JP4053579B2 - Biosensor - Google Patents

Description

  The present invention relates to a biosensor, a method for producing the same, and a method for analyzing an interaction between biomolecules using the biosensor. In particular, the present invention relates to a biosensor for use in a surface plasmon resonance biosensor and a method for producing the biosensor. And a method of analyzing an interaction between biomolecules using the biosensor.

  Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations, etc., but conventional methods require complicated operations and labeling substances, so measurement without the need for labeling substances Several techniques that can detect a change in the amount of a substance bound with high sensitivity are used. For example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, and measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. SPR measurement technology detects adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a fixed wavelength. It is a method to do. The QCM measurement technique is a technique that can detect the adsorption / desorption mass at the ng level from the change in the oscillation frequency of the oscillator due to the adsorption / desorption of a substance on the gold electrode (device) of the crystal oscillator. In addition, by functionalizing the surface of gold ultrafine particles (nm level), immobilizing a physiologically active substance on the surface, and performing a specific recognition reaction between the physiologically active substances, it is possible to obtain a living body from the sedimentation and arrangement of gold fine particles. Related substances can be detected.

  In any of the above techniques, the surface on which the physiologically active substance is immobilized is important. Hereinafter, the surface plasmon resonance (SPR) most used in this technical field will be described as an example.

  A commonly used measurement chip is composed of a transparent substrate (eg, glass), a deposited metal film, and a thin film having a functional group capable of immobilizing a physiologically active substance thereon, and the metal surface is interposed through the functional group. Immobilize physiologically active substances. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance.

  As a detection surface having a functional group capable of immobilizing a physiologically active substance, for example, Patent Document 1 describes a surface in which a hydrogel matrix is bound via a compound (SAM compound) that forms a self-assembled film. . Specifically, a barrier layer is formed by bonding a 16-mercaptohexadecanol layer to a gold film. On this gold film, the hydroxyl group of the barrier layer is epoxy activated by treating with epichlorohydrin. In the next step, dextran is attached to the barrier layer via an ether linkage. Next, a carboxymethyl group is introduced by reacting the dextran matrix with bromoacetic acid.

  However, in order to mass-produce a sensor using a SAM compound having a carbon chain of more than 10 as described in Patent Document 1, (1) the SAM compound is insoluble in water, so a large amount of film is formed. The use of this solvent increases the burden on the global environment, and there is a concern about the impact on the health of production personnel. (2) Since raw materials are not distributed, it is necessary to synthesize in a multi-step synthesis step. There is a problem that the supply property is difficult, the cost is high, and (3) when the sensor substrate is made of plastic, modification of the plastic is promoted.

Japanese Patent No. 2815120

  The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a biosensor that can immobilize a large amount of protein and has less nonspecific adsorption by using an SMA compound having high water solubility and good supply.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors can immobilize a large amount of protein by binding a hydrogel through an SMA compound having high water solubility and good supply ability. The present inventors have found that a biosensor with less specific adsorption can be produced and have completed the present invention.

That is, according to the present invention, a hydrophilic polymer having a reactive group capable of binding a physiologically active substance is immobilized via a compound represented by the following formula (1) dissolved in water at 25 ° C. at a concentration of 1 mM. There is provided a biosensor comprising an integrated substrate.
X-L-Y (1)
(In the formula, X represents a group capable of binding to a metal, L represents a linking group, and Y represents a functional group for binding a hydrophilic polymer.)

According to another aspect of the present invention, a biosensor comprising a substrate on which a hydrophilic polymer having a reactive group capable of binding a physiologically active substance is immobilized via a compound represented by the following formula (1) Is provided.
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y represents a hydrophilic polymer. Indicates a functional group for bonding.)

  Preferably, X is an asymmetric or symmetric sulfide group, sulfide group, diselenide group, selenide group, thiol group, nitrile group, isonitrile group, nitro group, selenol group, trivalent phosphorus compound, isothiocyanate group, xanthate group, thiocarbamate Group, phosphine group, thioacid group or dithioacid group.

Preferably, Y is a carboxy group, a hydroxyl group, an amino group, an aldehyde group, a hydrazide group, a carbonyl group, an epoxy group, or a vinyl group.
Preferably, X is a thiol group and Y is an amino group.

Preferably, the reactive group capable of binding a physiologically active substance in the hydrophilic polymer is an active carboxylic acid ester group.
Preferably, the substrate surface is coated with a mixture of an alkanethiol having an amino group and an alkanethiol having a hydrophilic group, and has a hydrophilic group having a reactive group capable of binding a physiologically active substance via the alkanethiol having an amino group. Functional polymer is immobilized.

Preferably, the hydrophilic group of the alkanethiol having a hydrophilic group is a hydroxyl group or an oligoethylene glycol group.
Preferably, the molar ratio in the mixture of alkanethiol having an amino group and alkanethiol having a hydrophilic group is in the range of 1/1 to 1 / 1,000,000.
Preferably, the hydrophilic polymer is a polysaccharide.

Preferably, the substrate is a metal surface or a metal film.
Preferably, the metal is gold, silver, copper, platinum or aluminum.
Preferably, the substrate is placed on a plastic support.

  Preferably, the biosensor of the present invention is used for non-electrochemical methods, more preferably for surface plasmon resonance analysis.

According to still another aspect of the present invention, a reactive group capable of binding a physiologically active substance is attached to a substrate coated with a compound represented by the following formula (1) dissolved in water at 25 ° C. at a concentration of 1 mM. A method for producing the biosensor of the present invention is provided, which comprises the step of binding the hydrophilic polymer to the compound represented by the formula (1) by contacting the hydrophilic polymer.
X-L-Y (1)
(In the formula, X represents a group capable of binding to a metal, L represents a linking group, and Y represents a functional group for binding a hydrophilic polymer.)

According to still another aspect of the present invention, a substrate coated with a compound represented by the following formula (1) is contacted with a hydrophilic polymer having a reactive group capable of binding a physiologically active substance, There is provided a method for producing a biosensor of the present invention, which comprises a step of binding a hydrophilic polymer to a compound represented by the formula (1).
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y represents a hydrophilic polymer. Indicates a functional group for bonding.)

Preferably, the substrate is coated with a mixture of an alkanethiol having an amino group and an alkanethiol having a hydrophilic group, and the hydrophilic polymer is bonded to the alkanethiol having an amino group.
Preferably, the hydrophilic group of the alkanethiol having a hydrophilic group is a hydroxyl group or an oligoethylene glycol group.
Preferably, the molar ratio in the mixture of alkanethiol having an amino group and alkanethiol having a hydrophilic group is in the range of 1/1 to 1 / 1,000,000.

Preferably, a hydrophilic polymer having a reactive group capable of binding a physiologically active substance is reacted with the compound represented by the formula (1) in a state where a thin film is formed on a substrate.
Preferably, a thin film is formed on the substrate by spin coating or spray coating.

According to still another aspect of the present invention, there is provided a biosensor manufactured by the above-described method of the present invention.
According to still another aspect of the present invention, a bioactive substance is immobilized on a biosensor comprising the step of bringing the biosensor of the present invention into contact with a bioactive substance and binding the bioactive substance to the biosensor. A method is provided.

According to still another aspect of the present invention, the bioactive substance interacts with the bioactive substance, comprising the step of bringing the biosensor of the present invention in which the bioactive substance is covalently bonded to the surface into contact with the test substance. A method for detecting or measuring a substance is provided.
Preferably, a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method, more preferably detected or measured by surface plasmon resonance analysis.

  In the present invention, by using a SAM compound that is easily dissolved in water, the amount of solvent used can be reduced, the global environmental load is reduced, and the production environment is improved. In addition, since the amount of solvent used is small, there is no problem of promoting the modification of the plastic even when the sensor substrate is plastic, thereby suppressing noise during the surface resonance plasmon measurement. Furthermore, a SAM compound having a short carbon chain that is easily dissolved in water is easily available and can be obtained at low cost. The biosensor of the present invention can immobilize a large amount of protein and has less nonspecific adsorption.

  The biosensor referred to in the present invention is interpreted in the broadest sense, and means a sensor that measures and detects a target substance by converting an interaction between biomolecules into a signal such as an electrical signal. A normal biosensor is composed of a receptor site for recognizing a chemical substance to be detected and a transducer site for converting a physical change or chemical change generated therein into an electrical signal. In the living body, there are enzymes / substrates, enzymes / coenzymes, antigens / antibodies, hormones / receptors and the like as substances having affinity for each other. Biosensors use the principle that one of these substances having affinity with each other is immobilized on a substrate and used as a molecular recognition substance, thereby selectively measuring the other substance to be matched.

  In the biosensor of the present invention, a metal surface or a metal film can be used as a substrate. The metal constituting the metal surface or metal film is not particularly limited as long as surface plasmon resonance can occur when, for example, a surface plasmon resonance biosensor is considered. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal.

  Although the thickness of the metal film is arbitrary, for example, when considering use for a surface plasmon resonance biosensor, the thickness is preferably 0.1 nm to 500 nm, particularly preferably 1 nm to 200 nm. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

  The metal film may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like.

  The metal film is preferably arranged on the support. Here, “arranged on the support” means not only when the metal film is arranged so as to be in direct contact with the support, but also with other layers without the metal film being in direct contact with the support. It is meant to include the case where it is arranged via. As a support that can be used in the present invention, for example, when considering use for a surface plasmon resonance biosensor, generally optical glass such as BK7 or synthetic resin (plastic support), specifically, polymethyl A material made of a material transparent to laser light such as methacrylate, polyethylene terephthalate, polycarbonate, cycloolefin polymer, or the like can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

In the present invention, a hydrophilic polymer having a reactive group capable of binding a physiologically active substance is immobilized on a substrate via a compound represented by the following formula (1) dissolved in water at 25 ° C. at a concentration of 1 mM. Has been.
X-L-Y (1)
(In the formula, X represents a group capable of bonding to a metal, L represents a linking group, and Y represents a functional group for bonding a hydrophilic polymer.)

