JP4921088B2 - Analytical device manufacturing method and coating composition - Google Patents

Description

  The present invention relates to a method for producing an analytical device used for detection and analysis of various physiologically active substances in a biological sample, a coating composition containing a capture substance-binding polymer material used for producing the analytical device, and the analytical device The present invention relates to a method for analyzing physiologically active substances.

  Analysis of physiologically active substances such as DNA, RNA, proteins, peptides, hormones, lipids, glycoproteins is important in clinical and industrial fields. In the clinical field, diagnosis of inherited diseases is performed by analyzing DNA and RNA sequences and amounts, and infectious diseases and metabolism by qualitatively or quantitatively determining proteins, peptides, hormones, lipids, glycoproteins, etc. Various diseases such as abnormalities are diagnosed. In the industrial field, for example, in the food industry, food safety is maintained by qualitatively or quantitatively determining the presence of allergens in foods that cause allergic symptoms.

  In order to analyze such a physiologically active substance, a method of capturing the physiologically active substance on an analysis device is used. In sandwich immunoassay, one of the most widely used analytical methods, a capture substance (capture antibody) for capturing a physiologically active substance is immobilized on a substrate serving as a reaction site, and the physiological substance is immobilized on this substrate. The physiologically active substance is captured by the capturing substance by contacting a sample containing the active substance. Subsequently, after washing away the uncaptured substance, a labeled substance (labeled antibody) having a binding property (different epitope) different from that of the captured substance is reacted with the captured physiologically active substance, and then an excessive amount of the labeled substance The amount of the physiologically active substance is measured by detecting the labeling substance (labeled antibody) after washing off. Examples of the reaction site in such a sandwich immunoassay include, for example, a reaction vessel wall surface formed on the substrate surface, a channel wall surface of a device having a minute channel called a microchip, or a flat substrate such as a slide glass. Surface.

  In addition, as a technology for profiling the fluctuations of all proteins (proteomes), microfluidics technology that enables the manipulation of ultra-trace amounts of proteins, ultra-trace amounts of solutions such as several nanoliters to several microliters, and chips There are “lab-on-chip” technologies with the goal of pretreatment, separation and detection above. In these technologies, a physiologically active substance such as a protein as a sample specifically reacts with and captures a capture substance (capture antibody) immobilized in a channel formed at the interface between two substrates. It is necessary to suppress non-specific adsorption to the inner wall of the flow path other than the substance immobilization part.

  In such a measurement system, nonspecific adsorption of a labeling substance (labeled antibody) to a reaction site, for example, a reaction vessel wall surface formed on the substrate surface, a microchip channel wall surface, or a flat substrate surface such as a slide glass is As a result, the signal-to-noise ratio is lowered and the detection accuracy is lowered. For this reason, in a normal sandwich immunoassay system, in order to prevent non-specific adsorption of the labeling substance (labeled antibody), the capture substance (capture antibody) for capturing the physiologically active substance on the substrate serving as the reaction site is used. After immobilization, the adsorption inhibitor is coated (for example, Non-Patent Document 1). As such an adsorption inhibitor, proteins such as bovine serum albumin and casein, and polymer substances containing phospholipids are generally used.

  However, in this method, since the capture substance (capture antibody) fixed on the substrate is covered with the adsorption inhibitor, there is a problem that the physiologically active substance is hardly captured by the capture substance (capture antibody) and sensitivity is lowered. In addition, since a process for coating the adsorption inhibitor is required, there is a problem that the entire process increases.

To improve the above-mentioned problems, it has a phosphorylcholine group for preventing non-specific adsorption of a physiologically active substance or a labeling substance (labeled antibody), and an activity for immobilizing the capture substance (capture antibody) A method for producing a biochip substrate, which is obtained by coating a substrate with a polymer substance having an ester group, has been developed (Patent Document 1). According to the method for producing a biochip substrate of Patent Document 1, a phosphorylcholine group for preventing nonspecific adsorption of a labeled substance (labeled antibody) and a capture substance (captured antibody) are immobilized in a coating solution of a polymer substance. Since the active ester group is included, the active ester group is exposed and fixed on the coating surface without being covered with the adsorption inhibitor by coating the polymer substance on the substrate, A capture substance (capture antibody) can be efficiently immobilized on the substrate via the active ester group. For this reason, according to the method of the biochip substrate of Patent Document 1, since the capture substance (capture antibody) is not coated with the adsorption inhibitor, the phenomenon that the binding ability with the physiologically active substance is reduced is avoided. Can do. Furthermore, since the phosphorylcholine group is exposed and fixed on the biochip substrate, nonspecific adsorption of non-analytical substances such as proteins and labeled substances (labeled antibodies) can be prevented. There is no need to provide a step of coating the inhibitor.
WO2005-029095 Acta Med Okayama Vol.48., No.6, .pp299-304

  However, in the method of Patent Document 1, in order to immobilize a capture substance for capturing a physiologically active substance on a substrate, a coating composition containing a polymer substance having a phosphorylcholine group and an active ester group is used. After the polymer substance is immobilized by coating on the substrate, the capture substance is immobilized. For this reason, in the method of Patent Document 1, the number of steps required for manufacturing an analytical device such as a biochip is 2 as compared with the conventional method (2 steps) of coating an adsorption inhibitor after immobilizing a capture substance. Since they are individual, the number of steps is the same as that of the conventional method, and the manufacturing process is not simplified.

