JP4369295B2 - Measuring method using biosensor - Google Patents

Description

  The present invention relates to a measurement method using a biosensor. More specifically, the present invention relates to a method for performing detection or measurement of a substance that interacts with a physiologically active substance immobilized on a biosensor and measurement of biological activity of the physiologically active substance in the same biosensor. .

  Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations, etc., but conventional methods require complicated operations and labeling substances, so measurement without the need for labeling substances Several techniques that can detect a change in the amount of a substance bound with high sensitivity are used. For example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, and measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. SPR measurement technology detects adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a fixed wavelength. It is a method to do. The QCM measurement technique is a technique capable of detecting 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.

  A measurement chip generally used in surface plasmon resonance (SPR) analysis includes a transparent substrate (for example, glass), a deposited metal film, and a thin film having a functional group capable of immobilizing a physiologically active substance thereon. A physiologically active substance is immobilized on the metal surface via a functional group. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance. As a thin film having a functional group capable of immobilizing a physiologically active substance, a functional group capable of binding to a metal, a linker having a chain length of 10 or more atoms, and a compound having a functional group capable of binding to a physiologically active substance, A measurement chip in which an active substance is immobilized has been reported (see Patent Document 1). In addition, a measurement chip comprising a metal film and a plasma polymerization film formed on the metal film has been reported (see Patent Document 2).

  As described above, in the conventional surface plasmon resonance (SPR) measurement, a substance that interacts with a physiologically active substance immobilized on a measurement chip has been detected or measured. Since the activity was not measured, it was not possible to analyze whether a substance that interacts with the physiologically active substance affects the biological activity of the physiologically active substance.

Japanese Patent No. 2815120 JP-A-9-264843

  The present invention has been made to solve the above-described problems of the prior art. That is, the present invention provides a method for detecting or measuring a substance that interacts with a physiologically active substance immobilized on a biosensor and simultaneously analyzing the influence of the substance on the biological activity of the physiologically active substance. It was a problem to be solved.

  As a result of intensive studies to solve the above problems, the present inventors have used the same biosensor in which a physiologically active substance is immobilized on a substrate to detect or measure a substance that interacts with the physiologically active substance. The present inventors have found that the above problems can be solved by measuring the biological activity of the physiologically active substance, and have completed the present invention.

  That is, according to the present invention, in a measurement method using a biosensor in which a physiologically active substance is immobilized on a substrate, detection or measurement of a substance that interacts with the physiologically active substance, and biological activity of the physiologically active substance The above-described measurement method is characterized in that the measurement is performed in the same biosensor as described above.

Preferably, the physiologically active substance is a protein. More preferably, the physiologically active substance is an enzyme.
Preferably, detection or measurement of a substance that interacts with a physiologically active substance is performed by surface plasmon resonance analysis.

  Preferably, the dielectric block, a metal film formed on one surface of the dielectric block, a light source for generating a light beam, and the light beam to the dielectric block, the dielectric block and the metal film The surface plasmon resonance state is detected by measuring the intensity of the light beam totally reflected at the interface and the optical system that makes the incident so that the total reflection condition can be obtained at the interface and including various incident angle components. A biosensor for use in a surface plasmon resonance measuring apparatus comprising a light detection means, comprising the dielectric block and the metal film, wherein the dielectric block includes an incident surface and an exit surface of the beam. And a biosensor in which the metal film is formed as one block including all of the one surface on which the metal film is formed, and the metal film is integrated with the dielectric block. Performing a surface plasmon resonance analysis.

  Preferably, the biological activity of the physiologically active substance is measured by spectroscopic measurement.

The measurement method of the present invention preferably comprises (1) a step of bringing a test substance into contact with a biosensor in which a physiologically active substance is immobilized on a substrate;
(2) detecting or measuring an interaction between a physiologically active substance and a test substance; and (3) measuring a biological activity of the physiologically active substance in the presence of the test substance.

  According to the measurement method of the present invention, two types of measurement, binding and enzyme reaction, can be performed with a small amount of protein. Further, according to the measurement method of the present invention, it is possible to determine whether or not the bound compound is binding to the active site by measuring the activity.

Embodiments of the present invention will be described below.
The present invention relates to a measurement method using a biosensor in which a physiologically active substance is immobilized on a substrate, the detection or measurement of a substance that interacts with the physiologically active substance, and the measurement of the biological activity of the physiologically active substance. The method is performed in the same biosensor as described above. By detecting or measuring a substance interacting with a physiologically active substance and measuring the biological activity of the physiologically active substance in the same biosensor, for example, a test substance bound to the physiologically active substance It becomes possible to determine whether or not it has bound to the active site, and the influence of the test substance on the biological activity of the bioactive substance can be analyzed simultaneously.

