JP2006090948A - Screening method using biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method carrying out screening materials to be examined by detecting or measuring a material which interacts with a physiologically active substance immobilized on a biosensor, and by analyzing the influence of the material on the biological activity of the physiologically active substance. <P>SOLUTION: The method for screening the materials to be examined comprises a step (1) of bringing the materials to be examined to contact with the biosensor, on which the physiologically active substance is immobilized and detecting the material to be examined which interacts with the physiologically active substance, by using a surface plasmon resonance analysis or a local plasmon resonance analysis; and a step (2) of measuring the biological activity of the physiologically active substance under the presence of the material to be examined. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a screening method using a biosensor. More specifically, the present invention is characterized in that both 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 are performed. The present invention relates to a test substance screening method.

  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.

  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 detects or measures a substance that interacts with a physiologically active substance immobilized on a biosensor, and analyzes the influence of the substance on the biological activity of the physiologically active substance, thereby analyzing the test substance. Providing a screening method was an issue to be solved.

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

That is, according to the present invention, (1) by bringing a test substance into contact with a biosensor in which a physiologically active substance is immobilized on a substrate, the test substance that interacts with the physiologically active substance is subjected to surface plasmon resonance analysis or local plasmon resonance. A step of detecting by analysis; and (2) a step of measuring the biological activity of the physiologically active substance in the presence of the test substance:
A method for screening a test substance is provided.

Preferably, 1 to 10 molecules of a test substance binds to one molecule of a physiologically active substance.
Preferably, detection of a plurality of test substances by surface plasmon resonance analysis or local plasmon resonance analysis is performed by parallel detection.
Preferably, immobilization of the physiologically active substance on the biosensor and detection of a test substance that interacts with the physiologically active substance are performed at different positions.

  Preferably, the test substance that interacts with the physiologically active substance is detected by surface plasmon resonance analysis or local plasmon resonance analysis based on any of the binding amount, the binding constant, the binding rate constant, or the dissociation rate constant.

  Preferably, the biological activity of the physiologically active substance is any of receptor activity, enzyme activity, antibody activity, metabolic activity, membrane potential, ion release, or gene expression.

  Preferably, before the step (1), a virtual screening is further performed on the test substance to reduce the number of test substances to be subjected to the screening.

  Preferably, screening is automatically performed using a screening apparatus in which a reference value is input in advance.

  According to the screening method of the present invention, it is possible to measure two types of binding and enzymatic reaction with a small amount of protein. Further, according to the screening 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.

Hereinafter, embodiments of the present invention will be described.
The test substance screening method according to the present invention comprises: (1) surface plasmon resonance analysis or local plasmon resonance analysis of a test substance that interacts with the bioactive substance by bringing the test substance into contact with a biosensor in which the bioactive substance is immobilized on a substrate. Detecting by plasmon resonance analysis; and (2) measuring the biological activity of the physiologically active substance in the presence of the test substance. In the present invention, by performing both detection or measurement of a substance that interacts with a physiologically active substance and measurement of the biological activity of the physiologically active substance, for example, a test substance bound to the physiologically active substance becomes a physiologically active substance. It is possible to determine whether or not the active substance has bound to the active site, and the influence of the test substance on the biological activity of the bioactive substance can be analyzed. Note that local plasmons (local surface plasmons) are plasmons that exist at the tips of metal fine particles and metal needles. The localized region is about the extent of the fine particles and the tip of the needle, and can be made sufficiently smaller than the wavelength of light. Since the frequency of local plasmons strongly depends on the shape of fine particles and the type of metal atom, the scattering spectrum changes for each fine particle. A physiologically active substance (protein or the like) is immobilized on the surface of the fine particles, and a test substance that binds to the physiologically active substance can be analyzed by changing the scattering spectrum.

  In the present invention, the physiologically active substance is immobilized. As a solid phase immobilization method, a known method is used. Moreover, it is preferable that the unit for immobilizing a physiologically active substance such as protein is separated from the unit for measuring the interaction between the immobilized physiologically active substance and the test substance. An example of an apparatus configuration for performing the screening of the present invention is shown in FIG. By separating the unit for immobilizing the protein and the unit for measuring the interaction, the measurement processing capability is greatly improved. As shown in FIG. 1, in the present invention, preferably, detection of a plurality of test substances by surface plasmon resonance analysis or local plasmon resonance analysis can be performed by parallel detection.