  Accordingly, the present invention provides a method for producing an analytical device used for detection and analysis of many types of physiologically active substances in a biological sample. The phenomenon that the binding ability is reduced can be avoided, and non-specific adsorption of non-analyzed protein and other contaminants and labeling substances (labeled antibodies) can be prevented. The object is to provide a simplified method.

  The present invention is for coating the surface of a substrate used in an analyzer, coating the wall surface of a reaction vessel formed on the substrate surface as the analyzer, or coating the wall surface of a channel of the analyzer. A coating composition comprising a capture agent-bound polymeric material is provided. That is, in the coating composition of the present invention, a capturing substance for capturing a physiologically active substance is contained in a polymer substance containing a first unit having a phosphorylcholine group and a second unit having a functional group. It is characterized by including a trapping substance binding polymer substance bonded between a functional group contained and a functional group of a second unit contained in the polymer substance.

  The first method for producing an analyzer according to the present invention is characterized in that phosphorylcholine and a capturing substance are present on the surface of the substrate by coating the surface of the substrate with the coating composition.

  The second method for producing an analyzer according to the present invention is characterized in that phosphorylcholine and a capturing substance are present on the reaction vessel wall surface by coating the coating composition on the reaction vessel wall surface formed on the substrate surface. To do.

  Furthermore, a third method for producing an analyzer according to the present invention is a method for producing an analyzer in which a channel is formed at the interface between two planar substrates, and an inlet and an outlet of the channel are formed. By coating the coating composition on the channel wall surface of the analyzer, phosphorylcholine and a capturing substance are present on the channel wall surface.

  Each of the analytical devices obtained by the first to third production methods of the present invention is a capture substance fixed on the substrate surface, channel wall surface, or reaction vessel wall surface coated with the coating composition of the present invention. Captures physiologically active substances in the sample, and the immobilized phosphorylcholine group has the property of suppressing nonspecific adsorption of contaminants such as proteins and labeled substances (labeled antibodies) that are not intended for analysis. Since the fixed capture substance is not covered with an adsorption inhibitor or the like, the capture substance has a characteristic that the binding ability to the physiologically active substance does not decrease. The analyzer obtained by the method of the present invention is useful as a microchip.

  A first analysis method of the present invention is a method for analyzing a physiologically active substance in a sample using an analysis device obtained by the method for manufacturing the first analysis device, wherein the physiological analysis is performed on a substrate of the analysis device. A liquid sample containing an active substance is supplied, and the presence / absence or amount of a physiologically active substance is detected.

  A second analysis method of the present invention is a method for analyzing a physiologically active substance in a sample using an analysis device obtained by the method for manufacturing the second analysis device, on a reaction vessel wall surface of the analysis device. A liquid sample containing a physiologically active substance is supplied, and the presence or amount of the physiologically active substance is detected.

  A third analysis method of the present invention is a method for analyzing a physiologically active substance in a sample using an analyzer obtained by the method for producing the third analyzer, wherein a microchip as the analyzer is analyzed. A liquid sample containing a physiologically active substance is supplied from the inlet of the channel to the wall surface of the channel, and the presence or amount of the physiologically active substance is detected.

  According to the method for producing a coating composition and an analysis apparatus of the present invention, non-specific adsorption of a labeled substance (labeled antibody, etc.) or a physiologically active substance not intended for analysis is applied to the capture substance-binding polymer substance in the coating composition. Since the phosphorylcholine group for prevention and the capture substance for capturing the physiologically active substance are contained, the coating composition is coated on the substrate surface, the channel wall surface, or the reaction vessel wall surface in one step. The capture substance can be immobilized, and at the same time, nonspecific adsorption of a labeled substance (labeled antibody or the like) or a physiologically active substance not intended for analysis can be prevented. Conventionally, the present invention requires a two-step process of coating a polymer substance to immobilize the capture substance and then immobilizing the capture substance. The manufacturing process of the physiologically active substance analyzer can be simplified.

  In addition, since the coating composition of the present invention contains a polymer substance to which a capture substance is bound, if a large amount is prepared at a time, the necessary amount of the capture substance can be coated only when necessary. Immobilization can be performed.

