JP2008209378A - Biosensor board - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor board useful for suppressing the nonspecific adsorption of a protein or compound having a positive charge. <P>SOLUTION: The biosensor board is modified with a hydrophilic high polymer containing dissociative groups with a pKa (acid dissociation constant) of up to 8. A ligand is fixed to the hydrophilic high polymer to bond to a tag molecule at Kd (binding constant) of up to 10<SP>-12</SP>M, and thereafter, the biosensor board is blocking-treated substantially not to be electrically charged. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

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

  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.

  Japanese Patent No. 3071823 describes that a biotinylated protein is immobilized on the biosensor surface via avidin. Japanese Patent No. 2815120 describes that a carboxyl group is introduced into dextran by binding dextran to a gold substrate via a self-assembled film (SAM) and further reacting with bromoacetic acid repeatedly. . Since this protein-immobilized film forms a three-dimensional matrix, it has an advantage that a large amount of protein can be immobilized. However, it contains a large amount of carboxyl groups that are dissociable groups, and a certain amount of carboxyl groups remains even after ordinary blocking treatment. In general, when immobilizing a biotinylated protein via avidin, and when measuring the binding of an analyte to the biotinylated protein, the carboxyl group of the immobilized membrane is dissociated and the substrate surface is negatively charged. Will have. For this reason, biotinylated protein having a positive charge, analyte itself, or various contaminants may cause non-specific adsorption on the substrate, thereby hindering accurate binding measurement.

Japanese Patent No. 3071823 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 substrate in which nonspecific adsorption of a positively charged protein or compound is suppressed. In particular, an object of the present invention is to provide a biosensor substrate with small negative baseline fluctuation and capable of detecting binding of a low-molecular-weight analyte.

As a result of intensive studies to solve the above problems, the present inventors have found that the hydrophilicity of a substrate modified with a hydrophilic polymer compound containing a dissociable group having a pKa (acid dissociation constant) of 8 or less. After immobilizing a ligand that forms a bond with a tag molecule and a Kd (binding constant) of 10 ^-12 M or less on a polymer, the biosensor substrate is subjected to a blocking treatment so as not to have a substantial charge. The present inventors have found that a biosensor substrate capable of immobilizing an active substance can be produced, and the present invention has been completed.

That is, according to the present invention, in a biosensor substrate comprising a substrate modified with a hydrophilic polymer compound containing a dissociable group having a pKa (acid dissociation constant) of 8 or less, the hydrophilic polymer contains tag molecules. And a Kd (binding constant) of a ligand that forms a bond of 10 ^-12 M or less is fixed, and then the biosensor substrate is subjected to a blocking treatment so as not to have a substantial charge. Is provided.

Preferably, the tag molecule is biotin and the ligand is a biotin binding protein.
Preferably, the biotin binding protein is an avidin.
Preferably, the dissociable group is a carboxyl group.

  Preferably, the biosensor substrate of the present invention is used for non-electrochemical detection, more preferably used for optical detection, and particularly preferably used for surface plasmon resonance analysis.

  According to another aspect of the present invention, there is provided the biosensor substrate of the present invention described above, wherein a physiologically active substance modified with a tag molecule is bound to the ligand.

  According to still another aspect of the present invention, the method includes a step of bringing the biosensor substrate of the present invention, in which a physiologically active substance modified with a tag molecule is bound to the ligand, into contact with a test substance. A method is provided for detecting or measuring a substance that interacts with an active substance.

  Preferably, the substance that interacts with the physiologically active substance is detected or measured by a non-electrochemical method, and more preferably, the substance that interacts with the physiologically active substance is detected or measured by surface plasmon resonance analysis.

  According to still another aspect of the present invention, there is provided a biosensor including the above-described biosensor substrate of the present invention.

  Since the biosensor substrate of the present invention has been treated so as not to have a substantial charge, nonspecific adsorption of various proteins and compounds is suppressed. In particular, according to the biosensor substrate of the present invention, the negative baseline fluctuation is small and the binding detection of a low molecular weight analyte is possible.

Hereinafter, embodiments of the present invention will be described.
The biosensor substrate of the present invention comprises a substrate modified with a hydrophilic polymer compound containing a dissociable group having a pKa (acid dissociation constant) of 8 or less, and a tag molecule and Kd (bonding) are attached to the hydrophilic polymer. After fixing a ligand that forms a bond with a constant) of 10 ⁻¹² M or less, the biosensor substrate is blocked so as not to have a substantial charge.

  Examples of the dissociable group having a pKa (acid dissociation constant) of 8 or less in the present invention include carboxylic acid (carboxyl group), sulfonic acid, phosphoric acid and the like, preferably carboxylic acid (carboxyl group).

  Specific examples of hydrophilic polymers containing a dissociable group having a pKa (acid dissociation constant) of 8 or less used in the present invention include proteins (albumin, casein, etc.), cellulose compounds (carboxymethylcellulose, hydroxymethylcellulose, etc.), Polysaccharides and their derivatives (agarose, dextran, carrageenan, alginic acid, sodium alginate, starch, starch derivatives, chitin, chitosan, etc.), synthetic hydrophilic polymers (polyvinyl alcohol, poly-N-vinylpyrrolidone, polyacrylamide, polyacrylic acid) Etc.).