  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 a 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 used in 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 the use for a surface plasmon resonance biosensor, it is preferably 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 2000 angstrom or less. If it exceeds 5000 angstroms, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 1 angstrom or more and 100 angstrom or less.

  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 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 substrate is preferably a metal surface or metal film coated with a hydrophobic polymer compound or a polyhydroxy polymer compound, or a metal surface or metal film having a self-assembled film. Hereinafter, the hydrophobic polymer compound, the polyhydroxy polymer compound, and the self-assembled film will be described.

  The hydrophobic polymer compound used in the present invention is a polymer compound having no water absorption, and its solubility in water (25 ° C.) is 10% or less, more preferably 1% or less, most preferably 0.1% or less. It is.

  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, values defined 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 value of the hydrophobic polymer compound and solvent used in the examples is as follows: Polymethyl methacrylate-polystyrene copolymer (1: 1): 21.0 against solvent 2-phenoxyethanol: 25.3, polymethyl methacrylate : Solvent acetonitrile with respect to 20.3: 22.9, Solvent toluene with respect to polystyrene: 21.6: 18.7.

  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 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 3000 angstrom or less.

  Examples of the polyhydroxy polymer compound used in the present invention include polysaccharides (eg, agarose, dextran, carrageenan, alginic acid, starch, cellulose), or synthetic polymer compounds (eg, polyvinyl alcohol). In the present invention, polysaccharides are preferably used, and dextran is most preferred.

  In the present invention, a polyhydroxy polymer compound having an average molecular weight of 10,000 to 2,000,000 is preferably used. Preferably, a polyhydroxy polymer compound having a molecular weight of 20,000 to 2,000,000, more preferably 30,000 to 1,000,000, and most preferably 200,000 to 800,000 can be used.

  The polyhydroxy polymer compound can be carboxylated, for example, by reacting with bromoacetic acid under basic conditions. By controlling the reaction conditions, it is possible to carboxylate a certain proportion of the hydroxy groups that the polyhydroxy compound contains in the initial state. In the present invention, for example, 1 to 90% of hydroxy groups can be carboxylated. In addition, about the surface coat | covered with arbitrary polyhydroxy polymer compounds, a carboxylation rate is computable with the following method, for example. The membrane surface was gas-phase modified with trifluoroethanol at 50 ° C. for 16 hours using a di-tert-butylcarbodiimide / pyridine catalyst, and the amount of fluorine derived from trifluoroethanol was measured by ESCA (electron spectroscopy for chemical analysis). A ratio with the amount of oxygen on the film surface (hereinafter referred to as F / O value) is calculated. Carboxylation rate at that time by measuring the F / O value when carboxylation is carried out under arbitrary conditions with the theoretical F / O value when all hydroxy groups are carboxylated as 100% carboxylation rate Can be calculated.

The polyhydroxy polymer compound can be attached to the metal film via the organic molecule X ¹ —R ¹ —Y ¹ . The organic molecule X ¹ —R ¹ —Y ¹ will be described in detail.

X ¹ is a group having a binding property to the metal film. Specifically, asymmetric or symmetric sulfide (—SSR ¹¹ Y ¹¹ , —SSR ¹ Y ¹ ), sulfide (—SR ¹¹ Y ¹¹ , —SR ¹ Y ¹ ), diselenide (—SeSeR ¹¹ Y ¹¹ , —SeSeR ¹ Y) ¹ ), selenide (SeR ¹¹ Y ¹¹ , —SeR ¹ Y ¹ ), thiol (—SH), nitrile (—CN), isonitrile, nitro (—NO ₂ ), selenol (—SeH), trivalent phosphorus compound, iso Thiocyanate, xanthate, thiocarbamate, phosphine, thioacid or dithioacid (-COSH, -CSSH) is preferably used.

R ¹ (and R ¹¹ ) is optionally interrupted by a heteroatom, preferably straight-chain (unbranched) for reasonably close packing, and optionally carbon containing double and / or triple bonds It is a hydrogen chain. The chain length is preferably greater than 10 atoms. The carbon chain can optionally be perfluorinated.

Y ¹ and Y ¹¹ are groups for binding the polyhydroxy polymer compound. Y ¹ and Y ¹¹ are preferably the same and have the property that they can be bonded directly or after activation to the polyhydroxy polymer compound. Specifically, hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, epoxy, vinyl group, or the like can be used.

