydrophilic surface. 25 spots were spotted in an area of 2 cm × 2 cm. After leaving for 30 minutes, it was immersed in pure water for cleaning. Subsequently, after leaving to stand at room temperature for 1 hour in a blocking solution in which BSA is dissolved in PBS (−) at a concentration of 1 μg / ml, it is immersed in pure water and washed to detect rat albumin. It used for the sensitivity evaluation test as an object detection board | substrate.

(Sensitivity evaluation test)
Rat albumin was dissolved in PBS (−) to a concentration of 0.1 μg / ml and 0.01 μg / ml to prepare a rat albumin solution. A control solution in which only PBS (−) was dissolved was prepared. 50 μl of each concentration of rat albumin solution and control solution was dispensed into the spot area of the biological substance detection substrate for detecting rat albumin of Examples 1 to 4 and Comparative Examples 1 to 4, and for a DNA chip provided with a 20 μm gap Covered with a hybrid cover and left in an incubator maintained at 100% humidity. Wash with shaking in PBS (-) containing 0.05% TWEEN except for the cover, then spot the rhodamine-labeled anti-rat albumin antibody solution (dissolved in PBS (-) at 1 μg / ml). 50 μl was dispensed into the area, covered with a DNA chip hybrid cover having a gap of 20 μm, and left in an incubator kept at 100% humidity. Thereafter, the cover was removed, and washing was performed while shaking in PBS (-) containing 0.05% TWEEN. Finally, after lightly washing with ultrapure water, water droplets on the substrate were removed by centrifugation. The intensity of the fluorescent spot on the substrate was measured with a confocal laser scanner. Table 1 shows the relative intensities of each concentration of the rat albumin solution, with the spot fluorescence intensity of the substrate of Example 1 being 100. For each concentration of the rat albumin solution, Table 2 shows a comparison of SN ratios with the SN ratio of the fluorescence intensity of the spots measured on the substrate of Example 1 being 100. Here, the S / N ratio is obtained by dividing the fluorescence intensity using each rat albumin concentration solution as the antigen solution by the fluorescence spot intensity on each substrate when using the PBS (−) solution as a control as the antigen solution. Calculated.

  From Table 1, it is apparent that the substrates of Examples 1 to 4 have high fluorescence intensity at the spot and high sensitivity. From Table 2, it is clear that the substrates of Examples 1 to 4 have a high S / N ratio and high detection sensitivity.

  In supplying a biological substance detection substrate such as a protein microarray, a substrate with higher sensitivity can be supplied.

Claims (18)

A substrate used to capture and immobilize a biological substance, and to detect the captured and immobilized biological substance, at least one surface of which is coated with a polymer having a superhydrophilic group. A substrate for detecting a biological substance, wherein a molecule or supramolecule that specifically captures is immobilized on a superhydrophilic polymer chain and / or via a polymer that enters between the chains. The molecule or supramolecule that specifically captures a biological substance is at least one selected from nucleic acids, peptides, proteins, sugar chains, lipids, and steroids, or a complex containing at least one of these The substrate for detecting a biological substance according to claim 1, which is a body. The biological substance detection substrate according to claim 1 or 2, wherein the molecule or supramolecule that specifically captures the biological substance is an antibody. The substrate for detecting a biological substance according to claim 1 or 2, wherein the molecule or supramolecule that specifically captures the biological substance is a lectin protein. The biological substance detection substrate according to claim 1 or 2, wherein the molecule or supramolecule that specifically captures the biological substance is an aptamer. The biological substance detection substrate according to claim 1 or 2, wherein the molecule or supramolecule that specifically captures the biological substance is an enzyme. The biological substance detection substrate according to claim 1 or 2, wherein the molecule or supramolecule that specifically captures the biological substance is a part of a cell constituent or a cell. The substrate for detecting a biological substance according to any one of claims 1 to 7, wherein the superhydrophilic group is derived from phospholipid. The biological substance detection substrate according to claim 8, wherein the polymer having a superhydrophilic group is a 2-methacryloyloxyethyl phosphorylcholine polymer and / or a copolymer of 2-methacryloyloxyethyl phosphorylcholine-containing components. The biological material is at least one selected from nucleic acids, peptides, proteins, sugar chains, lipids, and steroids, or a complex containing at least one of these. A substrate for detecting a biological substance. The biological substance detection substrate according to any one of claims 1 to 9, wherein the biological substance is a part of a cell structure or a cell. The biological substance detection substrate according to any one of claims 1 to 11, wherein a molecule or supramolecule that specifically captures a biological substance is fixed to the polymer by electrostatic bonding or covalent bonding. Immobilization of a molecule or supramolecule that specifically captures a biological substance is made by introducing a functional group having an electrostatic bond or a covalent bond into a copolymer of 2-methacryloyloxyethyl phosphorylcholine-containing component Item 13. The biological substance detection substrate according to Item 12. The biological substance detection substrate according to claim 12, wherein a molecule or supramolecule that specifically captures a biological substance is immobilized on a polycationic polymer. 15. The biological substance detection substrate according to claim 14, wherein the polycationic polymer is at least one selected from polyethyleneimine, polyvinylamine, polyallylamine, polyornithine, and polylysine. The biological substance detection substrate according to any one of claims 1 to 15, wherein molecules or supramolecules that specifically capture biological substances are fixed in spots on the substrate and arranged with regularity. . 2. A step of bringing a solution containing and / or dissolving a molecule that specifically captures a biological substance or a polymer immobilizing a supramolecule into contact with a substrate surface coated with a polymer having a superhydrophilic group. The manufacturing method of the biological substance detection board | substrate in any one of -16. (1) a step of dissolving a polymer having an active group in a solution;
(2) A step of dissolving a molecule or supramolecule that captures a biological substance in the solution of (1),
(3) Inactivating the active group of the polymer in the solution of (2), and (4) contacting the solution of (3) with the substrate surface coated with the polymer having a superhydrophilic group. The method for producing a biological substance detection substrate according to any one of claims 1 to 16.