  Preferably, a carboxyl group-containing synthetic polymer and a carboxyl group-containing polysaccharide can be used as the hydrophilic polymer containing a dissociable group having a pKa (acid dissociation constant) of 8 or less. 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 carboxyl group-containing polymer is preferably a carboxyl group-containing polysaccharide, and more preferably carboxymethyldextran.
The molecular weight of the carboxyl group-containing polymer used in the present invention is not particularly limited, but the average molecular weight is preferably 1000 to 5000000, the average molecular weight is more preferably 10,000 to 3000000, and the average molecular weight is 100,000 to 2000000. Further preferred. 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 for activating the carboxyl group-containing polymer, it is activated by a known method such as 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) which are water-soluble carbodiimides, or only EDC. And a method described in JP-A-2006-58071 (that is, a method of activating a carboxyl group using a uronium salt, phosphonium salt, or triazine derivative having a specific structure) And the method described in JP-A-2006-90781 (ie, a nitrogen-containing heteroaromatic compound having a hydroxyl group, activated with a carbodiimide derivative or a salt thereof, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group The method of activating the carboxyl group using any one of the compounds That.

下記一般式（Ｉａ）又は（Ｉｂ）で表される含窒素化合物

[式中、Ｒ₁及びＲ₂は、互いに独立して置換基を有しても良いカルボニル基、炭素原子、窒素原子を表し、Ｒ１及びＲ２は結合により５〜６員環を形成しても良く、Ａは置換基を有する炭素原子またはリン原子を表し、Ｍは（n-1）価の元素を表し、Ｘはハロゲン原子を表す]
下記一般式（ＩＩＩ）で表される含窒素化合物

[式中、Ａは置換基を有する炭素原子またはリン原子を表し、Ｙ及びＺは、互いに独立してＣＨ、または窒素原子を表し、Ｍは（n-1）価の元素を表し、Ｘはハロゲン原子を表す]
下記式で表される電子吸引性基を有するフェノール誘導体（好ましくは電子吸引性基のσ値が0.3以上）

を用いて活性化することができる。
これらの手法で活性化されたカルボキシル基含有ポリマーを、アミノ基を有する基板と反応させることで、本発明のバイオセンサーを製造することが可能となる。 For example, the following carbodiimide derivatives are compounds 1 to 3

Nitrogen-containing compounds represented by the following general formula (Ia) or (Ib)

[Wherein, R ₁ and R ₂ each independently represent a carbonyl group, a carbon atom or a nitrogen atom which may have a substituent, and R ₁ and R ₂ may form a 5- to 6-membered ring by a bond. Well, A represents a carbon atom or a phosphorus atom having a substituent, M represents an (n-1) valent element, and X represents a halogen atom.
Nitrogen-containing compounds represented by the following general formula (III)

[In the formula, A represents a carbon atom or a phosphorus atom having a substituent, Y and Z each independently represent CH or a nitrogen atom, M represents an (n-1) -valent element, and X represents Represents a halogen atom]
A phenol derivative having an electron withdrawing group represented by the following formula (preferably an σ value of the electron withdrawing group is 0.3 or more)

Can be used to activate.
By reacting the carboxyl group-containing polymer activated by these methods with a substrate having an amino group, the biosensor of the present invention can be produced.

  In the present invention, a known method can be used as a method for bonding the hydrophilic polymer as the binding matrix to the metal surface. A method of binding a binding matrix to a metal surface via a hydrophobic polymer as described herein below (see JP 2004-271514 A), X 2815120, A method of bonding the binding matrix to the metal surface through a tightly packed monolayer of -R-Y (X is bonded to the metal and Y is bonded to the binding matrix) can be applied. Preferably, after forming a dense layer on the metal surface using alkanethiol having a reactive group at the terminal (or disulfide which is an oxidant thereof), the reactive group at the terminal of the alkanethiol and the hydrophilic polymer are reacted. This is a coupling method.

In the biosensor substrate of the present invention, a ligand that forms a bond with a tag molecule and a Kd (binding constant) of 10 ⁻¹² M or less is fixed to the hydrophilic polymer. Examples of tag molecule / ligand combinations include biotin / biotin-binding protein, digoxigenin / digoxigenin antibody, and D-Ala-D-Ala derivative / vancomycin trimer derivative described in SCIENCE, 280,708-711 (1998). However, it is not limited to these. Specific examples of the biotin-binding protein include avidins (such as avidin, streptavidin, or a modified form thereof).

  According to the present invention, the hydrophilic polymer is a hydrophilic polymer containing a dissociable group having a pKa (acid dissociation constant) of 8 or less, and the ligand is converted to the hydrophilic polymer via the dissociable group. Can be fixed.

In the biosensor substrate of the present invention, the biosensor substrate is substantially charged after immobilizing a ligand that forms a bond with a tag molecule and a Kd (binding constant) of 10 ⁻¹² M or less on the hydrophilic polymer. Blocking treatment is performed so as not to have. The blocking process is not particularly limited as long as the biosensor substrate can be substantially free of charge. For example, a method of chemically changing a dissociable group having a charge so as not to have a charge by a chemical reaction, dissociation The method of neutralizing the charge of a dissociative group by ion-bonding the compound which has a charge opposite to a sex group is mentioned.