Specific examples of the organic molecule X ¹ -R ¹ -Y ¹ include 10-carboxy-1-decanethiol, 4,4′-dithiodibutylic acid, 11-hydroxy-1-undecanethiol, 11-amino-1- And undecanethiol.

  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, 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.

The substrate used in the present invention may further have a linker for bonding the compound represented by the general formula (A) to the substrate.
Specific examples of the linker used in the present invention include a compound represented by the following formula (4).
X ²⁰ -L ²⁰ -Y ²⁰ (4)
(Wherein X ²⁰ represents a group capable of reacting with a functional group in the hydrophobic polymer compound, polyhydroxy polymer compound or self-assembled film, L ²⁰ represents a divalent linking group, and Y ²⁰ represents a general group. A group capable of reacting with the compound represented by the formula (A) to form a covalent bond.

In the formula (4), X ²⁰ represents a group capable of reacting with a functional group in a hydrophobic polymer compound, a polyhydroxy polymer compound or a self-assembled film, preferably protected by a halogen atom, an amino group, or a protecting group. Amino group, carboxyl group, or carbonyl group having a leaving group, hydroxyl group, hydroxyl group protected by a protecting group, aldehyde group, —NHNH ₂ , —N═C═O, —N═C═S, epoxy group Or a vinyl group.

  The protecting group here is a group that can be deprotected in the reaction system to form a functional group. For example, the protecting group for amino group includes tertbutyloxycarbonyl group (Boc), 9-fluorene group. Examples include nylmethyloxycarbonyl group (Fmoc), nitrophenylsulfenyl group (Nps), dithiasuccinyl group (Dts) and the like.

Moreover, an acyl group etc. are mentioned as a hydroxyl-protecting group.
The leaving group here is a halogen atom, alkoxy group, aryloxy group, alkylcarbonyloxy group, arylcarbonyloxy group, halogenated alkylcarbonyloxy group, alkylsulfonyloxy group, halogenated alkylsulfonyloxy group, arylsulfonyloxy Groups and the like.
As the leaving group, an ester group produced by combining a carboxylic acid, a known dehydration condensation reagent (for example, carbodiimides) and an N-hydroxy compound is also preferably used.

In Formula (4), L ²⁰ represents a divalent linking group, and the total number of atoms of L is preferably 2 to 1000. In addition, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyleneoxy group, a substituted or unsubstituted aryleneoxy group, or X ^{20 of} formula (4) and Y ^{20 of} another molecule are bonded to form a continuous structure. It is preferable that it is a bivalent coupling group.

In the formula (4), Y ²⁰ represents a group capable of reacting with the compound represented by the general formula (A) to form a covalent bond, preferably a halogen atom, an amino group, or an amino group protected by a protecting group , A carbonyl group having a carboxyl group or a leaving group, a hydroxyl group, a hydroxyl group protected by a protecting group, an aldehyde group, —NHNH ₂ , —N═C═O, —N═C═S, an epoxy group, or a vinyl group It is.
As the protecting group and the leaving group, the same as those described above can be used.

  Specific examples of the compound of formula (4) are shown below, but the compound of formula (4) that can be used in the present invention is not limited thereto.

  The biosensor according to the present invention preferably has a functional group capable of immobilizing a physiologically active substance on the outermost surface of the substrate. Here, the “outermost surface of the substrate” means “the side farthest from the substrate”, and more specifically, “the side farthest from the substrate in the hydrophobic polymer compound coated on the substrate”. Meaning.

  The sensor substrate obtained as described above has a physiologically active substance bonded to the metal surface or metal film by covalently bonding the physiologically active substance via Y which is a functional group in the general formula (A). Can be immobilized.

  The physiologically active substance immobilized on the biosensor is not particularly limited as long as it interacts with the measurement target and itself has some physiological activity. For example, immune proteins, enzymes, microorganisms, nucleic acids, small molecules Examples include organic compounds, non-immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand-binding ability. Among these, proteins are preferable, and enzymes are particularly preferable.

  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, and 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 antigen 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, etc. 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.

  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 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 that enters 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 used in the present invention is used for surface plasmon resonance analysis, it can be applied as a part of the various surface plasmon measuring devices as described above.

  Further, in the measurement method of the present invention, the biologically active substance is biologically detected simultaneously with or after the substance that interacts with the physiologically active substance is detected or measured by a technique such as surface plasmon resonance analysis as described above. Activity measurements are performed on the same biosensor.