  When the substrate was coated after the polymer and the trapping substance were bonded in advance, it was initially expected that a part of the trapping substance would be caught in the polymer and buried inside the polymer and could not function. In addition, the trapping substance buried between the polymer and the substrate was originally expected to have some influence on the coating of the polymer on the substrate, for example, uneven coating, easy peeling of the coating, etc. Despite these bad expectations, as demonstrated in the examples below, the reactivity is unexpectedly equal to or better than the conventional method of pre-coating the polymer material and then binding the capture material. was gotten.

(Coating composition)
The capture substance-binding polymer substance contained in the coating composition of the present invention is a capture substance for capturing a physiologically active substance in a polymer substance comprising a first unit having a phosphorylcholine group and a second unit having a functional group. Are covalently bonded between the functional group contained in the capture substance and the functional group of the second unit contained in the polymer substance. Alternatively, the capture substance-binding polymer substance contained in the coating composition of the present invention is a polymer substance containing a first unit having a phosphorylcholine group, a second unit having a functional group, and a third unit having a butyl methacrylate group. The capturing substance for capturing the physiologically active substance is covalently bonded between the functional group contained in the capturing substance and the functional group of the second unit contained in the polymer substance.

  The polymer substance containing a first unit having a phosphorylcholine group and a second unit having a functional group can be obtained by copolymerization of a monomer having a phosphorylcholine group and a monomer having a functional group. The polymer substance comprising a first unit having a phosphorylcholine group, a second unit having a functional group, and a third unit having a butyl methacrylate group includes a monomer having a phosphorylcholine group, a monomer having a functional group, butyl It can be obtained by copolymerization with a monomer having a methacrylate group.

  The monomer having the phosphorylcholine group used for the production of the polymer substance in the coating composition of the present invention may have a methacrylic group or an acryl group.

  The monomer having the functional group used for the production of the polymer substance in the coating composition of the present invention may be a monomer having a carboxylic acid derivative group, and further a single monomer having an active ester group. Further, it may be a monomer, and more specifically, a monomer having a p-nitrophenyl group or an N-hydroxysuccinimide group.

Examples of the phosphorylcholine group contained in the first unit in the polymer material used in the coating composition of the present invention include (meth) acryloyloxy such as 2-methacryloyloxyethyl phosphorylcholine group and 6-methacryloyloxyhexyl phosphorylcholine group. An alkylphosphorylcholine group;
(Meth) acryloyloxyalkoxyalkylphosphorylcholine groups such as 2-methacryloyloxyethoxyethyl phosphorylcholine group and 10-methacryloyloxyethoxynonylphosphorylcholine group;
A group having an alkenylphosphorylcholine group such as an allylphosphorylcholine group, a butenylphosphorylcholine group, a hexenylphosphorylcholine group, an octenylphosphorylcholine group, and a decenylphosphorylcholine group, wherein the phosphorylcholine group is included in these groups Can be mentioned.

  Of these phosphorylcholine groups, a 2-methacryloyloxyethyl phosphorylcholine group is preferred.

  In the polymer material used in the coating composition of the present invention, examples of the functional group of the second unit include an ester group represented by the following formula (1).

（ただし、上記式（１）において、Ａは水酸基を除く一価の脱離基である。）

(However, in the above formula (1), A is a monovalent leaving group excluding a hydroxyl group.)

  As shown in the formula (1), since the leaving group A exists in the second unit via a carbonyl group, a capture substance that captures a physiologically active substance is more reliably introduced into the polymer substance more reliably. be able to.

  The monovalent group represented by the formula (1) may be any group selected from the following formula (p) or formula (q). By doing so, the leaving group A can be activated more reliably and the reactivity can be further improved. In addition, following formula (p) or formula (q) can also be set as the structure from which H was lose | eliminated from the cyclic compound N containing N, respectively, and the structure from which C was H from the cyclic compound containing C.

(However, in the above formulas (p) and (q), R ¹ and R ² are each independently a monovalent organic acid, which may be linear, branched or cyclic) In the formula (p), R ¹ may be a divalent group that forms a ring together with C. In the formula (q), R ² is a divalent group that forms a ring together with N. It may be a group.)

  Examples of the group represented by the formula (p) include groups represented by the following formulas (r), (s), and (w). Moreover, as group shown by said formula (q), the group shown by following formula (u) is mentioned, for example.

The group represented by the above formula (1) is, for example, a group derived from an acid anhydride represented by the following formula (r), (s) or the like;
A group derived from an acid halide represented by the following formula (t);
The group can be derived from an activated amide represented by the following formula (v).