  As an example of the former, there is a method of forming a covalent bond with an amino compound by an amine coupling method on a hydrophilic polymer substrate having a negatively charged carboxyl group as a dissociable group. Preferably, a primary amine compound is used as a blocking agent, and a linear primary amine compound is more preferably used. Specific examples include ethanolamine, propanolamine, and polyethylene glycolamine. As a specific blocking operation, for example, a liquid mixture of 1-ethyl-2,3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide is added to a hydrophilic polymer substrate having a carboxyl group for a certain period of time. After the reaction, washing may be performed, and then an ethanolamine / HCl solution may be added and the reaction may be performed after the reaction for a certain time.

  As an example of the latter, a hydrophilic compound having a cationic group is preferably used for a hydrophilic polymer substrate having a negatively-dissociable group such as a carboxyl group, a sulfonic acid group, and a phosphoric acid group. Preferably, a hydrophilic compound having an ammonium group, a pyridinium group, a phosphonium group, or a sulfonium base is used. Specific examples include O- [2-hydroxy-3- (trimethylammonio) propyl] dextran chloride and tetrabutylammonium chloride. As a specific blocking operation, an operation of adding an aqueous solution of a cationic hydrophilic compound, washing after reacting for a predetermined time can be exemplified.

  “Substantially free of charge” means that a protein having surface charge is not electrostatically concentrated with respect to the hydrophilic polymer substrate as described above. Specifically, using the optical biosensor of the present invention, the degree of concentration of a protein on a hydrophilic polymer substrate can be measured with a buffer system adjusted to a pH such that the protein is charged. In the comparison between the pH at which the protein is not charged and the pH at which the protein is charged, if there is no significant difference in the protein concentration, the hydrophilic polymer substrate can be regarded as having substantially no charge.

  By bringing a bioactive substance modified with a tag molecule into contact with the surface of the biosensor substrate of the present invention, the ligand of the biosensor substrate and the tag molecule bound to the bioactive substance interact, It becomes possible to immobilize the physiologically active substance on the biosensor substrate.

  As a buffer used when immobilizing a physiologically active substance modified with a tag molecule, or a buffer used when measuring the binding of a low molecular weight analyte to a physiologically active substance modified with a tag molecule, a buffer in a neutral region is used. It is preferable to use it. The pH is preferably 6.0 to pH 8.0, and more preferably pH 6.5 to pH 7.5. Specifically, the following buffer agents are preferably used. Phosphoric acid, MES (2-Morpholinoethanesulfonic acid, momohydrate), Bis-Tris (Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane), PIPES (Piperazine-1,4-bis (2-ethanesulfonic acid), MOPSO (2- Hydroxy-3-morpholinopropanesulfonic acid), BES (N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), HEPES (2- [4- (2-Hydroxyetyl) -1- piperazinyl] ethanesulfonic acid), POPSO (Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid), dihydrate), HEPPSO (2-Hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid, monohydrate), EPPS (3- [4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid). The concentration of the buffer agent is preferably 1 mM to 200 mM, more preferably 5 mM to 100 mM. The NaCl concentration is preferably 10 mM to 500 mM, more preferably 50 mM to 200 mM, and a chelating reagent such as EDTA can be added. For adjustment may be added Ca, K, Na, Mn, Fe, metal ions such as Zn.

  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 disposed on the substrate. Here, “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not directly in contact with the substrate, but through other layers. This also includes the case where they are arranged. As a substrate that can be used in the present invention, for example, in the case of a surface plasmon resonance biosensor, generally, optical glass such as BK7, or synthetic resin, specifically polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer 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, the hydrophilic polymer is bonded to the metal layer directly or through an intermediate layer. As the intermediate layer, a layer made of a hydrophobic polymer compound or a self-assembled film can be used. Hereinafter, the hydrophobic polymer compound and the self-assembled film will be described.

  The hydrophobic polymer compound is a polymer compound that does not absorb water, and has a solubility in water (25 ° C.) of 10% or less, more preferably 1% or less, and most preferably 0.1% or less.

  Hydrophobic monomers that form hydrophobic polymer compounds include vinyl esters, acrylic acid esters, methacrylic acid esters, olefins, styrenes, crotonic acid esters, itaconic acid diesters, and maleic acid diesters. , Fumaric acid diesters, allyl compounds, vinyl ethers, vinyl ketones and the like. The hydrophobic polymer compound may be a homopolymer composed of one type of monomer or a copolymer composed of two or more types of monomers.

  Examples of the hydrophobic polymer compound preferably used in the present invention include polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate, polyester, and nylon.

  Coating of the hydrophobic polymer compound on the substrate can be performed by a conventional method, for example, spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, spraying, vapor deposition, casting, It can be performed by an immersion method or the like.

  In the dipping method, coating is performed by bringing the substrate into contact with a hydrophobic polymer solution and then bringing the substrate into contact with a liquid not containing the hydrophobic polymer solution. Preferably, the solvent of the hydrophobic polymer compound solution and the solvent of the liquid not containing the hydrophobic polymer compound are the same solvent.

  In the dipping method, a hydrophobic polymer compound layer having a uniform coating thickness can be obtained on the surface of the substrate by appropriately selecting a solvent for coating the hydrophobic polymer compound, regardless of the unevenness, curvature, and shape of the substrate.

  The coating solvent for the dipping method is not particularly limited, and any solvent can be used as long as it dissolves a part of the hydrophobic polymer compound. For example, a formamide solvent such as N, N-dimethylformamide, a nitrile solvent such as acetonitrile, an alcohol solvent such as phenoxyethanol, a ketone solvent such as 2-butanone, and a benzene solvent such as toluene can be used. However, it is not limited to these.