  The biological activity referred to in the present invention includes enzyme activity, antibody activity, receptor activity and the like. Enzyme is a general term for proteins that act as a catalyst that smoothly promotes various chemical reactions that occur in the body, including digestion of food. A catalyst generally has a mediating function to efficiently advance a chemical reaction.

  The principle of measuring enzyme activity is described in Chapter IV of basic experimental methods for proteins and enzymes (Takeichi Horio and Nihei Yamashita). As detection methods, there are (1) spectroscopic measurement method, (2) fluorescence method, (3) electrode method, (4) luminescence method, etc., for example, New Chemistry Experiment Course Protein V, Enzyme Immunoassay 3rd edition Enzyme immunoassays The methods described in biochemical experimental methods and the like can also be used.

  For example, when the enzyme activity is measured by a fluorescence method, the enzyme activity is measured by reacting with a substrate specific to the enzyme (a substrate in which only the degradation product of the enzyme emits fluorescence) and measuring the fluorescence of the degradation product. be able to.

When enzyme activity is measured by spectroscopic measurement, the temporal change is measured using the difference in spectroscopic properties between the substrate and the product, and the initial rate of the enzyme reaction is measured. The enzyme activity can be measured by obtaining.
When measuring enzyme activity by the electrode method, it is possible to electrically detect the pH change of the sample solution due to acids or bases generated by chemical reactions including enzymatic reactions by a method using an automatic titrator. To measure enzyme activity.
When enzyme activity is measured by the luminescence method, the antibody or antigen is enzyme-labeled, and after the antigen-antibody reaction, the enzyme-specific chemiluminescent substrate (substrate that chemiluminescents only the enzyme degradation product) is reacted. The enzyme activity can be measured by measuring the chemiluminescence of the degradation product.

  When measuring the antibody activity, the target antigen is bound to the inner wall of each hole of the plastic plate for ELISA, and a test substance containing the antibody to be measured is added and reacted. The antibody binds to the solid phase antigen. The amount of binding increases as the amount of antibody in the specimen increases. In addition, an enzyme-labeled anti-immunoglobulin antibody is caused. The labeled antibody binds to the antibody that is bound to the solid phase. The amount of labeled antibody binding increases as the amount of antibody increases. The unreacted labeled antibody is removed, and a substrate that develops color under the action of the enzyme is added. Since the substrate develops color depending on the amount of enzyme, the more the enzyme-labeled antibody is bound, the stronger the color tone. By measuring the color intensity of this solution with a colorimeter, the antibody amount of the test substance can be known. By measuring each of serially diluted antibodies of a known amount, the amount of antibody in the test substance can be quantified by comparing with that value.

  When measuring receptor activity, one or more kinds of receptors or ligands immobilized on a support are contacted with an antigen-labeled ligand or a specimen containing the receptor, and then immobilized on the support. An antigen-labeled ligand or receptor bound to one or more receptors or ligands and an antibody against the antigen (for example, an antibody labeled with an enzyme for detection) are contacted to perform an antigen-antibody reaction, thereby By detecting the presence of the bound antibody, the ligand or receptor in the sample can be detected.

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

(1) Surface Plasmon Resonance Measuring Device and Dielectric Block The following experiment was performed using the device shown in FIG. 22 of Japanese Patent Laid-Open No. 2001-330560 (hereinafter referred to as the surface plasmon resonance measuring device of the present invention) (in this specification). 1) and the dielectric block (hereinafter referred to as the dielectric block of the present invention) (shown as FIG. 2 in this specification) described in FIG. 23 of the publication.

  The surface plasmon resonance measuring apparatus shown in FIG. 1 slidably engages two guide rods 400, 400 arranged in parallel with each other as a support for supporting a measurement unit, and an arrow Y in FIG. A slide block 401 that is linearly movable in the direction is used. The slide block 401 is screwed with a precision screw 402 arranged in parallel with the guide rods 400, 400 so that the precision screw 402 is rotated forward and backward by a pulse motor 403 constituting the support driving means together with the precision screw 402. It has become.

  The driving of the pulse motor 403 is controlled by a motor controller 404. That is, an output signal S40 of a linear encoder (not shown) that is incorporated in the slide block 401 and detects the position of the slide block 401 in the longitudinal direction of the guide rods 400, 400 is input to the motor controller 404. Based on this signal S40, the drive of the pulse motor 403 is controlled.

  A laser light source 31, a condensing lens 32, and a light detector 40 are disposed below the guide rods 400, 400 so that slide blocks 401 that move along the guide rods 400 are sandwiched from the left and right. The condensing lens 32 condenses the light beam 30. In addition, a photodetector 40 is installed.