In the present invention, a hydrophilic polymer having a reactive group capable of binding a physiologically active substance may be immobilized on a substrate via a compound represented by the following formula (1).
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y represents a hydrophilic polymer. Indicates a functional group for bonding.)

  The solubility of the compound of formula (1) in water is preferably 0.1 mM, and most preferably 1.0 mM, at 25 ° C. In the present invention, the degree of dissolution is defined by the transmitted light turbidity method. In a measurement cell with an optical path length of 10 mm, take a baseline with water and measure the absorbance of the compound solution at 660 nm. It is defined that the compound is dissolved when the absorbance (660 nm) of the compound solution is 0.005 or less.

  The solvent for dissolving the compound of the formula (1) is preferably water, but an organic solvent such as ethanol, methanol, butyl alcohol or the like may be mixed with water. The mixing ratio of the organic solvent is preferably 50% or less, more preferably 20% or less, and further preferably 5% or less. Most preferably, it dissolves in water alone. The organic solvent to be mixed preferably has water miscibility, specifically, alcohols are preferable, and ethanol is most preferable.

In the formula (1), X is a group capable of bonding to a metal. Specifically, asymmetric or symmetric sulfide (-SSR'Y ", -SSRY), sulfide (-SR'Y", -SRY), diselenide (-SeSeR'Y ", -SeSeRY), selenide (SeR'Y" , -SeRY), thiol (-SH), nitrile (-CN), isonitrile, nitro (-NO ₂ ), selenol (-SeH), trivalent phosphorus compound, isothiocyanate, xanthate, thiocarbamate, phosphine, thioacid or Dithioic acid (—COSH, —CSSH) is preferably used.

  In formula (1), L (as well as R and R ′) is a linking group, preferably a straight chain (not branched) for dense packing, optionally interrupted by a heteroatom. A hydrocarbon chain optionally containing double and / or triple bonds. The chain length is usually 2 carbon atoms or more, preferably 10 carbon atoms or less, and more preferably 9 carbon atoms or less. The carbon number of the hydrocarbon chain is more preferably 4 to 10 carbon atoms, and particularly preferably 4 to 8 carbon atoms. The carbon chain can optionally be perfluorinated.

  Y and Y ″ are groups for bonding a hydrophilic polymer. Y and Y ″ are preferably the same, and can be bonded directly or after activation to a hydrophilic polymer (eg, hydrogel). Has properties. Specifically, carboxyl, hydroxyl, amino, aldehyde, hydrazide, carbonyl, epoxy, vinyl group, or the like can be used.

  The compound represented by X-L-Y in the form of a tightly packed monolayer can be attached to the metal surface by bonding of the group represented by X to the metal.

  The self-assembled film (SAM) referred to in this specification is a monomolecular film having a structure with a certain order formed by a mechanism of the film material itself without fine control from the outside. An ultra-thin film such as an LB film. This self-organization forms ordered structures and patterns over long distances in a non-equilibrium situation.

  For example, the self-assembled film can be formed of the compound represented by the formula (1) as described above. For example, Nuzzo RG et al. (1983), J Am Chem Soc, 105, 4481-4482, Porter MD et al. (1987), J Am Chem Soc, 109, 3559-3568, Troughton EB et al. (1988), Langmuir, 4, 365-385, etc.

For the synthesis of the compound represented by the formula (1), for example, Current Organic Chemistry, 2004, 8, 1763-1797, Applications, Properties and Synthesis of ω-Functionalized N-Alkanethiols and Disulfides-the Building Blocks of Self-Assembled Monolayers
Author: Dariusz Witt et al. Can be performed according to the synthesis route from the raw materials described in Scheme 1 on page 10.

Moreover, the coating method of the metal film using the self-assembled film (SAM) has been vigorously developed by Professor Whitesides et al. Of Harvard University. The details are described in Chemical Review, 105, 1103-1169 (2005), for example. It has been reported. When gold is used as the metal, by using an alkanethiol derivative represented by the following formula 1 (wherein n represents an integer of 2 to 10), the Au-S bond and the van der Waals force between the alkyl chains are used. A monomolecular film having orientation is formed in a self-organized manner. The self-assembled film is prepared by an extremely simple technique in which a gold substrate is immersed in a solution of an alkanethiol derivative. By forming a self-assembled film using a compound where X = NH ₂ in the following formula 1, it is possible to cover the gold surface with an organic layer having an amino group.

The alkanethiol having an amino group at the terminal is a compound (general formula 1-1 ) in which the thiol group and the amino group are linked via an alkyl chain (in the general formula 1-1 , n represents an integer of 3 to 20). And alkanethiol having a carboxyl group at the terminal (general formulas 1-2 and 1-3 ) (in formula 1-2 , n represents an integer of 3 to 20, and in formula 4, each n is independently 1 Or a compound obtained by reacting a large excess of hydrazide or diamine. The reaction between the alkanethiol having a carboxyl group at the terminal and a large excess of hydrazide or diamine may be carried out in a solution state, or after binding the alkanethiol having a carboxyl group at the terminal to the substrate surface, a large excess of hydrazide. Or you may make diamine react.

The repeating number of the alkyl group of 1-1 to 1-3 is preferably 3 or more and 20 or less, more preferably 3 or more and 16 or less, and most preferably 4 or more and 8 or less. When the alkyl chain is short, it is difficult to form a self-assembled film, and when the alkyl chain is long, water solubility is lowered and handling becomes difficult.

As the diamine used in the present invention, any compound can be used, but when used on the biosensor surface, a water-soluble diamine is preferred. Specific examples of water-soluble diamines include aliphatic diamines such as ethylenediamine, tetraethylenediamine, octamethylenediamine, decamethylenediamine, piperazine, triethylenediamine, diethylenetriamine, triethylenetetraamine, dihexamethylenetriamine, and 1.4-diaminocyclohexane. , Paraphenylenediamine, metaphenylenediamine, paraxylylenediamine, metaxylylenediamine, 4,4'-diamonobiphenyl, 4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenyl ketone, 4,4'- Aromatic diamines such as diaminodiphenylsulfonic acid can be mentioned. From the viewpoint of improving the hydrophilicity of the biosensor surface, it is also possible to use a compound (general formula 1-4 ) in which two amino groups are linked by an ethylene glycol unit. The diamine used in the present invention is preferably a compound represented by ethylenediamine or general formula 1-4 (in general formula 1-4 , n and m each independently represents an integer of 1 to 20), and more Ethylenediamine or 1,2-bis (aminoethoxy) ethane (in formula 1-4 , n = 2, m = 1) is preferable.

The alkanethiol having an amino group can form a self-assembled film alone, or can be mixed with other alkanethiols to form a self-assembled film. When used on the biosensor surface, it is preferable to use a compound capable of suppressing nonspecific adsorption of a physiologically active substance as the other alkanethiol. The self-assembled membrane that can suppress non-specific adsorption of physiologically active substances has been studied in detail by the aforementioned Professor Whitesides et al. The self-assembled membrane formed from alkanethiol having a hydrophilic group is non-specifically adsorbed It is reported to be effective for suppression (Langmuir, 17,2841-2850, 5605-5620, 6336-6343 (2001)). In the present invention, as the alkanethiol that forms a mixed monomolecular film with an alkanethiol having an amino group, the compounds described in the above paper can be preferably used. As alkanethiol that forms a mixed monomolecular film with an alkanethiol having an amino group is excellent in non-specific adsorption inhibiting ability and is easily available, alkanethiol having a hydroxyl group (general formula 1-5 ) or ethylene group in alkanethiol (general formula 1-6) (general formula 1-5 with a call unit, n represents an integer of from 3 to 20, in the general formula 1-6, n and m are from 1 to 20 independently It is preferable to use an integer.

When an alkanethiol having an amino group is mixed with another alkanethiol to form a self-assembled film, the repeating number of the alkyl group of 1-1 to 1-3 is preferably 4 or more and 20 or less, and more preferably 4 or more and 16 The following is preferable, and 4 or more and 10 or less are most preferable. The number of repeating alkyl groups 1-5 and 1-6 is preferably 3 or more and 16 or less, more preferably 5 or more and 12 or less, and most preferably 5 or more and 10 or less.

  In the present invention, the alkanethiol having an amino group and the alkanethiol having a hydrophilic group can be mixed at an arbitrary ratio, but activated when the ratio of the alkanethiol having an amino group is small. When the binding amount of the carboxyl group-containing polymer (carboxyl group-containing polymer) is reduced and the proportion of alkanethiol having a hydrophilic group is small, the nonspecific adsorption inhibiting ability is reduced. Therefore, the mixing ratio of the alkanethiol having an amino group and the alkanethiol having a hydrophilic group is preferably in the range of 1/1 to 1 / 1,000,000, and preferably 1/4 to 1 / 10,000. Is more preferable, and the range of 1/10 to 1/1000 is more preferable. From the viewpoint of reducing steric hindrance when reacting with a polymer containing an activated carboxyl group, the molecular length of the alkanethiol having an amino group is preferably longer than the molecular length of the alkanethiol having a hydrophilic group.

  The alkanethiol used in the present invention may be a compound synthesized based on a review by Prof. Grzybowski et al. Of Northwestern University (Curr. Org. Chem., 8, 1763-1797 (2004).) And its cited references. Commercially available compounds may also be used. These compounds can be purchased from Dojindo Co., Ltd., Aldrich, SensoPath Technologies, Frontier Scientific Inc., etc. In the present invention, the disulfide compound that is an oxidation product of alkanethiol can be used in the same manner as alkanethiol.

  Next, a hydrophilic polymer having a reactive group capable of binding a physiologically active substance used in the present invention will be described. As the hydrophilic polymer used in the present invention, a polymer hardener for photographic silver halide, a hydrophilic polymer having an acetoacetyl group, and an active esterified carboxyl group-containing polymer can be preferably used. Hereinafter, the polymer hardener, the hydrophilic polymer having an acetoacetyl group, and the active esterified carboxyl group-containing polymer that can be used in the present invention will be described.