  Examples of the functional group represented by the formula (1) include an activated carboxylic acid derivative group. The activated carboxylic acid-derived group is a carboxylic acid having a leaving group via C═O, wherein the carboxyl group of the carboxylic acid is activated. Examples of the activated carboxylic acid-derived group include carboxyl groups such as acrylic acid, methacrylic acid, crotonic acid, maleic acid, and fumaric acid, which are carboxylic acids, acid anhydrides, acid halides, active esters, or active groups. And compounds converted to amides. Carboxylic acid-derived groups are activated groups derived from such compounds, such as p-nitrophenyl groups, N-hydroxysuccinimide groups, succinimide groups, phthalimide groups, 5-norbornene-2,3. -An active ester group such as a dicarboximide group; a halogen such as -Cl and -F; and the like.

  Of the carboxylic acid-derived groups, an active ester group is preferably used because of its excellent reactivity under mild conditions. As mild conditions, for example, neutral or alkaline conditions, specifically, pH 7.0 or more and 10.0 or less, preferably pH 7.6 or more, 9.0 or less, more preferably pH 7.6 or more, 8 0.0 or less.

  The “active ester group” referred to in the present invention is not strictly defined as to its definition, but is generally used as a conventional expression. The term “active ester group” is known to mean an ester group having an electron-attracting group with high acidity on the alcohol side of the ester group to activate a nucleophilic reaction, that is, an ester group having a high reaction activity. ing. In practice, phenol esters, thiophenol esters, N-hydroxyamine esters, esters of heterocyclic hydroxy compounds, etc. are generally known as active ester groups having much higher activity than alkyl esters. It has been.

  Examples of the capture substance bonded to the polymer substance used in the coating composition of the present invention include biotin, avidin, streptavidin, estradiol, hapten, hormone, oligonucleotide, nucleic acid, sugar, lipid, peptide, antibody, enzyme And the like in which two or more of these substances are bound, such as proteins, oligopeptides, glycoproteins, and the like in which physiologically active substances are artificially modified. One or more of these capture substances may be bound to a polymer protein to constitute a capture substance-bound polymer substance.

  The physiologically active substance captured by the capture substance in the present invention is a substance that binds to the capture substance with biochemical affinity, for example, biotin, avidin, streptavidin, estradiol, hapten, hormone, oligonucleotide , Nucleic acids, sugars, lipids, peptides, antibodies, proteins such as enzymes, oligopeptides, glycoproteins, bioactive substances artificially modified, etc., two or more of these substances bound, immune complex The body is mentioned.

  The conditions for covalently binding the capture substance to the polymer substance can be neutral or alkaline conditions, preferably pH 7.6 or higher.

  The polymer substance used in the coating composition of the present invention may contain a third unit, and a monomer containing a butyl methacrylate group is used as the monomer constituting the third unit.

  The production of the polymer substance used in the coating composition of the present invention comprises the monomer constituting the first unit (referred to as the first monomer) and the monomer constituting the second unit (the second unit). Or the first monomer, the second monomer, and the monomer constituting the third unit (referred to as a third monomer). be able to. Preferably, each monomer can be mixed and performed by a known polymerization method such as radical polymerization. For example, solution polymerization can be performed in an inert gas atmosphere such as Ar. Each monomer may be block copolymerized or may be copolymerized randomly.

  Specific examples of the polymer substance used in the present invention include a first monomer having a 2-methacryloyloxyethyl phosphorylcholine (MPC: abbreviation) group and a p-nitrophenyloxycarbonyl polyethylene glycol methacrylate (NPMA: abbreviation) group. And a second monomer having a butyl methacrylate (BMA) group. Poly (MPC-co-BMA-co-NPMA) (PMBN), which is a copolymer of these monomers, is schematically represented by the following general formula (2).

  In the general formula (2), a, b and c each independently represent a positive integer. In the above general formula (2), the first, second and third monomers may be block copolymerized, or these monomers may be copolymerized randomly. n represents an arbitrary integer.

  The copolymer represented by the general formula (2) has a more excellent structure due to the balance between appropriate hydrophobicity of the polymer substance, the property of suppressing non-specific adsorption, and the property of immobilizing the capture substance. . Therefore, by reacting the copolymer with a capture substance, a covalent bond is formed between the functional group of the second unit and the functional group contained in the capture substance, thereby obtaining a capture substance-bound polymer substance. Can do.

  As another specific example of the polymer substance used in the present invention, a first monomer having a 2-methacryloyloxyethyl phosphorylcholine (MPC: abbreviation) group, and p-nitrophenyloxycarbonyl polyethylene glycol methacrylate (NPMA: abbreviation) ) And a second monomer having a group. Poly (MPC-co-NPMA), which is a copolymer of these monomers, is schematically represented by the following general formula (3).

  In the general formula (3), a and c each independently represent a positive integer. In the general formula (3), the first and second monomers may be block copolymerized, or these monomers may be randomly copolymerized. n represents an arbitrary integer.