  Here, in the present embodiment, as an example, a stick-like unit connection body 410 formed by connecting and fixing eight measurement units 10 is used, and the eight measurement units 10 are set on the slide block 401 in a state of being arranged in a row. It has come to be.

  FIG. 2 shows the structure of the unit connector 410 in detail. As shown here, the unit connection body 410 is formed by connecting eight measurement units 10 by connection members 411.

  The measurement unit 10 is formed by integrally molding the dielectric block 11 and the sample holding frame 13 from, for example, a transparent resin, and constitutes a measurement chip that can be exchanged for the turntable. In order to make the exchange possible, for example, the measurement unit 10 may be fitted and held in a through hole formed in the turntable. In this example, the sensing substance 14 is fixed on the metal film 12.

(2) Measurement chip In the following experiment, the measurement chip (hydrogel-coated chip) described in Japanese Patent Application No. 2003-330927 was used. Specifically, a measurement chip coated with a hydrogel was prepared by the following procedure.

  11-hydroxy dissolved in ethanol / water (80/20) after treatment for 30 minutes with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) On the dielectric block of the present invention in which 50 nm gold is deposited as a metal film A 5.0 mM solution of -1-undecanethiol was added so as to contact the metal film, and surface treatment was performed at 25 ° C. for 18 hours. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water.

  Next, a 10% by weight epichlorohydrin solution (solvent: 1: 1 mixed solution of 0.4M sodium hydroxide and diethylene glycol dimethyl ether) is brought into contact with the surface coated with 11-hydroxy-1-undecanethiol at 25 ° C. The reaction was allowed to proceed for 4 hours in a shaking incubator. Thereafter, the surface was washed twice with ethanol and five times with water. Next, 4.5 ml of 1 M sodium hydroxide was added to 40.5 ml of a 25 wt% aqueous dextran (T500, Pharmacia) solution and the solution was contacted on the epichlorohydrin treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. The surface was washed 10 times with 50 ° C. water. Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated for 16 hours in a shaking incubator at 28 ° C. The surface was washed with water and then the above procedure was repeated once. This sample was used as a measuring chip coated with hydrogel.

In addition, a biosensor chip (PMMA measurement chip) coated with a hydrophobic polymer was prepared as follows.
(I) Fabrication of biosensor chip coated with polymethylmethacrylate 1cm (1cm cover glass model-208UV-ozone cleaning system (TECHNOVISION INC.) With gold deposited to a thickness of 500Å 30 minutes, and then set in a spin coater (MODEL ASS-303, manufactured by ABLE) and rotated at 1000 rpm. 50 μl of a methyl ethyl ketone solution of polymethyl methacrylate (2 mg / ml) was dropped into the center of the gold vapor deposition cover glass. The rotation was stopped after 2 minutes and the film thickness was measured by ellipsometry (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab), and the thickness of the polymethyl methacrylate film was 200 Å. Called PMMA surface chip.

(Ii) Introduction of COOH group onto PMMA surface The cover glass coated with polymethyl methacrylate prepared as described above was immersed in an aqueous NaOH solution (1N) at 40 ° C. for 16 hours, and then washed with water three times. This sample is called a PMMA / COOH surface chip.

(Iii) Preparation of surface having linker 2 ml of mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) from the PMMA / COOH surface chip prepared in (ii) above. And then immersed in 2 ml of an α-amino-polyethyleneoxy-ω-carboxylic acid (average molecular weight 5000) aqueous solution (10 mM) for 16 hours. Finally, it was washed 5 times with water. This sample is called a PMMA / PEO-C surface chip.

Example 1 Measurement of Trypsin and Leupeptin Trypsin (manufactured by Worthington) was immobilized on a measurement chip, and interaction measurement was performed using a surface plasmon resonance measuring apparatus.
(1) Immobilization of trypsin A mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added to the hydrogel-coated chip, and after standing for 20 minutes, 10 mM CaCl ₂ Washed with HBS-N buffer. Next, trypsin (1 mg / ml, 10 mM CaCl ₂ -containing HBS-N buffer) was added, allowed to stand for 30 minutes, and then washed with 10 mM CaCl ₂ -containing HBS-N buffer. The composition of the HBS-N buffer used above is HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4) and NaCl 0.15 mol / l.

  Furthermore, after adding it to an ethanolamine / HCl solution (1M, pH 8.5) measuring chip, it was washed with HBS-N buffer (Biacore) to block COOH groups remaining without reacting with trypsin.