  The polymer hardener is a polymer compound having a plurality of reactive functional groups in the molecule that bind and react with a hydrophilic colloid such as gelatin. JP-A-56-66841, British Patent 1, No. 322,971, US Pat. No. 3,671,256, JP-A-7-64226, JP-A-7-140596, JP-A-10-111545, JP-A-2000-62629, JP Patent Literature such as 2004-20919, and The Theory of the Photographic Process by James (James, The Theory of the Photographic Process), 4th edition, page 84 (1977, Macmillan), Campbell Other authors Polymeric Amine and Ammonium Salts (Campbelleta) ; Polymeric Amine and Ammonium Salts), pp 321-332 (1979, are described in textbooks par Gammon Press, Inc.) and the like.

The polymer hardener used in the present invention is a polymer compound having a reactive functional group capable of binding to a functional group on the biosensor surface (that is, a group represented by Y in formula (1)), A polymer compound having a reactive functional group represented by the following formulas (1) to (9) is preferable.
Equation _{(1): -SO 2 CH =} CH 2
Equation _{(2): -SO 2 CH 2} CH 2 X
In the formula (2), X represents a group (for example, —Cl, —OSO ₂ CH, etc.) which is removed by a substitution reaction or elimination reaction when the functional group represented by the formula (2) reacts with a nucleophile or a base _{3, -OSO 2 C 6 H 4} CH 3, -OCOCH 3, -OSO 3 -, a pyridinium).
Formula (3)

  In formula (3), X represents a single bond, —O—, or —NR—, and R represents a hydrogen atom, an alkyl group, or an aralkyl group. Y and Z represent a halogen atom (for example, chlorine atom, bromine atom), an alkoxy group (for example, methoxy), a hydroxyl group and a salt thereof, and an optionally substituted amino group, and at least one of Y and Z is a halogen atom. .

Formula (4): -CHO
Formula (5):

Formula (6): -NCO
Formula (7): —NHCONHCOCH═CH ₂
Formula (8): —NHCONHCOCH ₂ CH ₂ X
In formula, X is synonymous with X of Formula (2).
Formula (9): -COX
In the formula, X is a group (for example, easily leaving when the functional group represented by the formula (9) reacts with an amino group)

And formula (9) represents what is commonly known as an active ester group or mixed acid anhydride.

  In producing the polymer hardener used in the present invention, the polymerization method is not particularly limited. For example, the polymer hardener may be produced by a condensation polymerization method, and may be a radical using a compound having an ethylenically unsaturated bond. It may be produced by a method such as polymerization or anionic polymerization. Furthermore, it may be produced by introducing the reactive functional group into a natural polymer (for example, starch, dextran, gelatin, etc.). There is no particular restriction on the method of introducing the functional group capable of reacting with the hydrophilic colloid used in the present invention (the functional group represented by the above formula (1) to formula (9), hereinafter referred to as the reactive functional group). A polymer may be produced by carrying out a polymerization reaction using a monomer having a reactive functional group, or the reactive functional group may be introduced by a so-called polymer reaction after the polymer has been produced in advance. . It is also effective to perform a polymerization reaction using a monomer compound having a precursor of a reactive functional group and then generate a reactive functional group by an appropriate method.

  The polymer hardener used in the present invention may be produced by radical polymerization of a monomer having the reactive functional group (or its precursor) and an ethylenically unsaturated bond in the same molecule. The following compounds are typical examples of the monomer having a reactive functional group.

  The polymer used in the present invention may be a homopolymer of a monomer having a reactive functional group, or may be a copolymer with one or more other monomers. In the case of a copolymer, the proportion of the monomer having a reactive functional group is 1% by mass or more, and preferably 5% by mass or more. In the case of radical copolymerization, other monomers are not particularly limited as long as radical polymerization is possible, but the following monomers can be given as specific examples. In addition, when other monomers have a functional group capable of reacting, a combination of monomers is appropriately selected within a range in which no reaction occurs during the copolymerization with the functional groups represented by the formulas (1) to (9). Is preferred.

Acrylic acid, methacrylic acid and esters thereof: for example, acrylic acid, methyl acrylate, butyl acrylate, benzyl acrylate, hydroxyethyl acrylate, CH ₂ ═CHCOO (CH ₂ CH ₂ O) _n R (R is a hydrogen atom, an alkyl group , N is an integer of 1 or more), methacrylic acid, methyl methacrylate, ethyl methacrylate, benzyl methacrylate, hydroxyethyl methacrylate, 2-ethylhexyl methacrylate, 2-methoxyethyl methacrylate, N, N-dimethylaminoethyl methacrylate, 2-sulfoethyl methacrylate etc,
Amides of ethylenically unsaturated carboxylic acids: acrylamide, methacrylamide, N-acryloylmorpholine, N, N-dimethylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid (or a salt thereof), etc.
Aromatic monomers: styrene, vinyl toluene, pt-butyl styrene, p-vinyl benzoic acid, vinyl naphthalene, etc.
Other vinyl monomers: ethylene, propylene, vinyl chloride, vinylidene chloride, trifluoroethylene, trifluorochloroethylene, vinyl acetate, vinyl propionate, vinyl alcohol, N-vinyl pyrrolidone, N-vinyl acetamide, acrylonitrile, methacrylo Nitrile etc.

  Specific examples of the polymer hardener used in the present invention are shown below, but the present invention is not limited thereto. The copolymerization ratio of each compound represents a mass percentage ratio.

P-1: M-1 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (10/90)
P-2: M-1 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (30/70)
P-3: M-1 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (50/50)
P-4: M-1 / methyl methacrylate copolymer (20/80)
P-5: M-2 / sodium acrylate copolymer (30/70)
P-6: M-2 / 2-hydroxyethyl methacrylate copolymer (20/80)
P-7: M-3 / butyl acrylate copolymer (60/40)
P-8: M-4 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (30/70)
P-9: M-6 / ethyl acrylate copolymer (60/40)
P-10: M-7 / N-vinylpyrrolidone copolymer (20/80)

P-11: M-7 / diacetone acrylamide copolymer (10/90)
P-12: M-10 / sodium methacrylate copolymer (15/85)
P-13: M-10 / methyl acrylate / methyl methacrylate copolymer (20/40/40)
P-14: M-12 / ethyl methacrylate copolymer (33/67)
P-15: M-12 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (15/85)
P-16: M-13 / methyl methacrylate copolymer (33/67)
P-17: M-13 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (20/80)
P-18: M-13 / N-acryloylmorpholine copolymer (20/80)
P-19: M-13 / methoxy polyethylene glycol (23-mer) monomethacrylate copolymer (50/50)
P-20: M-18 / N, N-dimethylacrylamide copolymer (5/95)

P-21: M-18 / butyl methacrylate copolymer (30/70)
P-22: M-18 / styrene / butyl acrylate copolymer (20/30/50)
P-23: M-19 / 2-acrylamido-2-methylpropanesulfonic acid soda copolymer (20/80)
P-24: M-23 / methyl acrylate copolymer (20/80)
P-25: M-24 / ethyl acrylate / styrene copolymer (20/50/30)
P-26: M-26 / acrylamide copolymer (25/75)
P-27: M-26 / N, N-dimethylaminoethyl methacrylate copolymer (30/70)

  The reactive functional group of the polymer hardener used in the present invention is preferably an active olefin polymer hardener, s-triazine polymer hardener, active halogen polymer hardener, aldehyde. Type polymer hardeners, glycidyl type polymer hardeners, etc. are used, more preferably active olefin type polymer hardeners or precursors thereof, s-triazine type polymer hardeners, glycidyl type polymer hardeners. Filming agents are used, and vinylsulfone type polymer hardening agents or precursors thereof, and dichlorotriazine type polymer hardening agents are particularly preferred.

  In the present invention, the polymer hardener is intended to be immobilized on the biosensor surface to form a hydrogel. Therefore, it is desirable to have a hydrophilic group in addition to the reactive functional group. Specific examples of hydrophilic groups include nonionic groups such as hydroxyl groups and ethylene glycol groups, anionic groups such as sulfonic acid groups, carboxylic acids, and phosphoric acid groups, and cationic groups such as quaternary ammonium groups and pyridinium groups. Examples include zwitterionic groups such as groups.

In the present invention, examples of the monomer unit having a hydrophilic group include the following monomers.
Monomers having a nonionic group: 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, 2-hydroxy-3-chloropropyl acrylate, β-hydroxyethyl-β′-acryloyloxyethyl phthalate, 1,4-butylene glycol monoacrylate, hydroxystyrene, allyl alcohol, methallyl alcohol, isopropenyl alcohol, 1-butenyl alcohol and the like.

Monomers having an anionic group: vinyl sulfonic acid, methallyl sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, sulfoethyl methacrylate, styrene sulfonic acid, acrylic acid, methacrylic acid, 2- (phosphonoethyloxy) ethyl Methacrylate and the like.

Monomers having a cationic group: [2- (acryloyloxy) ethyl] trimethylammonium chloride, [2- (methacryloyloxy) ethyl] trimethylammonium chloride, etc.

Monomers having zwitterionic groups: [2- (methacryloyloxy) ethyl] dimethyl- (3-sulfopropyl) ammonium hydroxide, [2- (methacryloyloxy) ethyl] phosphorylcholine and the like.

  By introducing a cationic group or an anionic group into the polymer hardener, it is possible to concentrate a physiologically active substance having an opposite charge to the detection surface using electrostatic interaction. For example, in the case of a protein dissolved in a buffer solution having a pH higher than the isoelectric point, since the polymer hardener having a cationic group is electrostatically concentrated on the bonded hydrogel surface, the reactive functional It becomes possible to combine with a group. On the other hand, in the case of a protein dissolved in a buffer solution having a pH lower than the isoelectric point, it is electrostatically concentrated on the surface of the hydrogel to which a polymer hardener having an anionic group is bonded, so that the reactive functional group can be efficiently used. It becomes possible to combine with a group. Specific examples of the compound having such a structure include the following compounds. The compounds described in JP-A-54-65033, JP-A-56-142524, and JP-A-60-61742 can also be preferably used.