  The copolymer represented by the general formula (3) has a more excellent structure due to the balance between the property of suppressing nonspecific adsorption and the property of immobilizing the capture substance. Therefore, by reacting the copolymer with a capture substance, a covalent bond is formed between the functional group of the second unit and the functional group contained in the capture substance, thereby obtaining a capture substance-bound polymer substance. Can do.

  In a preferred embodiment of obtaining the capture substance-binding polymer substance in the present invention, 14 parts by weight or more and 20 parts by weight or less of biotin into which an amino group is introduced are mixed in 10 parts by weight of the active ester group-containing polymer, The reaction is performed in a state in which the solution is sufficiently mixed for 4 hours or more while maintaining the reaction solution temperature at 10 ° C or more and 60 ° C or less. If the amount of biotin introduced with an amino group is less than 14 parts by weight, the reaction does not proceed sufficiently, and if it exceeds 20 parts by weight, the amount of biotin is more than necessary for the reaction, which is wasteful and industrially undesirable. When the reaction temperature is lower than 10 ° C., the reaction proceeds at a slower rate and is industrially preferable.

  The diluting solvent that can be included in the coating composition containing the capture substance-binding polymer substance is not particularly limited as long as it can dissolve the active ester group-containing polymer and the biotin introduced with the amino group. Examples include water, dipotassium hydrogen phosphate aqueous solution, potassium dihydrogen phosphate aqueous solution, sodium carbonate aqueous solution, sodium hydrogen carbonate aqueous solution, sodium tetraborate borax aqueous solution, and the like, but are not limited thereto. However, an organic solvent system is preferable because the active ester group may be hydrolyzed. Further, by adding trimethylamine, triethylamine or the like, the active ester can be easily decomposed and the reaction can be promoted.

(substrate)
The substrate used in the analyzer of the present invention is used as constituting at least a part of the analyzer. As the material for the substrate used in the present invention, for example, glass, plastic, metal or the like can be used. Of these, plastics are preferable from the viewpoint of easy surface treatment and mass productivity, and thermoplastic resins are particularly preferable. As the thermoplastic resin, one having a small amount of fluorescence generation is preferable in order to reduce the background in the detection reaction of the physiologically active substance and increase the detection sensitivity. As the thermoplastic resin having a small amount of fluorescent light emission, for example, linear polyolefin such as polyethylene, polypropylene, polypentene, polycarbonate, polystyrene, polyamide, saturated cyclic polyolefin, fluorine-containing resin, etc. are preferably used. It is more preferable to use a saturated cyclic polyolefin that is particularly excellent in properties, low fluorescence, and moldability. Here, the saturated cyclic polyolefin refers to a polymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin.

(Analysis equipment)
The shape of the substrate used in the analyzer of the present invention is not particularly limited.

  FIG. 1 shows an example of an embodiment of an analyzer according to the present invention, and is a plan view showing a configuration of the analyzer when the substrate shape is a flat substrate having a slide glass shape, and FIG. 2 is a sectional view thereof. Reference numeral 1 denotes an analyzer comprising a flat substrate 2 for analyzing a physiologically active substance in a liquid sample. A capture zone 3 is formed by coating a specific portion of the flat substrate 2 with the coating composition of the present invention. Yes. The capture zone 3 may be part or all of the surface of the flat substrate 2. 1 and 2 can be used for detection or quantification of a physiologically active substance in a liquid sample captured by a capture substance by bringing the liquid sample into contact with the capture zone 3.

  FIG. 3 shows an example of another embodiment of the analyzer of the present invention, in which a capture zone 13 formed by coating the coating composition of the present invention on the reaction vessel wall surface formed on the surface of the flat substrate 12 is formed. FIG. 4 is a cross-sectional view showing the configuration of the analyzer 11.

  3 and 4 can be used for detection or quantification of a physiologically active substance in a liquid sample captured by the capture substance by bringing the liquid sample into contact with the capture zone 13.

  FIG. 5 is a plan view showing a configuration of an analyzer as a microchip according to an example of an embodiment of the present invention, and FIG. 6 is a cross-sectional view thereof. Reference numeral 21 denotes a microchip for analyzing a physiologically active substance in a liquid sample, and is configured by joining a first member 25 and a second member 26. The first member 25 is formed with a groove having a cross-sectional dimension of 1 μm to 5000 μm in width and 1 μm to 5000 μm in depth, and forms the flow path 22 when joined to the second member 26. The flow path 22 functions as a fine flow path capable of circulating a liquid. A channel inlet 23 is provided at one end of the channel 22 and a channel outlet 24 is provided at the other end. The channel inlet 23 functions as a liquid sample introduction part. Further, since the channel outlet 24 communicates with the outside air, the channel outlet 24 also functions as an air guide hole through which the liquid in the channel 22 flows. One or more inlets for introducing a liquid sample, a reagent, or the like may be provided between the channel inlet 23 and the channel outlet 24. Further, it is possible to provide another flow path connected to such a flow path according to the purpose.