  By the above operation, trypsin was covalently fixed to the measurement chip surface. The amount of change in resonance signal (RU value) before addition of trypsin and after washing was defined as the amount of trypsin fixation (RU value). The amount of change in the resonance signal due to trypsin fixation was 1360 RU.

(2) Detection of a substance interacting with a physiologically active substance After the measurement chip is washed with an HBS-N buffer, it is placed on a surface plasmon resonance measuring apparatus, and a leupeptin solution (0.002 mM, HBS-N buffer) is added for 10 minutes. After standing, the amount of change in resonance signal (RU value) was defined as the binding amount of leupeptin (manufactured by ICN) to trypsin.

(3) Measurement of biological activity of physiologically active substance The enzyme activity of trypsin was measured using the chip after measuring the binding amount of leupeptin in (2) above. Specifically, a substrate specific for trypsin (benzoyl-L-arginine-4 methylcoumarin-7 amide, hereinafter referred to as Bz-Arg-MCA; Peltide Laboratory Co., Ltd.) was reacted to measure fluorescence of the degradation product.

(4) Reaction of Bz-Arg-MCA The HBS-N buffer in the measurement chip was removed, and a leupeptin solution (0.002 mM, HBS-N buffer) +0.1 mM Bz-Arg-MCA was added. The reaction was performed for 60,120 minutes.

(5) Fluorescence measurement Fluorescence measurement was performed for each time using a BMGLab Technologies FLUOstar measuring device (Excitation 380 nm, Emission 460 nm).

Example 2 Measurement of Trypsin and Nitrofurantoin Trypsin (manufactured by Worthington) was immobilized on a measurement chip, and the interaction measurement was performed using a surface plasmon resonance measuring apparatus.
(1) Immobilization of trypsin A mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added to the hydrogel-coated chip, and after standing for 20 minutes, 10 mM CaCl ₂ Washed with HBS-N buffer. Next, trypsin (1 mg / ml, 10 mM CaCl ₂ -containing HBS-N buffer) was added, allowed to stand for 30 minutes, and then washed with 10 mM CaCl ₂ -containing HBS-N buffer.

  Furthermore, after adding it to an ethanolamine / HCl solution (1M, pH 8.5) measuring chip, it was washed with HBS-N buffer (Biacore) to block COOH groups remaining without reacting with trypsin.

  By the above operation, trypsin was covalently fixed to the measurement chip surface. The amount of change in resonance signal (RU value) before addition of trypsin and after washing was defined as the amount of trypsin fixation (RU value). The amount of change in resonance signal due to trypsin fixation was 1500 RU.

(2) Detection of a substance interacting with a physiologically active substance After the measurement chip was washed with an HBS-N buffer, it was placed on a surface plasmon resonance measuring apparatus, and a nitrofurantoin solution (Sigma, 0.002 mM, HBS-N95: DMSO 5 Buffer) was added and allowed to stand for 10 minutes, and the amount of change in resonance signal (RU value) was defined as the binding amount of nitrofurantoin to trypsin.

(3) Measurement of biological activity of physiologically active substance The enzyme activity of trypsin was measured using the chip after measuring the binding amount of nitrofurantoin in (2) above. Specifically, a substrate specific for trypsin (benzoyl-L-arginine-4 methylcoumarin-7 amide, hereinafter referred to as Bz-Arg-MCA; Peltide Laboratory Co., Ltd.) was reacted to measure fluorescence of the degradation product.

(4) Reaction of Bz-Arg-MCA HBS-N95: DMSO 5 buffer in the measurement chip is removed, and nitrofrint-in solution (0.002 mM, HBS-N buffer) +0.1 mM Bz-Arg-MCA is added. And allowed to react for 10, 30, 60, 120 minutes.

(5) Fluorescence measurement The fluorescence measurement of each time was performed using the BMGLab Technologies FLUOstar measuring apparatus (Excitation 380 nm, Emission 460 nm).

Example 3 Measurement of Trypsin and Chymostatin Trypsin (manufactured by Worthington) was immobilized on a measurement chip, and interaction measurement was performed using a surface plasmon resonance measurement apparatus.
(1) Immobilization of trypsin A mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added to the hydrogel-coated chip, and after standing for 20 minutes, 10 mM CaCl ₂ Washed with HBS-N buffer. Next, trypsin (1 mg / ml, 10 mM CaCl ₂ -containing HBS-N buffer) was added, allowed to stand for 30 minutes, and then washed with 10 mM CaCl ₂ -containing HBS-N buffer.

  Furthermore, after adding it to an ethanolamine / HCl solution (1M, pH 8.5) measuring chip, it was washed with HBS-N buffer (Biacore) to block COOH groups remaining without reacting with trypsin.