  Next, a hydrophilic polymer having an acetoacetyl group will be described. The acetoacetyl group-containing water-soluble polymer has a property of curing by reacting with a crosslinking agent having a plurality of aldehyde groups, amino groups, hydrazide groups, and the like at room temperature. Therefore, by applying a water-soluble polymer containing an acetoacetyl group on one side of a substrate and a cross-linking agent on one side of another substrate and pressing both sides, the two substrates are bonded instantaneously. It becomes possible. Further, by adding and mixing a crosslinking agent to an aqueous solution of an acetoacetyl group-containing water-soluble polymer, it becomes possible to easily obtain a gel having a very high water content and not easily broken at room temperature. As described above, the water-soluble polymer containing an acetoacetyl group has extremely excellent properties as a water-soluble adhesive, as disclosed in JP-A-512771, JP-A-5156220, JP-A-2002-285117, etc. It is disclosed.

  A method for producing an acetoacetyl group-containing water-soluble polymer will be described. As a method for producing acetoacetylated polyvinyl alcohol, which is a typical compound of water-soluble polymer containing acetoacetyl group, a method of adding diketene gas in a state where a polyvinyl alcohol resin is dispersed in acetic acid, dimethylformamide , A method of adding diketene gas in a state where polyvinyl alcohol resin is dissolved in a solvent such as dioxane, a method of directly reacting polyvinyl alcohol powder and diketene gas, a method of transesterifying by reacting polyvinyl alcohol and acetoacetate in a solution, Etc. are known. The acetoacetylated polyvinyl alcohol in the present invention can be synthesized, for example, by the method described in JP-A-2002-285117, and commercially available acetoacetylated polyvinyl alcohol, for example, Nippon Synthetic Chemical Industry Co., Ltd. It is also possible to purchase and use Goseifamer Z100, Z200, Z200H, Z210, Z320, etc.

  The acetoacetyl group-containing water-soluble polymer can also be produced by copolymerizing an acetoacetyl group-containing monomer with a water-soluble monomer. As the acetoacetyl group-containing monomer, acetoacetoxyethyl acrylate, acetoacetoxyethyl methacrylate, acetoacetoxyethyl crotonate, acetoacetoxypropyl acrylate, acetoacetoxypropyl methacrylate, acetoacetoxypropyl crotonate, 2-cyanoacetoacetoxyethyl methacrylate, N- (2-acetoxyethyl) acrylamide, N- (2-acetoxyaminoethyl) methacrylamide, allyl acetoacetate, vinyl acetoacetate and the like. These monomers are produced by a reaction between a functional group-containing ethylenically unsaturated monomer and a diketene, an acetoacetoxyalkyl ester in the monomer, and a transesterification reaction. The acetoacetylated water-soluble polymer in the present invention can be synthesized, for example, by the technique described in Japanese Patent No. 2777732.

  In the present invention, the acetoacetyl group-containing water-soluble polymer can use copolymers with various monomers in addition to the acetoacetyl group-containing monomer. Examples of such copolymerization monomer units include the following monomers.

Acrylic acid, methacrylic acid and esters thereof: for example, acrylic acid, methyl acrylate, butyl acrylate, benzyl acrylate, hydroxyethyl acrylate, CH ₂ ═CHCOO (CH ₂ CH ₂ O) _n R (R is a hydrogen atom, an alkyl group , N is an integer of 1 or more), methacrylic acid, methyl methacrylate, ethyl methacrylate, benzyl methacrylate, hydroxyethyl methacrylate, 2-ethylhexyl methacrylate, 2-methoxyethyl methacrylate, N, N-dimethylaminoethyl methacrylate, 2-sulfoethyl methacrylate etc,

Amides of ethylenically unsaturated carboxylic acids: acrylamide, methacrylamide, N-acryloylmorpholine, N, N-dimethylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid (or a salt thereof), etc.

Aromatic monomers: styrene, vinyl toluene, pt-butyl styrene, p-vinyl benzoic acid, vinyl naphthalene, etc.

Other vinyl monomers: ethylene, propylene, vinyl chloride, vinylidene chloride, trifluoroethylene, trifluorochloroethylene, vinyl acetate, vinyl propionate, vinyl alcohol, N-vinyl pyrrolidone, N-vinyl acetamide, acrylonitrile, methacrylo Nitrile etc.

  In the present invention, the acetoacetyl group-containing hydrophilic polymer is intended to be immobilized on the biosensor surface to form a hydrogel. Therefore, it is desirable to have a hydrophilic group in addition to the reactive functional group. Specific examples of hydrophilic groups include nonionic groups such as hydroxyl groups and ethylene glycol groups, anionic groups such as sulfonic acid groups, carboxylic acids, and phosphoric acid groups, and cationic groups such as quaternary ammonium groups and pyridinium groups. Examples include zwitterionic groups such as groups.

In the present invention, examples of the monomer unit having a hydrophilic group include the following monomers.
Monomers having a nonionic group: 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, 2-hydroxy-3-chloropropyl acrylate, β-hydroxyethyl-β′-acryloyloxyethyl phthalate, 1,4-butylene glycol monoacrylate, hydroxystyrene, allyl alcohol, methallyl alcohol, isopropenyl alcohol, 1-butenyl alcohol and the like.

Monomers having an anionic group: vinyl sulfonic acid, methallyl sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, sulfoethyl methacrylate, styrene sulfonic acid, acrylic acid, methacrylic acid, 2- (phosphonoethyloxy) ethyl Methacrylate and the like.

Monomers having a cationic group: [2- (acryloyloxy) ethyl] trimethylammonium chloride, [2- (methacryloyloxy) ethyl] trimethylammonium chloride, etc.

Monomers having zwitterionic groups: [2- (methacryloyloxy) ethyl] dimethyl- (3-sulfopropyl) ammonium hydroxide, 2-[(methacryloyloxy) ethyl] phosphorylcholine and the like.

  By introducing a cationic group or an anionic group into the acetoacetyl group-containing water-soluble polymer, it becomes possible to concentrate a physiologically active substance having an opposite charge to the detection surface using electrostatic interaction. For example, in the case of a protein dissolved in a buffer solution having a pH higher than the isoelectric point, since it is electrostatically concentrated on the surface of the hydrogel to which the acetoacetyl group-containing hydrophilic polymer having a cationic group is bound, It becomes possible to combine with a reactive functional group. On the other hand, in the case of a protein dissolved in a buffer solution having a pH lower than the isoelectric point, since it is electrostatically concentrated on the hydrogel surface to which the water-soluble polymer containing an acetoacetyl group having an anionic group is bound, An acetoacetyl group or an acetoacetyl group can be combined with an introduced carboxylic acid by reacting with an amino acid.

  Next, the polymer containing an active esterified carboxyl group used in the present invention will be described. The polymer containing an active esterified carboxyl group used in the present invention can be prepared by subjecting the carboxyl group of a polymer containing a carboxyl group to active esterification. As the polymer containing a carboxyl group, a carboxyl group-containing synthetic polymer and a carboxyl group-containing polysaccharide can be used. Examples of the carboxyl group-containing synthetic polymer include polyacrylic acid, polymethacrylic acid, and copolymers thereof, for example, JP-A-59-44615, JP-B-54-34327, JP-B-58-12777, JP-B-54- No. 25957, JP-A-59-53836, JP-A-59-71048, methacrylic acid copolymer, acrylic acid copolymer, itaconic acid copolymer, crotonic acid copolymer , Maleic acid copolymers, partially esterified maleic acid copolymers, and those obtained by adding an acid anhydride to a polymer having a hydroxyl group. The carboxyl group-containing polysaccharide may be any one of an extract from a natural plant, a product of microbial fermentation, an enzymatic synthesis, or a chemical synthesis. Specifically, hyaluronic acid, chondroitin sulfate, heparin, Examples include dermatanic acid sulfuric acid, carboxymethylcellulose, carboxyethylcellulose, ceurouronic acid, carboxymethylchitin, carboxymethyldextran, and carboxymethyl starch. As the carboxyl group-containing polysaccharide, a commercially available compound can be used. Specifically, CMD, CMD-L, CMD-D40 (manufactured by Meisho Sangyo Co., Ltd.), sodium carboxymethylcellulose (Japanese) Kogaku Pharmaceutical Co., Ltd.), sodium alginate (Wako Pure Chemicals Co., Ltd.), and the like.

  The polymer containing a carboxyl group is preferably a polysaccharide containing a carboxyl group, more preferably carboxymethyl dextran.

  Although the molecular weight of the polymer containing a carboxyl group used in the present invention is not particularly limited, the average molecular weight is preferably 1,000 to 5,000,000, more preferably 10,000 to 2,000,000, and the average molecular weight is 100,000 to 1,000,000. More preferably it is. When the average molecular weight is smaller than this range, the fixed amount of the physiologically active substance becomes small, and when the average molecular weight is larger than this range, the handling becomes difficult due to the high solution viscosity.

  The hydrophilic polymer used in the present invention preferably has a film thickness in an aqueous solution of 1 nm to 300 nm. If the film thickness is thin, the amount of the physiologically active substance immobilized decreases, and the hydrated layer on the sensor surface becomes thin, so that the interaction with the analyte substance becomes difficult to detect due to the denaturation of the physiologically active substance itself. When the film thickness is thick, it becomes an obstacle that the analyte substance diffuses into the film, and the distance from the detection surface to the interaction forming part is particularly large when detecting the interaction from the opposite side of the hydrophilic polymer fixing surface of the sensor substrate. Longer and lower detection sensitivity. The hydrophilic polymer film thickness in the aqueous solution can be evaluated by AFM, ellipsometry, or the like.