  The solution of the hydrophobic polymer compound to be brought into contact with the substrate may be either a completely dissolved hydrophobic polymer compound or a suspension containing an insoluble component of the hydrophobic polymer compound. The liquid temperature is not particularly limited as long as a part of the hydrophobic polymer compound is dissolved, but is preferably −20 ° C. or higher and 100 ° C. or lower. The liquid temperature may be varied while the substrate is in contact with the hydrophobic polymer compound solution. Although there is no restriction | limiting in particular in the hydrophobic polymer compound density | concentration of a solution, Preferably it is 0.01% or more and 30% or less, More preferably, it is 0.1% or more and 10% or less.

  The time for bringing the solid substrate into contact with the hydrophobic polymer solution is not particularly limited, but is preferably 1 second to 24 hours, more preferably 3 seconds to 1 hour.

As a liquid not containing a hydrophobic polymer compound, the difference between the SP value of the solvent itself (unit: (J / cm ³ ) ^1/2 ) and the SP value of the hydrophobic polymer compound is 1 or more and 20 or less. Preferably, it is 3 or more and 15 or less. The SP value is represented by the square root of the cohesive energy density between molecules and is also called a solubility parameter. In the present invention, the SP value δ is calculated by the following formula. As the cohesive energy Ecoh and the molar volume V of each functional group, the values specified by Fedors were used (R. F. Fedors, Polym. Eng. Sci., 14 (2), P147, P472 (1974)).
δ = (ΣEcoh / ΣV) ^1/2
By way of example, the SP values of hydrophobic polymer compounds and solvents are as follows: Polymethylmethacrylate-polystyrene copolymer (1: 1): for 21.0 Solvent 2-phenoxyethanol: 25.3, for Polymethylmethacrylate: 20.3 Solvent acetonitrile: 22.9, polystyrene: 21.6 with respect to polystyrene: 21.6.

  The time for contacting the substrate with the liquid not containing the hydrophobic polymer compound is not particularly limited, but is preferably 1 second or longer and 24 hours or shorter, more preferably 3 seconds or longer and 1 hour or shorter. The liquid temperature is not particularly limited as long as the solvent is in a liquid state, but is preferably −20 ° C. or higher and 100 ° C. or lower. The liquid temperature may be varied while the substrate is in contact with the solvent. When using a solvent that is difficult to volatilize, for the purpose of removing the solvent, the solvent may be replaced with a volatile solvent that dissolves each other after contacting the solvent.

  The coating thickness of the hydrophobic polymer compound is not particularly limited, but is preferably 0.1 nm to 500 nm, particularly preferably 1 nm to 300 nm.

  Next, the self-assembled film will be described. Sulfur compounds such as thiols and disulfides spontaneously adsorb on a noble metal substrate such as gold to give an ultra-thin film of single molecular size. The aggregate is called a self-assembled film because it shows an arrangement depending on the crystal lattice of the substrate and the molecular structure of adsorbed molecules. In the present invention, for example, as a self-assembling compound, 6-hydroxy-1-hexanethiol, 7-carboxy-1-heptanethiol, 10-carboxy-1-decanethiol, 4,4′-dithiodibutylic acid, 11 -Hydroxy-1-undecanethiol, 11-amino-1-undecanethiol and the like can be used.

Further, as a self-assembling compound usable in the present invention, an alkanethiol derivative represented by the general formula A-1 (in the general formula A-1 , n represents an integer of 3 to 20 and X represents a functional group) It can be preferably used. In the general formula A-1 , for example, by forming a self-assembled film using a compound where X = NH2, the gold surface can be covered with an organic layer having an amino group.

When an alkanethiol having an amino group at the terminal is used, a compound in which the thiol group and the amino group are linked via an alkyl chain (general formula 1-1 ) (in general formula 1-1 , n is 3 to 20 An alkanethiol having a carboxyl group at the terminal (general formulas 1-2 and 1-3 ) (in general formula 1-2 , n represents an integer of 3 to 20, and in general formula 4, n represents each Independently represents an integer of 1 to 20) and a large excess of hydrazide or diamine may be used. 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.

These alkanethiols can form a self-assembled film alone, or can be mixed with other types of alkanethiols to form a self-assembled film. When used on the biosensor surface, it is preferable to use a compound capable of suppressing non-specific adsorption of a physiologically active substance as the other kind of 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)). For example, as the alkanethiol that forms a mixed monomolecular film with an alkanethiol having an amino group, the compounds described in the 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 case of a mixed monomolecular film, for example, an alkanethiol having an amino group and an alkanethiol having a hydrophilic group can be mixed at an arbitrary ratio, but when the ratio of the alkanethiol having an amino group is small. Decreases the binding amount of the activated carboxyl group-containing polymer, and the nonspecific adsorption suppression ability decreases when the proportion of alkanethiol having a hydrophilic group is small. 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.

  The physiologically active substance immobilized on the sensor substrate of the present invention is not particularly limited as long as it interacts with the measurement object. For example, immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-immune proteins And immunoglobulin binding proteins, sugar binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, polypeptides or oligopeptides having ligand binding ability, and the like.

  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 amount of change in the differential value (see Japanese Patent Application No. 2000-398309 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.
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 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.

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

  The following experiment was performed using the apparatus described in paragraphs 0025 to 0049 and FIGS. 1 to 8 of Japanese Patent Application No. 2006-167855. Specifically, the following apparatus is used.