  A capture zone 27 for capturing the target physiologically active substance in the liquid sample is provided in the flow path 22, and the capture zone 27 is formed by applying the coating composition of the present invention. The capture zone 27 may be formed on a part of or the entire surface of the flow path 22.

  At least one of the first member 25 and the second member 26 constituting the microchip 21 can be made of a resin that is transparent to the detection light. The transparent resin material is appropriately selected according to the wavelength of the detection light used for the detection reaction of the physiologically active substance. By making at least one of the first member 25 and the second member 26 transparent, the state of liquid feeding can be easily confirmed. Further, at least one of the first member 25 and the second member 26 may be appropriately colored. By doing so, an effect of increasing sensitivity can be expected when the reaction in the flow path 22 is optically observed.

  The microchip 21 shown in FIGS. 5 and 6 can be used for detection or quantification of a physiologically active substance in a liquid sample captured by the capture substance by flowing the liquid sample in the flow path 22.

  The diameter of the channel inlet 23 or the channel outlet 24 is appropriately designed according to the thickness of the first member 25 or the second member 26, the width of the channel 22, and the like. Moreover, about the groove | channel used as the flow path 22, it can be set as the following structures. In order to efficiently perform the detection reaction of the physiologically active substance in the flow path 22 of the microchip 21 in which the capture substance is fixed to the microchip substrate, a certain flow rate is required. Further, it is the surface portion of the flow path 22 where the capture substance is immobilized that contributes to the reaction. For these reasons, it is preferable that the cross-sectional area of the flow path 22 is small in order to make the reaction efficiently with a small amount of liquid sample.

  The microchip 21 shown in FIG. 5 and FIG. 6 can be used for detecting or quantifying a physiologically active substance in a liquid sample captured by a capture substance by flowing a liquid sample in a flow path 22.

Next, a manufacturing method of the microchip 21 shown in FIGS. 5 and 6 will be described.
First, a first member 25 having a groove to be a flow path 22 engraved on one surface, a flow path inlet 23 communicating with the flow path 22 and penetrating the other surface of the first member 25, and a flow path The first member 25 provided with the outlet 24 is produced.

  Next, when the first member 25 is joined, the second member 26 that covers the groove of the first member 25 and forms the flow path 22 is joined. At this time, the joint surface of both members, for example, the surface on the side where the flow path 22 is formed, is coated in advance with the coating composition of the present invention. The coating is performed, for example, by a method in which a coating composition is attached onto a substrate when a microchip substrate is manufactured, or a method in which a specific portion in a flow path forming part is coated with a pin spotter or an inkjet spot, or a flow path is formed. Examples of the method include a method in which the flow path is filled with a coating agent after the formation, and a part or the whole of the inner wall surface of the flow path can be coated by these methods.

  After the coating composition film is formed, washing is performed to remove excess coating composition.

  The capture zone in the analyzer of the present invention shown in FIGS. 1 to 6 has a first unit containing a phosphorylcholine group and a second unit containing a carboxylic acid derivative group, and captures a physiologically active substance in the second unit. The trapping substance to be coated is coated with a polymer substance formed by covalent bonding. A plurality of carboxylic acid-derived groups in the polymer compound are covalently bonded to the capturing substance or inactivated. Here, the carboxylic acid-derived group is inactivated means that a part of the group (leaving group) constituting the carboxylic acid-derived group is substituted with another group and the activity is lost. Say.

(How to use the analyzer)
Next, as one embodiment of the analyzer of the present invention, a method for using a microchip will be described by taking as an example a case where a primary antibody is immobilized on a chip as a capture substance.

  When using a microchip, first, a certain amount of liquid sample is fed using liquid feeding means such as a micropump or a microsyringe. In this step, the detection target protein is captured by the antibody. After the liquid sample is sent, cleaning is performed by sending a certain amount of the cleaning liquid.

  Next, a certain amount of a secondary antibody obtained by labeling a fluorescent substance or the like with an antibody against the protein to be analyzed is fed and washed. If the protein to be analyzed is present in the liquid sample, the label can be detected by a fluorescence detector or the like.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
Preparation of capture substance-bound polymer substance and reaction conditions As a capture substance having a functional group, biotin (Ez-link ^™ Amine-PEO ₃ -Biotin: registered trademark, manufactured by PIERCE) into which an amino group has been introduced is prepared, and water An aqueous solution (biotin aqueous solution) dissolved in was prepared. This biotin solution was mixed with 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate-p-nitrophenyloxycarbonyl polyethylene glycol methacrylate copolymer (active ester group-containing polymer substance) and 1.0, 1.2 in terms of theoretical functional group number, 1.4, 1.6, and 1.8 equivalents were mixed, respectively, and reacted at 37 ° C. for 2, 4, 6, 12, and 24 hours.