  As a method of activating a polymer containing a carboxyl group, a known method, for example, a method of activating with water-soluble carbodiimide 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) , A method of activating by EDC alone, a method described in Japanese Patent Application No. 2004-238396 (Japanese Patent Laid-Open No. 2006-58071) (that is, any compound of uronium salt, phosphonium salt, or triazine derivative having a specific structure) A method for activating a carboxyl group using an acid), a method described in Japanese Patent Application No. 2004-275012 (Japanese Patent Application Laid-Open No. 2006-90781) (ie, a nitrogen-containing heteroaromatic having a hydroxyl group activated by a carbodiimide derivative or a salt thereof) Of aromatic compounds having thiol groups, phenol derivatives having electron withdrawing groups Things a carboxyl group can be preferably used a method) or the like to activate with. By reacting a polymer containing a carboxyl group activated by these methods with a substrate having an amino group, the biosensor of the present invention can be produced.

  In the present invention, the hydrophilic polymer having a reactive group capable of binding a physiologically active substance may be reacted with the substrate as a solution, or a thin film on the substrate is formed using a technique such as spin coating. You may make it react. Preferably, the reaction is in a state where a thin film is formed.

  As a method for forming a thin film on a substrate, known methods can be used. Specifically, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spin coating method, and a spray coating method. , Slide bead coating method, slit and spin method, slit coating method, die coating method, dip coating method, knife coating method, blade coating method, flow coating method, roll coating method, wire bar coating method, transfer printing method, etc. It is possible. For these thin film formation methods, "Progress in coating technology" by Yuji Harasaki, Comprehensive Technology Center (1988), "Coating Technology" Technical Information Association (1999), "Aqueous Coating Technology" CMC (2001), "Evolution" Described in "Organic Thin Film Deposition", Sumibe Techno Research (2004), "Polymer Surface Processing" by Iwamori Satoshi, Gihodo Publishing (2005), etc. Since a coating film with a controlled film thickness can be easily produced, the method for forming a thin film on a substrate in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method.

  The spray coating method is a method in which a polymer solution is uniformly coated on a substrate by moving the substrate in a state where a fine polymer solution is sprayed on the substrate. When the trigger of the spray gun is pulled, the air valve and the needle valve are simultaneously opened, the polymer solution is ejected from the nozzle in a mist state, and the atomized polymer solution is further refined by the air ejected from the air cap at the tip of the nozzle. A polymer film with a controlled film thickness is easily prepared by forming a coating film of a fine polymer solution on the substrate surface and then evaporating the solvent. The film thickness of the polymer thin film can be controlled by the polymer solution concentration, the moving speed of the substrate and the like.

  The spin coating method is a method in which a polymer solution is dropped on a horizontally placed substrate and then rotated at a high speed, and the polymer solution is uniformly applied to the entire substrate by centrifugal force. A polymer film with a controlled film thickness is easily produced with the scattering of the polymer solution by the centrifugal force and the evaporation of the solvent. The film thickness of the polymer thin film can be controlled by the rotation speed, polymer solution concentration, solvent vapor pressure, and the like. In the present invention, the rotation speed during spin coating is not particularly limited, but if the rotation speed is too low, the solution remains on the substrate, and if the rotation speed is too high, usable devices are limited. Therefore, in this study, the rotation speed during spin coating is preferably 500 rpm to 10000 rpm, and more preferably 1000 rpm to 7000 rpm.

  In the present invention, the carboxyl group-containing polymer is activated by a known method, for example, 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS), which are water-soluble carbodiimides. It is possible to immobilize a physiologically active substance having As a method for activating carboxylic acid, the method described in Japanese Patent Application No. 2004-238396 (that is, any of uronium salt, phosphonium salt, or triazine derivative having a specific structure for the carboxyl group present on the surface of the substrate) A method of forming a carboxylic acid amide group by activating with a compound) and a method described in Japanese Patent Application No. 2004-275012 (that is, a carboxyl group present on the surface of a substrate is activated with a carbodiimide derivative or a salt thereof) And then esterifying with a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group, and then reacting with an amine to form a carboxylic acid amide group. The method of forming) can also be preferably used.

  The uronium salt, phosphonium salt, or triazine derivative having a specific structure in Japanese Patent Application No. 2004-238396 described above is a uronium salt represented by the following general formula 1, a phosphonium salt represented by the following general formula 2, Or the triazine derivative represented by the following general formula 3 is shown.

(In General Formula 1, R ₁ and R ₂ each independently represent an alkyl group having 1 to 6 carbon atoms, or together, form an alkylene group having 2 to 6 carbon atoms to form a ring together with an N atom. And R ₃ represents an aromatic ring group having 6 to 20 carbon atoms or a heterocyclic group containing at least one hetero atom, and X ⁻ represents an anion, wherein R ₄ and R ₅ are each independently Represents an alkyl group having 1 to 6 carbon atoms, or together with each other, an alkylene group having 2 to 6 carbon atoms is formed to form a ring together with an N atom, and R ₆ is an aromatic ring having 6 to 20 carbon atoms A group or a heterocyclic group containing at least one heteroatom, and X ⁻ represents an anion, wherein R ₇ represents an onium group, and R ₈ and R ₉ each independently represent an electron donating group. .)

The reactive group capable of binding the physiologically active substance possessed by the hydrophilic polymer is protected by a halogen atom, an amino group, or a protecting group as a group other than the carboxyl group (or carboxyl group formed by active esterification) as described above. Amino group, or a carbonyl group having a leaving group, a hydroxyl group, a hydroxyl group protected by a protecting group, an aldehyde group, —NHNH ₂ , —N═C═O, —N═C═S, an epoxy group, or vinyl It may be a group.

  In the present invention, the reactive group to be introduced into the polymer (polymer) as the immobilizing group of the physiologically active substance is, for example, a biotin-binding protein (avidin, streptavidin, As shown below, a physiologically active substance such as neutravidin), protein A, protein G, antigen, antibody (eg, known tag antibody such as anti-GST antibody) is immobilized in advance. An embodiment in which a physiologically active substance is immobilized is possible. In addition, if an immobilization layer in which alkane is introduced into a polymer is used, a physiologically active substance having a membrane structure such as lipid can be immobilized. In addition, by adjusting the length of the polymer chain, the thickness of the polymer, the density of the polymer, or the amount of reactive groups introduced into the polymer, it is possible to deal with various proteins depending on the application. If NTA (nitrilotriacetic acid) or the like is introduced into the polymer as a fixing group, the His-tag ligand or the like can be fixed via a metal chelate.

  The physiologically active substance immobilized on the biosensor of the present invention is not particularly limited as long as it interacts with the measurement target, and examples thereof include immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-immune proteins, Examples include immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand-binding ability.

  Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.

  The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme and the like can be used. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

  The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.

  As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.

  Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used.
As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor (RF) and the like can be used.
Examples of sugar-binding proteins include lectins.
Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.

  These physiologically active substances are preferably immobilized by applying a solution containing the physiologically active substance onto a substrate on which a hydrophilic polymer is immobilized and drying.

  The concentration of the solution (coating solution) containing the physiologically active substance is preferably higher in the concentration of the physiologically active substance immobilized on the substrate surface. Although it varies depending on the physiologically active substance, it is preferably 0.1 mg / mL to 10 mg / mL, more preferably 1 mg / mL to 10 mg / mL.

  In the drying process of a solution containing a physiologically active substance, the physiologically active substance tends to be deposited on the outer periphery of the applied solution or on the portion where the liquid remains until just before the coating solution is dried. This undesirably causes a distribution in the amount of the physiologically active substance immobilized on the substrate surface. In order to make the amount of the physiologically active substance immobilized on the substrate surface uniform, it is preferable to increase the viscosity of the coating solution as long as the binding of the physiologically active substance to the substrate surface is not inhibited. By increasing the viscosity of the coating solution, the horizontal movement of the physiologically active substance in the coating solution in the drying process with respect to the substrate surface can be suppressed, and as a result, variations in the amount of physiologically active substance fixed can be suppressed. The viscosity of the coating solution in the drying process is preferably kept at 0.9 cP or higher.

  The drying step of the present invention means that after application of a solution containing a physiologically active substance, the solution is naturally dried by standing, or the solution is dried by heating or blowing, and the solution is intentionally dried. Refers to a process. Here, increasing the drying rate of a solution (coating solution) containing a physiologically active substance is effective in suppressing variations in the amount of physiologically active substance immobilized. By increasing the drying speed and finishing the drying sufficiently quickly with respect to the horizontal movement speed of the physiologically active substance, it is possible to finish the drying before the physiologically active substance substantially moves and to suppress variations. Become. The method of increasing the drying speed is not particularly limited, but the coating solution temperature and drying environment temperature are increased, the evaporation energy is applied by irradiation with infrared rays, laser, etc., the solvent vapor pressure during drying is lowered by blowing, the solution is applied in a thin layer And a method of increasing the evaporation area with respect to the coating amount. In particular, when the coating solution contains water, the drying speed is increased by drying in an environment having a large difference between the dry bulb temperature and the wet bulb temperature. It is preferable to dry in an environment where the temperature difference between the dry bulb temperature and the wet bulb temperature is 7 ° C or higher, more preferably 10 ° C or higher, and further preferably 13.5 ° C or higher. Further, in the production process, the drying time is preferably within 10 minutes, more preferably within 5 minutes, and particularly preferably within 1 minute.

  Examples of a method for applying a solution containing a physiologically active substance include a method using a dispenser that dispenses a coating solution in a fixed amount. By operating the discharge port of the dispenser on the substrate at a constant speed and a constant interval, it is possible to uniformly apply to any place on the substrate. In the case of applying with a dispenser, the distance between the substrate and the discharge port is made as narrow as possible, and the thickness of the physiologically active substance can be made uniform by reducing the thickness of the coating solution, and the drying speed can be increased, which is preferable. A preferred method for applying a solution containing a physiologically active substance is spin coating. This method is particularly preferable when the coating film thickness is reduced. In order to dry after forming a solution having a uniform thickness, the spin coater preferably prevents evaporation of the solvent during rotation. For this reason, it is particularly preferable because the thin film formation during the rotation and the drying speed after the thin film formation can be controlled by maintaining the environment in which the solvent concentration around the substrate is high by a method such as placing the substrate in a sealed container during rotation. . After these coating steps, it is preferable to dry under conditions where the temperature and humidity are kept constant.