  The biosensor 10 as a screening device of this embodiment is a so-called surface plasmon sensor that measures the interaction between a physiologically active substance D and a compound using surface plasmons generated on the surface of a metal film. In the present embodiment, the biosensor 10 is used to select a compound that specifically binds to the physiologically active substance D from a large amount of the compound A.

  As shown in FIG. 1, the biosensor 10 includes a tray holding unit 12, a transport unit 14, a container mounting table 16, a liquid suction / discharge unit 20, an optical measurement unit 54, and a control unit 60.

  The tray holding unit 12 includes a mounting table 12A and a belt 12B. The mounting table 12A is attached to a belt 12B spanned in the arrow Y direction, and is movable in the arrow Y direction by the rotation of the belt 12B. Two trays T are positioned and mounted on the mounting table 12A. In the tray T, eight sensor sticks 40 are stored. The sensor stick 40 is a chip to which the physiologically active substance D is fixed, and details will be described later. Under the mounting table 12A, a push-up mechanism 12D for pushing up the sensor stick 40 to a position of a stick holding member 14C described later is disposed.

  As shown in FIGS. 2 and 3, the sensor stick 40 includes a dielectric block 42, a flow path member 44, and a holding member 46.

  The dielectric block 42 is made of transparent resin or the like that is transparent to the light beam, and has a prism portion 42A having a trapezoidal cross section, and the prism portion 42A integrally with both ends of the prism portion 42A. The formed held portion 42B is provided. A metal film 57 is formed on the upper surface on the wide side of the two parallel surfaces of the prism portion 42A. The dielectric block 42 functions as a so-called prism. When the measurement is performed by the biosensor 10, a light beam is incident from one of two opposing non-parallel sides of the prism portion 42A, and a metal film is formed from the other. The light beam totally reflected at the interface with 57 is emitted.

  A linker layer 57A is formed on the surface of the metal film 57 as shown in FIG. The linker layer 57A is a layer for immobilizing the physiologically active substance D on the metal film 57.

  On both side surfaces of the prism portion 42A, engaging convex portions 42C that are engaged with the holding member 46 are formed along the upper side edges. Further, a flange portion 42D that is engaged with a transport rail (not shown) is formed along the side end side below the prism portion 42A.

  As shown in FIG. 3, the flow path member 44 includes six base portions 44A, and four cylindrical members 44B are erected on each of the base portions 44A. In the base portion 44A, for each of the three base portions 44A, one upper portion of the standing cylindrical members 44B is connected by a connecting member 44D. The flow path member 44 is made of a soft and elastically deformable material, for example, an amorphous polyolefin elastomer.

  As shown in FIGS. 5 and 6, two substantially S-shaped channel grooves 44 </ b> C are formed on the bottom surface side of the base portion 44 </ b> A. Each of the channel grooves 44C communicates with the hollow portion of one cylindrical member 44B. The bottom surface of the base portion 44A is in close contact with the upper surface of the dielectric block 42, and the liquid channel 45 is configured by the space formed between the channel groove 44C and the upper surface of the dielectric block 42 and the hollow portion. The Two liquid channels 45 are formed in one base portion 44A. In each liquid channel 45, an inlet / outlet port 43 of the liquid channel 45 is formed on the upper end surface of the cylindrical member 44B.

  Here, one of the two liquid channels 45 is used as the measurement channel 45A, and the other one is used as the reference channel 45R. The physiologically active substance D is fixed on the metal film 57 of the measurement channel 45A, and the measurement is performed in a state where the physiologically active substance D is not fixed on the metal film 57 of the reference channel 45R. As shown in FIG. 5, light beams L1 and L2 are incident on the measurement channel 45A and the reference channel 45R, respectively. The light beams L1 and L2 are applied to an S-shaped bent portion disposed near the center of the base portion 44A. Hereinafter, the irradiation region of the light beam L1 in the flow channel 45A is referred to as a measurement region E1, and the irradiation region of the light beam L2 in the flow channel 45R is referred to as a reference region E2. The reference area E2 is an area for performing measurement for correcting data obtained from the measurement area E1 where the physiologically active substance D is fixed.

  The holding member 46 is long and has a shape in which an upper surface member 47 and two side plates 48 are formed in a lid shape. The side plate 48 is formed with an engagement hole 48C engaged with the engagement convex portion 42C of the dielectric block 42, and a window 48D at a portion corresponding to the optical path of the light beams L1 and L2. The holding member 46 is attached to the dielectric block 42 with the engagement hole 46 </ b> C and the engagement protrusion 42 </ b> C engaged. As will be described later, the flow path member 44 is integrally formed with the holding member 46 and is disposed between the holding member 46 and the dielectric block 42.

  A receiving portion 49 is formed on the upper surface member 47 at a position corresponding to the cylindrical member 44 </ b> B of the flow path member 44. As shown in FIG. 4, the receiving portion 49 has a substantially cylindrical shape, and a cylindrical member 44 </ b> B is disposed in a hollow lower portion. Further, a concave portion 49A communicating with the entrance / exit 43 is formed above the hollow cylindrical member 44B. The pipette tip 50 is inserted into the recess 49A.

  The holding member 46 is made of a material harder than the flow path member 44, for example, crystalline polyolefin.