  The reaction solution obtained in the above step was diluted 50 times with ethanol and the absorbance at 270 nm wavelength was measured to determine the amount of free functional groups in the remaining polymer substance (the amount of active ester groups not bound to amino groups). It was measured. The results are shown in FIG. 7 as a graph with the horizontal axis representing the binding reaction time and the vertical axis representing the absorbance. According to FIG. 7, it can be seen that when the biotin solution is added by 1.6 times equivalent or more, the amount of free active ester is minimized and biotin is efficiently bound to the polymer substance. Further, it can be confirmed that the reaction is almost completed in 5 hours.

[Example 2]
Storage stability of capture substance-bound polymer substance In Example 1, biotin having an amino group introduced therein (Ez-link ^™ Amine-PEO ₃ -Biotin: registered trademark, manufactured by PIERCE) was converted to 2-methacryloyloxyethyl phosphorylcholine- Refrigerate the mixture obtained by adding 1.8 times equivalent of the theoretical number of functional groups of butyl methacrylate-p-nitrophenyloxycarbonyl polyethylene glycol methacrylate copolymer (active ester group-containing polymer) and reacting at 37 ° C. for 6 hours. After storage, the change with time was examined.

  A flat substrate made of a saturated cyclic polyolefin resin was immersed in a 0.5 wt% ethanol solution of the mixed solution refrigerated in the above step, thereby introducing a polymer substance having a phosphorylcholine group and biotin on the substrate surface. A Cy3-labeled streptavidin solution was spotted on the substrate, washed, and then the fluorescence signal of Cy3 was measured to measure the amount of biotin on the substrate surface. The results are shown in FIG. 8 as a graph in which the horizontal axis represents Cy3-labeled streptavidin concentration (ng / ml) and the vertical axis represents Cy3 fluorescence intensity. According to FIG. 8, as a result of comparative examination with a storage period of 0 (immediately after the binding reaction), 1, 2, and 28 days, no decrease in signal was observed even in the mixed solution after 28 days, and biotin was stabilized. It turns out that it can confirm that it can introduce | transduce into the substrate surface.

[Example 3]
1. Comparison of the amount of biotin immobilized between the biotin-immobilized substrate by the conventional method and the biotin-immobilized substrate by the method according to the present invention . Preparation of biotin-immobilized substrate by a conventional method By immersing a flat substrate made of saturated cyclic polyolefin resin in a 0.5 wt% ethanol solution of an active ester-containing polymer substance, phosphorylcholine groups and active ester groups are formed on the substrate surface. Introduced polymer material. Subsequently, polydimethylsiloxane (PDMS, manufactured by Fluidware Technologies) having grooves (width 300 μm, depth 100 μm, length 6.5 cm) formed on the surface is formed so that a flow path is formed between the substrate and PDMS. Crimped to. Fill the flow path with biotin (Ez-link ^™ Amine-PEO ₃ -Biotin: registered trademark, manufactured by PIERCE) prepared to 50 μM with a spotting solution, and incubate at 37 ° C. for 5 hours. By reacting the amino group, a microchip having a phosphorylcholine group and biotin on the substrate side surface in the flow path was produced.

2. Preparation of biotin-immobilized substrate by the method according to the present invention A microchip having phosphorylcholine groups and biotin on the substrate surface by immersing in a 0.5 wt% ethanol solution of the mixed solution prepared by the method described in Example 2 Was made.

3. Comparison of biotin immobilization amount Cy5-labeled streptavidin (FluoroLink ^™ Cy ^{™ 5} Labeled Streptavidin: trade name, manufactured by Amersham Biosciences) adjusted to 100 ng / mL with PBS was sent to the flow path at 37 ° C. for 10 minutes, washed, and then Cy5 The fluorescence signal was detected with a microarray scanner (Biodetect 64Reader: trade name, manufactured by GeneScan). As a result, the fluorescence signal of Cy5 of the biotin-immobilized substrate by the conventional method was 20008, whereas the fluorescence signal of Cy5 of the biotin-immobilized substrate by the method of the present invention was 20142, and the coating composition of the present invention It was confirmed that a signal equivalent to or higher than that of the conventional method could be obtained even with the method using.