  When detecting the interaction between the physiologically active substance and the analyte, the variation in the amount of the physiologically active substance immobilized on the sensor surface causes an error in the quantitative and kinetic evaluation of the interaction. For the purpose of minimizing this error, it is preferable to make the fixed amount of the physiologically active substance uniform. The variation of the fixed amount of the physiologically active substance on the substrate surface used for the detection of the interaction is preferably 15% or less, more preferably 10% or less in terms of CV value (variation coefficient) (standard deviation / average value). The CV value can be calculated from a fixed amount of at least 2 points on the substrate surface, preferably 10 points or more, more preferably 100 points or more. Uniformity can also be evaluated by quantifying the amount of the substance on the sensor substrate before and after immobilizing the physiologically active substance, but this is done by fluorescently labeling a substance known to bind to the physiologically active substance. It is also possible to measure the fluorescence intensity using a fluorescence microscope after fixing the labeling substance to the sensor substrate. In addition, physiologically active substances can be quantified using an SPR imager, ellipsometer, TOF-SIMS, ATR-IR apparatus or the like.

  In the present invention, a compound having a residue capable of forming a hydrogen bond (hereinafter referred to as Compound S) may be used for the purpose of improving the storage stability of a physiologically active substance immobilized on a substrate. .

  In general, physiologically active substances such as proteins maintain a three-dimensional structure by coordinating water molecules in an aqueous solution. However, when dried, they cannot retain the three-dimensional structure and are deactivated. Moreover, when it is carried in the hydrophilic polymer on the surface of the substrate, the physiologically active substances come close to each other and aggregate due to drying. The compound S having a residue capable of forming a hydrogen bond according to the present invention suppresses inactivation by retaining the three-dimensional structure of the physiologically active substance instead of the water molecule, or covers the physiologically active substance to provide a steric effect. Can be used for the purpose of suppressing aggregation.

  In the present invention, the compound S having a residue capable of forming a hydrogen bond is preferably added to a layer in which a physiologically active substance on a substrate is fixed with an aqueous solution. As an addition method, it may be applied to the substrate surface as a mixed solution with a physiologically active substance, or may be added by overcoating after fixing the physiologically active substance on the substrate surface. When applied as a mixed solution of Compound S and a physiologically active substance, variation in the amount of the physiologically active substance immobilized can be suppressed. Compound S aqueous solution is preferably added to the substrate in a thin film state. As a method for forming a thin film on a substrate, known methods can be used. Specifically, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spin coating method, and a spray coating method. , Slide bead coating method, slit and spin method, slit coating method, die coating method, dip coating method, knife coating method, blade coating method, flow coating method, roll coating method, wire bar coating method, transfer printing method, etc. It is possible. Since a coating film with a controlled film thickness can be easily produced, the method for forming a thin film on a substrate in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method.

  The concentration of the coating solution of Compound S is not particularly limited as long as there is no problem of coating properties and penetration into a layer containing a physiologically active substance, but is preferably 0.1% by weight or more and 5% by weight or less. In addition, a surfactant, a buffering agent, an organic solvent, a salt, or the like may be added to the coating solution from the viewpoint of coating properties and pH adjustment.

  The compound S having a residue capable of forming a hydrogen bond is preferably a non-volatile compound at normal temperature and normal pressure, preferably having an average molecular weight of more than 350 and less than 5 million, more preferably 1200 or more and 2 million or less. Most preferably, it is 1200 or more and 70,000 or less. The compound S containing a hydroxyl group in the molecule is preferably a saccharide, and the saccharide may be a monosaccharide or a polysaccharide. In the case of n-saccharides, n is preferably 4 or more and 1200 or less, and more preferably n is 20 or more and 600 or less.

  If the average molecular weight of Compound S is low, it will crystallize on the surface of the substrate, causing the destruction of the hydrophilic polymer layer to which the physiologically active substance is immobilized and the three-dimensional structure of the physiologically active substance. Problems such as an obstacle to fixing the active substance to the substrate, impregnation into a layer containing a physiologically active substance, and occurrence of layer separation occur.

  For the purpose of suppressing the deterioration of the physiologically active substance immobilized on the substrate, the compound S having a residue capable of forming a hydrogen bond preferably has a dextran skeleton or a polyethylene oxide skeleton, thereby achieving the object of the present invention. Any substituent may be used as long as it is within the range. Moreover, it is preferable that it is a nonionic compound which does not have a dissociable group for the purpose of suppressing deterioration of the bioactive substance fixed on the board | substrate. The compound S having a residue capable of forming a hydrogen bond is preferably a compound having a high affinity for water molecules, and the partition coefficient LogP value between water and n-octanol is preferably 1 or more. The LogP value can be measured by a method described in JIS standard Z7260-107 (2000) "Measurement of distribution coefficient (1-octanol / water)-shaking method".

  Specific examples of the compound S having a residue capable of forming a hydrogen bond include polyhydric alcohols such as polyvinyl alcohol, proteins such as collagen, gelatin, and albumin, hyaluronic acid, chitin, chitosan, starch, cellulose, alginic acid, and dextran. Polysaccharides such as polysaccharides, polyethylene glycol, polyethylene oxide, polypropylene glycol, polypropylene oxide, pluronics, and other polyethers such as polyethylene xylene-polypropylene oxide condensates, two or more residues such as tween 20, tween 40, tween 60, tween 80 Or a derivative and a polymer of these compounds. Of these, polysaccharides and polyethers are preferable, and polysaccharides are more preferable. Specifically, dextran, cellulose, tween 20, tween 40, tween 60, and tween 80 are preferably used. Furthermore, the non-volatile monomer and non-volatile water-soluble oligomer described in JP-A-2006-170832 can also be used. For example, the non-volatile monomer may be tetrose, pentose, heptose, hexose and its glycoxide whose hydroxyl group may be protected with a protecting group, or methyl glucoside or cyclitols whose hydroxyl group may be protected with a protecting group. In addition, the nonvolatile water-soluble oligomer is represented by the formula (1), (2) or (3)-[CH2-CH (CONH2)-] n- (1)-[CH2-CH2-O-] n- (2)- [CH2-CH (OH)-] n- (3) (in the formulas (1) to (3), n represents any integer of 10 to 200), and the hydroxyl group is a protecting group. It is also possible to use oligosaccharides of n sugars (where 2 ≦ n ≦ 10) which may be protected. Furthermore, saccharides described in US 2003/0175827, DE 20306476A1, for example, trehalose, sucrose, maltose, lactose, xylitol, fructose, mannitol, glucose, xylol, maltodextran, saccharose, polyvinylpyrrolidone, and the like may be used. These compounds S are preferably substantially the same as the basic skeleton of the hydrophilic polymer used in the present invention. Here, the basic skeleton refers to, for example, a sugar ring structure, and is substantially the same if the ring structures are the same even if the functional groups and lengths are different.

  The content of the compound S having a residue capable of forming a hydrogen bond on the substrate is a ratio of the average molecular weight, and the ratio of the average molecular weight of the compound S to the average molecular weight of the hydrophilic polymer is 0.005 or more and 0. .2 or less is preferable. When the ratio is lower than this, the compound S is easily crystallized, and when it is higher, the penetration into the hydrophilic polymer layer is difficult. By setting the ratio within this range, these problems can be solved, and the physiologically active substance The deactivation suppression effect and the aggregation suppression effect can be obtained higher.

  The biosensor on which a physiologically active substance is immobilized as described above can be used for detection and / or measurement of a substance that interacts with the physiologically active substance.

  In the present invention, it is preferable to detect and / or measure the interaction between the physiologically active substance immobilized on the sensor substrate and the test substance by a non-electrochemical method. Non-electrochemical methods include surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles.

  According to a preferred aspect of the present invention, the biosensor of the present invention can be used as a surface plasmon resonance biosensor characterized by including a metal film disposed on a transparent substrate, for example.

  The surface plasmon resonance biosensor is a biosensor used in the surface plasmon resonance biosensor, and includes a part that transmits and reflects light emitted from the sensor, and a part that fixes a physiologically active substance. And may be fixed to the main body of the sensor or may be removable.

  The phenomenon of surface plasmon resonance is due to the fact that the intensity of monochromatic light reflected from the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the metal exit side. Yes, so the sample can be analyzed by measuring the intensity of the reflected monochromatic light.

  As a surface plasmon measuring device for analyzing the characteristics of a substance to be measured using a phenomenon in which surface plasmons are excited by light waves, there is an apparatus using a system called Kretschmann arrangement (for example, JP-A-6-167443). See the official gazette). A surface plasmon measuring apparatus using the above system basically includes a dielectric block formed in a prism shape, for example, and a metal film formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a sample liquid. A light source that generates a light beam; an optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the metal film; and It comprises light detecting means for detecting the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface.

  In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or a component incident on the light beam at various angles is included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes according to the change in the incident angle of the incident light beam is detected by a small photodetector that moves in synchronization with the change in the reflection angle, or the direction in which the reflection angle changes Can be detected by an area sensor extending along the line. On the other hand, in the latter case, it can be detected by an area sensor extending in a direction in which each light beam reflected at various reflection angles can be received.

  In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the measured substance in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, so that the entire energy is transferred to the interface between the dielectric block and the metal film. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means. The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

  If the wave number of the surface plasmon is known from the incident angle at which this total reflection attenuation (ATR) occurs, that is, the total reflection attenuation angle (θSP), the dielectric constant of the substance to be measured can be obtained. In this type of surface plasmon measurement apparatus, as shown in Japanese Patent Application Laid-Open No. 11-326194, in order to measure the total reflection attenuation angle (θSP) with high accuracy and a large dynamic range, It is considered to use detection means. This light detection means is provided with a plurality of light receiving elements arranged in a predetermined direction, and arranged so that different light receiving elements receive light beam components totally reflected at various reflection angles at the interface. Is.