  As shown in FIG. 6, the pipette tip 50 has a substantially conical cylinder shape, and includes a distal end portion 51, a main body portion 52, and a holding portion 53. The tip 51 is cylindrical, and an opening 51A for discharging or sucking liquid is formed at the forefront of the insertion direction. The main body 52 has a conical cylinder shape whose outer periphery is larger than that of the distal end portion 51, and an outer peripheral stepped portion 52 </ b> A is formed between the main body portion 52 and the distal end portion 51. 52 A of outer periphery level | step-difference parts are made into the taper shape where the front-end | tip part 51 side is small diameter. The holding portion 53 has a larger outer diameter than the main body portion 52, and a holding step portion 53 </ b> A is formed between the holding portion 53 and the main body portion 52. The holding step 53A is a portion used when holding the pipette tip 50 in a pipette tip stocker having an upper surface plate with holding holes (not shown).

  The recess 49A of the receiving portion 49 is configured to be surrounded by a first inner wall portion 49B and a second inner wall portion 49C on the flow path member 44 side. In the first inner wall portion 49B, the insertion direction Z of the pipette tip 50 is slightly longer than the tip portion 51 of the pipette tip 50, slightly larger than the outer diameter of the tip portion 51, and is along the tip portion 51. It is made into a shape.

  The second inner wall portion 49C includes an inner circumferential step portion 49D between the first inner wall portion 49B, a central inner wall portion 49E adjacent to the inner circumferential step portion 49D, and an uppermost upper inner wall portion 49F. The inner circumferential step portion 49D is continuous from the first inner wall portion 49B, and has a tapered shape with the upper portion having a large diameter along the outer circumferential step portion 52A of the pipette tip 50. The central inner wall portion 49E is continuous with the inner peripheral stepped portion 49D, and has a tapered shape with a large diameter above the pipette tip 50. The upper inner wall portion 49F is continuous with the central inner wall portion 49E, and has a tapered shape with a larger diameter above the pipette tip 50.

  The holding member 46 and the flow path member 44 are integrally molded by a so-called two-color molding method (double mold) in which different materials are molded in the same mold.

  As shown in FIG. 1, the transport unit 14 of the biosensor 10 includes an upper guide rail 14A, a lower guide rail 14B, and a stick holding member 14C. The upper guide rail 14 </ b> A and the lower guide rail 14 </ b> B are horizontally disposed in the arrow X direction perpendicular to the arrow Y direction, above the tray holding unit 12 and the optical measurement unit 54. A stick holding member 14C is attached to the upper guide rail 14A. The stick holding member 14C can hold the held parts 42B at both ends of the sensor stick 40 and can move along the upper guide rail 14A. The engagement groove 42E of the sensor stick 40 held by the stick holding member 14C and the lower guide rail 14B are engaged, and the stick holding member 14C moves in the arrow X direction, so that the sensor stick 40 is placed on the optical measurement unit 54. To the measurement unit 56. The measuring unit 56 is provided with a pressing member 58 that presses the sensor stick 40 during measurement. The pressing member 58 is movable in the Z direction by a driving mechanism (not shown), and presses the sensor stick 40 disposed in the measurement unit 56 from above.

  On the container mounting table 16, an analyte solution plate 17, a buffer liquid stock container 18, and a waste liquid container 19 are mounted. The analyte solution plate 17 is partitioned in a matrix shape, and various types of analyte solutions are stocked. The buffer liquid stock container 18 is composed of a plurality of containers, and a plurality of types of buffer liquids having different refractive indexes are stocked. The buffer solution stock container 18 is formed with an opening K into which a pipette tip 50 described later can be inserted. The waste liquid container 19 is composed of a plurality of containers, and an opening K into which the pipette tip 50 can be inserted is formed in the same manner as the buffer liquid stock container.

  As shown in FIG. 1, the liquid suction / discharge section 20 includes a head 24 and a suction / discharge drive section 26. The head 24 is movable in the direction of arrow Y (see FIG. 1) along a conveyance rail (not shown). The head 24 is also movable in the vertical direction (arrow Z direction) by a drive mechanism (not shown) inside the head 24.

  The supply of the liquid to the liquid flow path 45 by the head 24 is performed by inserting two pipette tips 50 into two openings of one liquid flow path 45 to discharge the liquid from one pipette tip 50 and the other. The pipette tip 50 is used to suck the liquid in the liquid channel 45.

  In this embodiment, the liquid is supplied to the sensor stick 40 by the pipette tip 50. Instead of the pipette tip, one end is connected to the solution plate and the other end can be connected to the sensor stick 40. An injection tube may be provided, and the liquid may be supplied by a liquid feed pump.

  As shown in FIG. 7, the optical measurement unit 54 includes a light source 54A, a first optical system 54B, a second optical system 54C, a light receiving unit 54D, and a signal processing unit 54E. A divergent light beam L is emitted from the light source 54A. The light beam L becomes two light beams L1 and L2 via the first optical system 54B, and is incident on the measurement region E1 and the reference region E2 of the dielectric block 42 arranged in the measurement unit 56. In the measurement region E1 and the reference region E2, the light beams L1 and L2 include various incident angle components with respect to the interface between the metal film 50 and the dielectric block 42 and are incident at an angle greater than the total reflection angle. The light beams L 1 and L 2 are totally reflected at the interface between the dielectric block 42 and the metal film 50. The totally reflected light beams L1 and L2 are also reflected with various reflection angle components. The totally reflected light beams L1 and L2 are received by the light receiving unit 54D through the second optical system 54C, are photoelectrically converted, and a light detection signal is output to the signal processing unit 54E. In the signal processing unit 54E, predetermined processing is performed based on the input light detection signal, and the measurement data G1 of the measurement region E1 and the reference data G2 of the reference region E2 are obtained. The measurement data G1 and reference data G2 are output to the control unit 60.