By the above operation, trypsin was covalently fixed to the measurement chip surface. The amount of change in resonance signal (RU value) before addition of trypsin and after washing was defined as the amount of trypsin fixation (RU value).
The amount of change in resonance signal due to trypsin fixation was 1400 RU.

(2) Detection of a substance interacting with a physiologically active substance After the measurement chip is washed with an HBS-N buffer, it is placed on a surface plasmon resonance measuring apparatus, and a chymostatin solution (0.002 mM, HBS-N buffer) is added for 10 minutes. After standing, the amount of change in resonance signal (RU value) was defined as the binding amount of chymostatin (manufactured by ICN) to trypsin.

(3) Measurement of biological activity of physiologically active substance The enzyme activity of trypsin was measured using the chip after measuring the amount of chymostatin bound in (2) above. Specifically, a substrate specific for trypsin (benzoyl-L-arginine-4 methylcoumarin-7 amide, hereinafter referred to as Bz-Arg-MCA; Peltide Laboratory Co., Ltd.) was reacted to measure fluorescence of the degradation product.

(4) Reaction of Bz-Arg-MCA The HBS-N buffer in the measurement chip was removed, and a chymostatin solution (0.002 mM, HBS-N buffer) +0.1 mM Bz-Arg-MCA was added. The reaction was performed for 60,120 minutes.

(5) Fluorescence measurement The fluorescence measurement of each time was performed using the BMGLab Technologies FLUOstar measuring apparatus (Excitation 380 nm, Emission 460 nm).

Example 4 Measurement of Trypsin and Measurement Buffer Trypsin (manufactured by Worthington) was immobilized on a measurement chip, and the interaction measurement was performed using a surface plasmon resonance measurement apparatus.
(1) Immobilization of trypsin A mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added to the hydrogel-coated chip, and the mixture was allowed to stand for 20 minutes and then contained 10 mM CaCl2. Washed with HBS-N buffer. Next, trypsin (1 mg / ml, 10 mM CaCl 2 -containing HBS-N buffer) was added, allowed to stand for 30 minutes, and then washed with 10 mM CaCl 2 -containing HBS-N buffer.

  Furthermore, after adding it to an ethanolamine / HCl solution (1M, pH 8.5) measuring chip, it was washed with HBS-N buffer (Biacore) to block COOH groups remaining without reacting with trypsin.

By the above operation, trypsin was covalently fixed to the measurement chip surface. The amount of change in resonance signal (RU value) before addition of trypsin and after washing was defined as the amount of trypsin fixation (RU value).
The amount of change in resonance signal due to trypsin fixation was 1450 RU.

(2) Detection of a substance interacting with a physiologically active substance After the measurement chip is washed with an HBS-N buffer, it is placed on a surface plasmon resonance measuring apparatus, an HBS-N buffer is added, and after standing for 10 minutes, a resonance signal ( The RU value was defined as the amount of HBS-N buffer binding to trypsin.

(3) Measurement of biological activity of physiologically active substance The enzyme activity of trypsin was measured using the chip after measuring the amount of HBS-N buffer bound in (2) above. Specifically, a substrate specific for trypsin (benzoyl-L-arginine-4 methylcoumarin-7 amide, hereinafter referred to as Bz-Arg-MCA; Peltide Laboratory Co., Ltd.) was reacted to measure fluorescence of the degradation product.

(4) Reaction of Bz-Arg-MCA The HBS-N buffer in the measurement chip was removed, and HBS-N buffer + 0.1 mM Bz-Arg-MCA was added and reacted for 10, 30, 60, 120 minutes. .

(5) Fluorescence measurement The fluorescence measurement of each time was performed using the BMGLab Technologies FLUOstar measuring apparatus (Excitation 380 nm, Emission 460 nm).

  The measurement results are shown in Table 1 below.

  In Table 1, the measurement result of the fluorescence intensity when no substance interacts with trypsin shows the level of enzyme activity when the interacting substance does not act. When leupeptin is used, leupeptin is bound to the enzyme active site of trypsin and the enzyme activity is inhibited. When nitrofurantoin is used, nitrofurantoin is bound to trypsin, but inhibition of the enzyme activity of trypsin is not observed, and therefore nonspecific binding. When chymostatin is used, trypsin enzyme activity inhibition is not observed because it is not bound to trypsin.

  From the results shown in Table 1, the measurement method of the present invention allows two types of measurement, binding and enzymatic reaction, with a small amount of protein. Whether the compound bound by the activity measurement is binding to the active site or not is determined. It was shown that it was possible to judge.