  In that case, there is provided differential means for differentiating the light detection signals output from the light receiving elements of the arrayed light detection means with respect to the arrangement direction of the light receiving elements, and based on the differential value output by the differential means. In many cases, the total reflection attenuation angle (θSP) is specified to obtain a characteristic related to the refractive index of the substance to be measured.

  Moreover, as a similar measuring device using total reflection attenuation (ATR), for example, “Spectroscopic Research” Vol. 47, No. 1, (1998), pages 21 to 23 and pages 26 to 27 are described. Devices are also known. This leakage mode measuring device is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be in contact with the sample liquid. Optical waveguide layer to be generated, a light source for generating a light beam, and the light beam to the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. The optical system includes an incident optical system and light detection means for detecting the excitation state of the waveguide mode, that is, the total reflection attenuation state by measuring the intensity of the light beam totally reflected at the interface.

  In the leakage mode measuring apparatus having the above-described configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle having a wave length propagates in the waveguide mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of guided light depends on the refractive index of the substance to be measured on the optical waveguide layer, knowing the specific incident angle at which total reflection attenuation occurs, the refractive index of the substance to be measured and the measurement object related thereto The properties of the substance can be analyzed.

  In this leakage mode measuring apparatus, the above-mentioned array-shaped light detecting means can be used to detect the position of the dark line generated in the reflected light due to the total reflection attenuation, and the above-described differentiating means is applied in conjunction therewith. Often done.

  In addition, the surface plasmon measurement device and the leakage mode measurement device described above may be used for random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research. In this case, the thin film layer A sensing substance is fixed on the sensing substance on the sensing substance (a metal film in the case of a surface plasmon measuring apparatus, a clad layer and an optical waveguide layer in the case of a leakage mode measuring apparatus), and various analytes are placed on the sensing substance. A sample solution dissolved in a solvent is added, and the total reflection attenuation angle (θSP) is measured every time a predetermined time elapses.

  If the analyte in the sample liquid binds to the sensing substance, the refractive index of the sensing substance changes with time due to this binding. Therefore, by measuring the total reflection attenuation angle (θSP) every predetermined time and measuring whether or not the total reflection attenuation angle (θSP) has changed, the binding state of the analyte and the sensing substance is determined. It is possible to determine whether or not the analyte is a specific substance that binds to the sensing substance based on the result. Examples of the combination of the specific substance and the sensing substance include an antigen and an antibody, or an antibody and an antibody. Specifically, a rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and a human IgG antibody can be used as the specific substance.

  Note that in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of total reflection attenuation (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the change amount of the differential value (see Japanese Patent Application Laid-Open No. 2003-106926 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

  Furthermore, a measuring apparatus using total reflection attenuation capable of measuring a large number of samples in a short time by sequentially measuring a plurality of measuring chips mounted on a turntable or the like is disclosed in JP-A-2001-2001. No. 330560.

  The biosensor of the present invention can be used as a biosensor that detects a change in refractive index using a waveguide, for example, having a waveguide structure on the substrate surface. In this case, the waveguide structure on the surface of the substrate has a diffraction grating and possibly an additional layer. The waveguide structure consists of a planar waveguide made of a thin dielectric layer. Light rays collected in the waveguide are guided into this thin layer by total reflection. The propagation speed of the guided light wave (hereinafter referred to as mode) takes the value of C / N. Here, C is the speed of light in vacuum, and N is the effective refractive index of the mode guided in the waveguide. The effective refractive index N is determined by the configuration of the waveguide on one side and the refractive index of the medium adjacent to the thin waveguide layer on the other side. Light wave conduction is performed not only in a thin planar layer, but also by another waveguide structure, in particular a strip-shaped waveguide. In that case, the waveguide structure is in the form of a strip-like film. An important factor for a biosensor is that the change in the effective refractive index N is caused by a change in the medium adjacent to the waveguide layer and a change in the refractive index and thickness of the waveguide layer itself or an additional layer adjacent to the waveguide layer. It is.

  Regarding the configuration of the biosensor of this system, for example, from Japanese Patent Publication No. 6-27703, page 4, line 48 to page 14, line 15 and FIG. 1 to FIG. 8, US Pat. No. 6,829,073, column 6, line 31 It is described in the 47th line of column7 and FIGS. 9A and B.

  For example, as one embodiment, there is a structure in which a waveguide layer having a flat thin layer is provided on a base material (for example, Pyrex (registered trademark) glass). The waveguide layer and the substrate together form a so-called waveguide. The waveguide layer is, for example, an oxide layer (SiO2, SnO2, Ta2O5, TiO2, TiO2-SiO2, HfO2, ZrO2, Al2O3, Si3N4, HfON, SiON, scandium oxide or a mixture thereof), a plastic layer (for example, polystyrene, polyethylene, A multilayer laminate such as polycarbonate) is possible. In order for light rays to propagate through the waveguide layer by total reflection, the refractive index of the waveguide layer must be greater than the refractive index of an adjacent medium (for example, a base material or an additional layer described later). A diffraction grating is disposed on the surface of the waveguide layer or the volume of the waveguide layer facing the substrate or the measurement substance. The diffraction grating can be formed in the substrate by embossing, holography or other methods. A thin waveguide film having a higher refractive index is then coated on the upper surface of the diffraction grating. The diffraction grating focuses incident light to the waveguide layer, emits a mode already guided in the waveguide layer, transmits part of the mode in the traveling direction, and reflects part of the mode. Has function. The waveguide layer covers the grating region with an additional layer. The additional layer can be a multilayer film as required. The additional layer can have a function that enables selective detection of a substance contained in the measurement substance. As a preferred embodiment, a layer having a detection function can be provided on the outermost surface of the additional layer. As a layer having such a detection function, a layer capable of immobilizing a physiologically active substance can be used.

  As another embodiment, a configuration in which an array of diffraction grating waveguides is incorporated in a well of a microplate is also possible (Japanese Patent Publication No. 2007-501432). That is, if the diffraction grating waveguides are arranged in an array on the bottom surface of the well of the microplate, it is possible to screen a drug or chemical substance with high throughput.

  In order to enable detection of a physiologically active substance on the upper layer (detection region) of the diffraction grating waveguide, the diffraction grating waveguide detects incident light and reflected light to detect a change in refractive characteristics. For this purpose, one or more light sources (eg lasers, diodes) and one or more detectors (eg spectrometers, CCD cameras or other photodetectors) can be used. . There are two different modes of operation—spectroscopy and angle methods for measuring refractive index changes. In spectroscopy, a broadband beam is sent as incident light to a diffraction grating waveguide and the reflected light is collected and measured, for example, with a spectrometer. By observing the spectral position of the resonance wavelength (peak), the refractive index change, that is, the coupling at or near the surface of the diffraction grating waveguide can be measured. Also, in the angle method, nominally single wavelength light is focused to produce a range of illumination angles and directed into the diffraction grating waveguide. The reflected light is measured by a CCD camera or other photodetector. By measuring the position of the resonance angle reflected by the diffraction grating waveguide, it is possible to measure the refractive index change, ie, coupling, at or near the surface of the diffraction grating waveguide.

  When the biosensor of the present invention is used for surface plasmon resonance analysis, it can be applied as a part of various surface plasmon measurement devices as described above.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1
About the following SAM compound, it mixed with water so that it might become the density | concentration shown in Table 1, and it observed after standing at 25 degreeC for 1 day. The case where visual precipitation or precipitation occurred was indicated as x, the case where white turbidity occurred was indicated as Δ, and the colorless and transparent one was indicated as ◯. The results are shown in Table 1.
16-Hydroxy-1-hexadecanethiol (manufactured by Frontier Scientific)
11-Hydroxy-1-undecanethiol (Aldrich)
8-Hydroxy-1-octanethiol (manufactured by Aldrich)
6-Hydroxy-1-hexanethiol (Aldrich)
11-Amino-1-undecanethiol, hydrochloride (manufactured by Dojin Chemical)
8-Amino-1-octanethiol, hydrochloride (manufactured by Dojindo)
6-Amino-1-hexanethiol, hydrochloride (manufactured by Dojin Chemical)

It can be seen that 8-Hydroxy-1-octanethiol, 6-Hydroxy-1-hexanethiol, 8-Amino-1-octanethiol, hydrochloride, and 6-Amino-1-hexanethiol, hydrochloride have good water solubility. On the other hand, 16-Hydroxy-1-hexadecanethiol, 11-Hydroxy-1-undecanethiol, and 11-Amino-1-undecanethiol, hydrochloride cannot form a self-assembled film (SAM) in the form of a metal film in an aqueous solution system.

Example 2
Hydrogel membranes that can immobilize proteins were prepared using SAM compounds with high water solubility, and the amount of immobilized proteins and nonspecific adsorption performance were evaluated.

(1) Creation of substrate
Mixed aqueous solution of 6-Hydroxy-1-undecanethiol (Aldrich) and 8-Amino-1-octanethiol, hydrochloride (Dojin Chemical) (6-Hydroxy-1-undecanethiol 4.995 mM / 8-Amino-1-octanethiol, hydrochloride 0.005 mM). This solution is called A solution.

Next, a gold thin film was formed on the upper surface of a plastic prism obtained by injection molding ZEONEX (manufactured by Nippon Zeon Co., Ltd.) by the following method. A prism is attached to the substrate holder of the sputtering device, and vacuum is applied (base pressure 1 × 10 ^-3 Pa or less), then Ar gas is introduced (1 Pa), and the substrate holder is rotated (20 rpm) while RF power is applied to the substrate holder. (0.5kW) is applied for about 9 minutes to plasma-treat the prism surface. Next, Ar gas was stopped and vacuum was drawn, Ar gas was introduced again (0.5 Pa), and DC power (0.2 kW) was applied to the 8-inch Cr target for about 30 seconds while rotating the substrate holder (10 to 40 rpm). This is applied to form a 2 nm Cr thin film. next. Stop Ar gas, evacuate again, introduce Ar gas again (0.5 Pa), rotate the substrate holder (20 rpm), and apply DC power (1 kW) to an 8-inch Au target for about 50 seconds to 50 nm A thin Au film is formed.