[Example 4]
Comparison of immunoassay results using a biotin-immobilized substrate by a conventional method and a biotin-immobilized substrate by a method according to the present invention B using two types of microchips produced by the conventional method and the method of the present invention in Example 3 above The surface antigen of hepatitis B virus (HBsAg) was detected by immunoassay, and the difference in signal was examined. Prepare two types of anti-HBsAg antibodies with different epitopes (first antibody: SCANTIBODIES LABORATORY, INC., Second antibody: manufactured by Special Immunology Laboratories) and use streptavidin (SA) -labeled anti-HBsAg first antibody by a known method. A horseradish peroxidase (HRP) -labeled anti-HBsAg second antibody was prepared. The SA-labeled first antibody, the HRP-labeled second antibody, and HBsAg were mixed at 2 μg / mL, 1 μg / mL, and 1 ng / mL, respectively, and sent to the channel at 37 ° C. for 10 minutes. PBS was fed into the channel and washed, and then SAT Blue, which is an HRP coloring substrate solution, was fed, and the degree of coloration was measured with a thermal lens microscope (GRINSpectra: trade name, manufactured by Nippon Sheet Glass Co., Ltd.). The result is shown in the graph of FIG. According to FIG. 9, it was confirmed that the biotin-immobilized substrate prepared by the method according to the present invention can obtain a signal-to-noise ratio equal to or higher than that of the conventional method.

  The analysis apparatus in which the substrate is coated with the coating composition of the present invention has a phosphorylcholine group for preventing nonspecific adsorption of a labeling substance (labeled antibody, etc.), and a capture substance (capture antibody) is immobilized. As a microchip, it is useful in the analysis field and clinical field of physiologically active substances.

It is a top view which shows the structure of the analyzer which concerns on an example of embodiment of this invention. It is sectional drawing of the analyzer of FIG. It is a top view which shows the structure of the analyzer which concerns on another example of embodiment of this invention. It is sectional drawing of the analyzer of FIG. It is a top view which shows the structure of the analyzer as a microchip based on another example of embodiment of this invention. It is sectional drawing of the analyzer of FIG. The reaction conditions of the capture substance-binding polymer substance contained in the coating composition of the present invention are shown as a graph with the horizontal axis representing the binding reaction time and the vertical axis representing the absorbance. The storage stability of the capture substance-binding polymer substance contained in the coating composition of the present invention is shown as a graph in which the horizontal axis represents Cy3 labeled streptavidin concentration (ng / ml) and the vertical axis represents Cy3 fluorescence intensity. It is a graph which shows the comparison of the immunoassay result using the biotin fixed board | substrate by a conventional method, and the biotin fixed board | substrate by the method concerning this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,11 Analyzer 2,12 Planar substrate 3,13 Capture zone 21 Microchip 22 Channel 23 Channel inlet 24 Channel outlet 25 First member 26 Second member 27 Capture zone

Claims (13)

  A trapping substance for trapping a physiologically active substance in a polymer substance containing a first unit having a phosphorylcholine group and a second unit having a functional group is included in the functional group contained in the trapping substance and the polymer substance. A coating composition comprising a capture substance-binding polymer substance bonded with a functional group of a second unit.   The coating composition according to claim 1, wherein the functional group contained in the polymer substance is a carboxylic acid derivative group.   The coating composition according to claim 2, wherein the carboxylic acid-derived group is an active ester group.   The coating composition according to any one of claims 1 to 3, wherein the functional group contained in the capture substance is an amino group.   The coating composition according to claim 1, wherein the polymer substance has a butyl methacrylate group as a third unit.   The coating composition according to claim 1, wherein the capture substance includes biotin.   7. A method for producing an analytical device, wherein the substrate surface is coated with the coating composition according to claim 1 to cause phosphorylcholine and a capturing substance to be present on the substrate surface.   An analysis characterized in that phosphorylcholine and a trapping substance are present on the reaction vessel wall surface by coating the coating composition according to any one of claims 1 to 6 on the reaction vessel wall surface formed on the substrate surface. Device manufacturing method.   A method of manufacturing an analyzer in which a flow path is formed at an interface between two planar substrates, and an inlet and an outlet of the flow path are formed, and the flow path wall surface of the analyzer is any one of claims 1 to 6. A method for producing an analytical device, characterized in that phosphorylcholine and a capturing substance are present on a wall surface of a flow path by coating the coating composition according to the item.   The method for producing an analyzer according to claim 7, 8 or 9, wherein the coated surface is washed after coating the coating composition according to any one of claims 1 to 6.   A sample analysis comprising: supplying a liquid sample containing a physiologically active substance on a substrate of an analyzer manufactured by the manufacturing method according to claim 7; and detecting the presence or amount of the physiologically active substance. Method.   A liquid sample containing a physiologically active substance is supplied to the reaction vessel wall surface of the analyzer manufactured by the manufacturing method according to claim 8, and the presence or absence or amount of the physiologically active substance is detected. Analysis method.   A liquid sample containing a physiologically active substance is supplied from the inlet of the flow path of the analyzer manufactured by the manufacturing method according to claim 9 to the wall surface of the flow path, and the presence or absence or amount of the physiologically active substance is detected. A method for analyzing a sample characterized by the following.

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