  The control unit 60 has a function of controlling the entire biosensor 10, and is connected to a light source 54A, a signal processing unit 54E, and a drive system (not shown) of the biosensor 10 as shown in FIG. As shown in FIG. 8, the control unit 60 includes a CPU 60A, a ROM 60B, a RAM 60C, a memory 60D, and an interface I / F 60E that are connected to each other via a bus B, and a display unit 62 that displays various types of information. These are connected to an input unit 64 for inputting various instructions and information.

  The memory 60D stores various programs for controlling the biosensor 10 and various data.

[Example 1]
(1) Creation of gold surface substrate
A 2 mM aqueous solution of 6-hydroxy-1-hexanethiol (Aldrich) was prepared. 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 About Au thin film was formed.

  The sample on which the obtained Au thin film was formed was immersed in a 2 mM aqueous solution of 6-hydroxy-1-hexanethiol (manufactured by Aldrich) at 25 ° C. for 16 hours and washed 10 times with ultrapure water.

(2) Preparation of hydrophilic polymer-modified substrate containing a dissociable group
After dissolving 4.95 ml of 0.1% by weight CMD (name sugar industry: 1 million molecular weight) solution, 2% of 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.

  On the gold surface side of the sample prepared in (1), 500 μl of the active esterified CMD solution prepared in (2) is dropped and spin coated at 1000 rpm for 45 seconds on the substrate having amino groups. An active esterified carboxymethyl dextran thin film was formed. After reacting at room temperature for 1 hour, a hydrophilic polymer-modified substrate sample containing a dissociable group was obtained by washing 5 times with 0.1 N NaOH and 5 times with ultrapure water.

(3) Immobilization of neutral avidin The above-mentioned sample was set in an SPR measuring apparatus, and neutral avidin (manufactured by PIERCE) was immobilized by the following procedure.
A neutral avidin 100 μg / ml solution was prepared using an acetate buffer (pH 5.5). Next, a running buffer (50 mM phosphate buffer (pH 7.4) / 150 mM NaCl) was added to take a baseline. Next, a mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added, allowed to stand for 7 minutes, and then washed with a running buffer. Next, the prepared neutral avidin solution was added and allowed to stand for 30 minutes, and then washed with a running buffer (pH 7.4). Next, in order to block the remaining active ester groups, an ethanolamine / HCl solution (1M, pH 8.5) was added and allowed to stand for 7 minutes, then washed 3 times with 10 mM NaOH running buffer, and further with running buffer. Washed 3 times.

(4) Blocking treatment of dissociable group The following dissociable group blocking treatment is performed on the sample on which the neutral avidin is immobilized. The number of times of this blocking treatment was carried out as shown in Table 1, and Samples 1 to 3 were obtained.

  Set the sample in the SPR measuring device, add a mixture of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM), let stand for 7 minutes, then wash with running buffer did. Next, an ethanolamine / HCl solution (1 M, pH 8.5) was added and the mixture was allowed to stand for 7 minutes, and then washed with a running buffer three times.

(5) Evaluation of surface charge Relative evaluation of the amount of negative charge on the sample surface was performed in the following procedure using Kanamycin showing positive charge in an acidic atmosphere.

  The sample on which neutral avidin was immobilized was set in an SPR measuring device, acetate buffer (pH 5.0) was added, and a baseline was taken. Subsequently, a Kanamycin solution (dissolved in acetate buffer (pH 5.0)) having a concentration of 0.1 mg / ml was added, and the mixture was allowed to stand for 2 minutes. The amount of signal change from the first baseline was defined as the surface charge (RU). Table 1 shows the charge amount of Sample 1 as 1, and the charge amounts of Samples 2 and 3 as relative values.

(6) Biotinylation of carbonic anhydrase Carbonic anhydrase (derived from Bovine, manufactured by SIGMA) was biotinylated by the following procedure. 1 x PBS (pH 7.4) of 0.045 mg / ml SNHS-LC-LC-Biotin (manufactured by PIERCE) in a 1.5 ml microtube containing 0.5 ml of 1 mg PBS (pH 7.4) of 2 mg / ml carbonic anhydrase. ) 0.5ml of the solution was added at 4 ° C and reacted at 4 ° C for 2 hours. 0.5 ml of the reaction solution was placed in a Microcon filter (YM-10 manufactured by Millipore) placed on a microtube and centrifuged at 4 ° C./14000 g / 60 minutes. The solution dropped in the microtube was discarded, 0.2 ml of 1 × PBS (pH 7.4) was placed on the filter, and centrifuged at 4 ° C./14000 g / 30 minutes. The solution dropped in the microtube was discarded, 0.2 ml of 1 × PBS (pH 7.4) was placed on the filter, and centrifuged at 4 ° C./14000 g / 30 minutes. The filter was turned upside down and set in a new microtube, and centrifuged at 4 ° C./1000 g / 3 minutes to recover the solution remaining on the filter. Further, 0.1 ml of 1 × PBS (pH 7.4) was added to the filter and centrifuged at 4 ° C./1000 g / 3 minutes. The collected solutions were combined to obtain a biotinylated carbonic anhydrase solution. Microreactor operation removes unreacted biotinylation reagent. About one biotinylation reagent was modified with respect to one carbonic anhydrase molecule (biotinylation ratio: about 1). Protein recovery was approximately 70%.