Example 5: Measurement of trypsin and leupeptin (measured with a Biacore 3000 using a PMMA membrane)
Trypsin (manufactured by Worthington) was immobilized on a PMMA measuring chip, and the interaction measurement was performed using a surface plasmon resonance measuring apparatus (Biacore 3000).
(1) Immobilization of trypsin
A PMMA measuring chip was set on a Biacore 3000, a mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysulfosuccinimide (100 mM) was added, reacted for 7 minutes, and then trypsin (0.05 mg / ml, 10 mM CaCl ₂ -containing HBS-N buffer) was added and reacted for 7 minutes.

  Further, an ethanolamine / HCl solution (1 M, pH 8.5) was added and reacted for 7 minutes to block COOH groups remaining without reacting with trypsin.

  Through the above operation, trypsin was covalently fixed to the surface of the PMMA measuring chip. The amount of change in resonance signal (RU value) before addition of trypsin and after washing was defined as the amount of trypsin fixation (RU value). The amount of change in resonance signal due to trypsin fixation was 2000 RU.

(2) Detection of a substance interacting with a physiologically active substance Leupeptin solution (0.002 mM, HBS-N buffer) was added, and after reacting for 10 minutes, the amount of change in resonance signal (RU value) was changed to leupeptin against trypsin (ICN). The amount of binding.

(3) Measurement of biological activity of physiologically active substance The enzyme activity of trypsin was measured using the PMMA chip after measuring the binding amount of leupeptin in (2) above. Specifically, a substrate specific for trypsin (benzoyl-L-arginine-4 methylcoumarin-7 amide, hereinafter referred to as Bz-Arg-MCA; Peltide Laboratory Co., Ltd.) was reacted to measure fluorescence of the degradation product.

(4) Reaction of Bz-Arg-MCA
After the inside of the PMMA measurement chip was washed with HBS-N buffer, leupeptin solution (0.002 mM, HBS-N buffer) +0.1 mM Bz-Arg-MCA was added and allowed to react for 10, 30, 60, 120 minutes.

(5) Fluorescence measurement Fluorescence measurement was performed for each time using a BMGLab Technologies FLUOstar measuring device (Excitation 380 nm, Emission 460 nm).

(6) Measurement results
Results equivalent to the values obtained with the CM5 cup (measurements of the amount of binding interacting with trypsin and fluorescence intensity) were obtained.

FIG. 1 shows a surface plasmon resonance measuring apparatus. FIG. 2 shows a dielectric block.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Measurement unit 11 Dielectric block 12 Metal film 13 Sample holding frame 14 Sensing substance 31 Laser light source 32 Condensing lens 40 Photo detector S40 Output signal 400 Guide rod 401 Slide block 402 Precision screw 403 Pulse motor 404 Motor controller 410 Unit connection body 411 connecting member

Claims (6)

In the measurement method using a biosensor in which a physiologically active substance is immobilized on a substrate, detection or measurement of a substance that interacts with the physiologically active substance and measurement of biological activity of the physiologically active substance are the same as described above. gastric line in a biosensor, and carrying out by surface plasmon resonance analysis to detect or measure a substance which interacts with the physiologically active substance, the measuring method described above. The measurement method according to claim 1, wherein the physiologically active substance is a protein. The measurement method according to claim 1, wherein the physiologically active substance is an enzyme. A dielectric block, a metal film formed on one surface of the dielectric block, a light source that generates a light beam, and the light beam with respect to the dielectric block at an interface between the dielectric block and the metal film An optical system that allows the total reflection condition to be obtained and includes various incident angle components, and a light detection means that detects the surface plasmon resonance state by measuring the intensity of the light beam totally reflected at the interface. A biosensor for use in a surface plasmon resonance measurement apparatus comprising the dielectric block and the metal film, wherein the dielectric block includes an incident surface, an output surface, and the metal of the beam. The surface plasm is formed by using a biosensor in which the metal film is integrated with the dielectric block, which is formed as one block including all of one surface on which the film is formed. Performing down resonance analysis, measuring method according to claim 1. To measure the biological activity of the physiologically active substance by spectrophotometry, measuring method according to any one of claims 1 to 4. (1) A step of bringing a test substance into contact with a biosensor having a physiologically active substance immobilized on a substrate;
(2) detecting or measuring the interaction between a physiologically active substance and a test substance; comprising the step of measuring the biological activity of the physiologically active substance in the presence of and (3) the test substance, claims 1-5 The measuring method in any one of.

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