  The sensor stick on which the Au thin film obtained above was formed was immersed in solution A for 1 hour at 40 ° C. and washed 10 times with ultrapure water.

(2) Active esterification of CMD (carboxymethyl dextran)
After dissolving 4.95 ml of 0.1% by weight CMD (manufactured by Sangyo Sangyo Co., Ltd .: molecular weight 1 million) solution, 2% of the carboxyl groups are activated when the whole amount is reacted (1-Ethyl-3- [ 3-Dimethylaminopropyl] carbodiimide hydrochloride (0.4M) / NHS (N-hydroxysulfosuccinimide) (0.1M) mixed solution (50 μl) was added and stirred at room temperature.

(3) Binding reaction of CMD to substrate Drop 500 μl of the active esterified CMD solution prepared in (2) on the substrate prepared in (1) and spin coat at 1000 rpm for 45 seconds. Then, an active esterified carboxymethyl dextran thin film was formed on a substrate having an amino group. After reacting at room temperature for 1 hour, Sample 1 was obtained by washing 5 times with 0.1 N NaOH and 5 times with ultrapure water.

Comparative Example 1
(I) Creation of substrate having OH group
A 5 mM solution (ethanol / water = 8/2) of 16-Hydroxy-1-hexadecanethiol (manufactured by Frontier Scientific) was prepared. This solution is called B solution. Next, a 50 nm gold film was formed on the sensor stick in the same manner as in Example 2, immersed in solution B at 40 ° C. for 60 minutes, 5 times with ethanol, and once with 50 ml of ethanol / water (80/20). Washed 5 times with 50 ml of ultrapure water. And washed 5 times.

(Ii) Treatment with epichlorohydrin
The substrate is immersed in a mixed solution of 20 ml of 0.4 M sodium hydroxide, 20 ml of diethylene glycol dimethyl ether and 2.0 ml of epichlorohydrin, reacted in a shaking incubator at 25 ° C. for 4 hours, and then twice with 50 ml of ethanol. And washed 5 times with 50 ml of water.

(Iii) Treatment with dextran The above substrate was immersed in a mixed solution of 40.5 ml of water, 13.5 g of dextran (T500, Pharmacia) and 4.5 ml of 1M sodium hydroxide, reacted for 20 hours in a shaking incubator at 25 ° C, Washed 15 times with water at 50 ° C.

(Iv) Treatment with bromoacetic acid The substrate is immersed in a mixed solution of 3.5 g of bromoacetic acid and 27 g of 2M sodium hydroxide solution.
After 16 hours of reaction in a shaking incubator at 0 ° C., the mixture was washed with water. Again, a reaction with a bromoacetic acid solution for 16 hours and washing with water were performed to obtain Sample 2.

Example 3
Example 3 relates to protein immobilization on the sensor samples obtained in Example 2 and Comparative Example 1. CA (Carbonic Anhydrase: manufactured by SIGMA) was used as the protein.

  Samples 1 and 2 prepared in Example 2 and Comparative Example 1 were set in a surface plasmon resonance apparatus. After injecting PBS buffer and confirming the baseline (with the resonance angle at this time as a reference point), 0.2 M EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide) and 50 mM NHS (N-Hydroxysuccinimide) ) And then let stand for 7 minutes, then inject PBS buffer and let stand for 1 minute, then inject 0.1 mg / ml CA solution (acetate buffer pH 5.0) and let stand for 15 minutes, then Inject PBS buffer and leave for 1 minute, then inject 1M ethanolamine solution (Biacore) for 7 minutes, then inject PBS buffer for 1 minute, then inject 10 mM NaOH for 1 minute. The solution was placed three times, and finally PBS buffer was injected and allowed to stand for 1 minute. The difference between the resonance angle at this time and the resonance angle at the origin was defined as the CA fixed amount. Here, the resonance angle 1/10000 degree is expressed as 1RU.

  The CA fixed amount was 5200 RU (Sample 1) and 5000 RU (Sample 2). It was found that a large amount of protein can be immobilized even on the surface prepared using a highly water-soluble SAM compound.

Example 4
Example 4 relates to non-specific adsorption of low molecular weight compounds to the sensor samples obtained in Example 2 and Comparative Example 1. As low molecular weight compounds, CGP74514, which is an inhibitor of Cyclin-Dependent Kinases, and Tween 20, which is a surfactant, were selected, and the ability to suppress nonspecific adsorption to the sensor sample surface was examined.

  Samples 1 and 2 prepared in Example 2 and Comparative Example 1 were set in a surface plasmon resonance apparatus. After injecting PBS buffer and confirming the baseline (with the resonance angle at this time as a reference point), inject CGP74514 in PBS buffer solution (50 μM) or Tween20 in PBS buffer solution (0.005 wt%) for 2 minutes. The sample was allowed to stand, and finally PBS buffer was injected and left to stand for 2 minutes. The difference between the resonance angle at this time and the resonance angle at the origin was defined as the nonspecific adsorption amount.

  The amount of non-specific adsorption for Sample 1 was 3RU for CGP74514 and 5RU for Tween20. The nonspecific adsorption amount for sample 2 was 5RU for CGP74514 and 9RU for Tween20. It was found that non-specific adsorption of low molecular weight compounds can be suppressed even on surfaces prepared using SAM compounds with high water solubility.

Claims (26)

A biosensor produced by a process comprising a step of coating a substrate with an aqueous solution of a compound represented by the following formula (1) and a step of bringing a polysaccharide having a reactive group capable of binding a physiologically active substance into contact with the substrate. .
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y binds a polysaccharide. For functional groups.) Comprising a substrate on which a polysaccharide having a reactive group capable of binding a physiologically active substance is immobilized via a compound represented by the following formula (1) dissolved in water at 25 ° C. at a concentration of 0.1 mM. The biosensor according to claim 1.
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y binds a polysaccharide. For functional groups.) It consists of a substrate on which a polysaccharide having a reactive group capable of binding a physiologically active substance is immobilized via a compound represented by the following formula (1) dissolved in water at 25 ° C. at a concentration of 1 mM. The biosensor according to 1.
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y binds a polysaccharide. For functional groups.) X is an asymmetric or symmetric sulfide group, sulfide group, diselenide group, selenide group, thiol group, nitrile group, isonitrile group, nitro group, selenol group, trivalent phosphorus compound, isothiocyanate group, xanthate group, thiocarbamate group, phosphine The biosensor according to any one of claims 1 to 3, which is a group, a thioacid group, or a dithioacid group. The biosensor according to any one of claims 1 to 4, wherein Y is a carboxy group, a hydroxyl group, an amino group, an aldehyde group, a hydrazide group, a carbonyl group, an epoxy group, or a vinyl group. The biosensor according to any one of claims 1 to 5, wherein X is a thiol group and Y is an amino group. The biosensor according to any one of claims 1 to 6, wherein the reactive group capable of binding a physiologically active substance in the polysaccharide is an active carboxylate group. The substrate surface is coated with a mixture of an alkanethiol having an amino group and an alkanethiol having a hydrophilic group, and a polysaccharide having a reactive group capable of binding a physiologically active substance is immobilized through the alkanethiol having an amino group. The biosensor according to any one of claims 1 to 7, wherein The biosensor according to claim 8, wherein the hydrophilic group of the alkanethiol having a hydrophilic group is a hydroxyl group or an oligoethylene glycol group. The biosensor according to claim 8 or 9, wherein a molar ratio in a mixture of alkanethiol having an amino group and alkanethiol having a hydrophilic group is in a range of 1/1 to 1 / 1,000,000. The biosensor according to any one of claims 1 to 10, wherein the polysaccharide is a carboxyl group-containing polysaccharide. The biosensor according to any one of claims 1 to 11, wherein the polysaccharide has an underwater film thickness of 1 nm or more and 300 nm or less. The biosensor according to any one of claims 1 to 12, wherein the polysaccharide has an average molecular weight of 10,000 to 2,000,000. The biosensor according to any one of claims 1 to 13, wherein the substrate is a metal surface or a metal film. The biosensor according to any one of claims 1 to 14, wherein the metal is gold, silver, copper, platinum, or aluminum. The biosensor according to claim 1, wherein a substrate is placed on the plastic support. The biosensor according to any one of claims 1 to 16, which is used for surface plasmon resonance analysis. The method according to claim 1, comprising a step of coating a substrate with an aqueous solution of a compound represented by the following formula (1), and a step of bringing a polysaccharide having a reactive group capable of binding a physiologically active substance into contact with the substrate. Biosensor manufacturing method.
X-L ¹ -Y ⁽¹⁾
(In the formula, X represents a group capable of bonding to a metal, L ¹ represents a linear alkylene group having 2 to 10 carbon atoms which may be interrupted by a hetero atom, and Y binds a polysaccharide. For functional groups.) The method according to claim 18, wherein the substrate is coated with a mixture of an alkanethiol having an amino group and an alkanethiol having a hydrophilic group, and the polysaccharide is bound to the alkanethiol having an amino group. The method according to claim 18 or 19, wherein the hydrophilic group of the alkanethiol having a hydrophilic group is a hydroxyl group or an oligoethylene glycol group. The method according to claim 19 , wherein the molar ratio in the mixture of alkanethiol having an amino group and alkanethiol having a hydrophilic group is in the range of 1/1 to 1 / 1,000,000. The polysaccharide having a reactive group capable of binding a physiologically active substance is reacted with the compound represented by the formula (1) in a state where a thin film is formed on a substrate. Method. A biosensor produced by the method according to any one of claims 18 to 22. 24. A biosensor comprising the step of bringing the biosensor according to any one of claims 1 to 17 and 23 into contact with a physiologically active substance, and binding the physiologically active substance to the biosensor. Method. A substance that interacts with the physiologically active substance is detected or measured, comprising the step of bringing the biosensor according to any one of claims 1 to 17 into contact with the test substance, wherein the physiologically active substance is covalently bound to the surface. how to. The method according to claim 25, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.