(7) Immobilization of biotinylated carbonic anhydrase Biotinylated carbonic anhydrase was immobilized on the above samples 1 to 3 by the following procedure.
Running buffer (50 mM phosphate buffer (pH 7.4) / 150 mM NaCl) was added to take a baseline. Next, the prepared biotinylated carbonic anhydrase solution was added and allowed to stand for 30 minutes, and then washed with a running buffer (pH 7.4). The amount of signal change from the initial baseline was defined as the fixed amount (RU) of biotinylated carbonic anhydrase. Table 1 shows the amount of biotinylated carbonic anhydrase immobilized on sample 1 as 1, and the amount of biotinylated carbonic anhydrase immobilized on samples 2 and 3 as relative values.

(8) Measurement of Baseline Variation Baseline variation was measured for samples 1 to 3 on which biotinylated carbonic anhydrase was immobilized by the following procedure.
Running buffer (50 mM phosphate buffer (pH 7.4) / 150 mM NaCl) was added and the baseline value (RU) was recorded. The baseline value (RU) was recorded again 10 minutes after standing still. The difference between the baseline values is defined as the baseline fluctuation value and is shown in Table 1.

Baseline fluctuation value (RU) = Baseline value after 10 minutes (RU) −Initial baseline value (RU)
When the baseline fluctuation for 10 minutes exceeds 5 RU, the binding measurement of the low molecular weight analyte is greatly hindered.

(9) Measurement of binding of low molecular weight analyte Indapamide (manufactured by SIGMA) was measured by the following procedure for samples 1 to 3 on which biotinylated carbonic anhydrase was immobilized. Indapamide is a small molecule inhibitor known to bind specifically to carbonic anhydrase. First, a running buffer (50 mM phosphate buffer (pH 7.4) / 150 mM NaCl / DMSO 10% by volume) was prepared. Indapamide was dissolved in the running buffer to a concentration of 10 μM.

Samples 1 to 3 on which biotinylated carbonic anhydrase was immobilized were set in a surface plasmon resonance measuring apparatus, a running buffer was added, and a baseline was taken. Next, the prepared Indapamide solution was added and allowed to stand for 10 minutes, and the signal change (RU) immediately after the running buffer was added was recorded. The Indapamide bond amount (RU) was calculated according to the following formula and listed in Table 1.
Indapamide binding amount (RU) = signal value after washing (RU) −initial baseline value (RU)

  From the results in Table 1, it can be seen that Sample 1 has a very large negative baseline fluctuation, and it is difficult to detect the binding of a low-molecular weight analyte. This baseline fluctuation is presumed to occur because biotinylated carbonic anhydrase is non-specifically adsorbed due to a large amount of carboxyl groups on the surface of the sample 1 and gradually dissociates. In Samples 2 and 3 of the present invention, the negative baseline fluctuation is extremely small, and binding detection of a low molecular weight analyte is possible.

It is a whole perspective view of the biosensor concerning this embodiment. It is a perspective view of the sensor stick concerning this embodiment. It is a disassembled perspective view of the sensor stick of this embodiment. It is sectional drawing of the 1 liquid flow path part of the sensor stick of this embodiment. It is a figure which shows the state in which the light beam is injecting into the measurement area | region and reference area | region of the sensor stick of this embodiment. It is a perspective view of the pipette concerning this embodiment. It is the schematic of the optical measurement part vicinity of the biosensor concerning this embodiment. It is a schematic block diagram of the control part concerning this embodiment and its periphery.

Explanation of symbols

10 Biosensor 40 Sensor Stick 54 Optical Measurement Unit 60D Memory 60 Control Unit D Bioactive Substance G1 Measurement Data G2 Reference Data

Claims (12)

In a biosensor substrate comprising a substrate modified with a hydrophilic polymer compound containing a dissociable group having a pKa (acid dissociation constant) of 8 or less, the hydrophilic polymer has a tag molecule and a Kd (binding constant) of 10 ^A biosensor substrate, wherein after fixing a ligand that forms a bond of ^-12 M or less, the biosensor substrate is subjected to a blocking treatment so as not to have a substantial charge. The biosensor substrate according to claim 1, wherein the tag molecule is biotin and the ligand is a biotin-binding protein. The biosensor substrate according to claim 2, wherein the biotin-binding protein is an avidin. The biosensor substrate according to any one of claims 1 to 3, wherein the dissociable group is a carboxyl group. The biosensor substrate according to any one of claims 1 to 4, which is used for non-electrochemical detection. The biosensor substrate according to any one of claims 1 to 5, which is used for optical detection. The biosensor substrate according to any one of claims 1 to 6, which is used for surface plasmon resonance analysis. The biosensor substrate according to any one of claims 1 to 7, wherein a physiologically active substance modified with a tag molecule is bound to the ligand. The biosensor substrate according to any one of claims 1 to 7, wherein a bioactive substance modified with a tag molecule is bound to the ligand, and a step of bringing the test substance into contact with the bioactive substance. A method for detecting or measuring substances to be detected. The method according to claim 9, wherein a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method. The method according to claim 9 or 10, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis. A biosensor comprising the biosensor substrate according to claim 1.

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