  The number of test substances subjected to screening in the present invention is preferably 1000 or more, more preferably 10,000 or more, and further preferably 100,000 or more.

  The test substance used in the present invention is not particularly limited as long as it is a substance that interacts with a physiologically active substance (ligand) as described below in the present specification, but a low molecular organic compound, a high molecular compound, DNA, etc. And nucleic acids, proteins (including antibodies), peptides or fragments thereof, sugars, amino acids and the like.

  In a preferred embodiment of the present invention, screening can be automatically performed using a screening apparatus in which a reference value is input in advance. That is, a reference value in a range to be selected in advance in the screening device (for example, in screening of a test substance that interacts with a physiologically active substance, a binding amount, a binding constant, a binding rate constant, a dissociation rate constant, etc .; In biological activity, receptor activity, enzyme activity, metabolic activity, membrane potential, ion release, gene expression, etc. are entered, and test substances that fall within that range are automatically selected. Is preferred. The selected test substance is preferably displayed on the display device so as to be visually distinguished. The visual distinction method is not particularly limited, and examples thereof include a method of character size, color, character blinking, character color, underline, and framed.

  In the present invention, a first step of selecting a test substance that interacts with a physiologically active substance, and a second step of measuring the biological activity of the physiologically active substance in the presence of the test substance selected in the first step Screening according to the process is performed. In the first step, it is preferable that test substances that fall within the selection range of the reference value input in advance in the apparatus are automatically selected, and the measurement in the second step is automatically performed on the selected test substances. Furthermore, in the second step, a reference value (receptor activity amount, enzyme activity amount, metabolic activity amount, membrane potential, ion release amount, gene expression amount, etc.) reflecting the biological activity previously input to the apparatus. It is preferable that test substances that fall within the selection range are automatically selected.

  Furthermore, in the present invention, virtual screening can be further performed on the test substance before the first step described above, and the number of test substances to be subjected to screening can be reduced.

  The biosensor 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 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, when considering use for 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 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 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 the liquid not containing the 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 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 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.

  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, 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, 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 test substance that interacts with the physiologically active substance.

  In the present invention, preferably, 1 to 10 molecules of a test substance can bind to one molecule of a 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, an apparatus using a system called Kretschmann arrangement can be cited (for example, Japanese Patent Laid-Open No. 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 substance to be measured 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 light energy is transferred to the surface plasmon. 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 amount of change in angle. If the above-described arrayed light detecting means and differentiating means are applied to a measuring apparatus 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.

  Furthermore, in the measurement method of the present invention, a substance that interacts with a physiologically active substance is detected or measured by a technique such as surface plasmon resonance analysis as described above, and the biological activity of the physiologically active substance is measured. .

  The biological activity referred to in the present invention includes enzyme activity, antibody activity, receptor activity, metabolic activity, membrane potential, ion release, gene expression, 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 measurement of metabolic activity, membrane potential, ion release, and gene expression can all be performed by known methods.

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

Example 1 Selection of Compounds by Affinity- Using the SPR system for screening shown in FIG. 1, ligand fixation, compound binding measurement, and compound selection were performed.
(1) Manufacture of sensor chip 1cm (1cm cover glass with gold deposited so that the thickness of the gold film is 500 angstroms is treated with Model-208UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, then spin It was set on a coater (MODEL ASS-303, manufactured by ABLE) and rotated at 1000 rpm, and 50 μl of a methyl ethyl ketone solution (2 mg / ml) of polymethyl methacrylate was dropped on the center of the gold vapor deposition cover glass, and the rotation was stopped after 2 minutes. When the film thickness was measured by ellipsometry (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab), the thickness of the polymethyl methacrylate film was 200 angstrom.This sample is called PMMA surface chip. The surface chip was immersed in an aqueous NaOH solution (1 N) at 40 ° C. for 16 hours and then washed with water three times.This sample is called a PMMA / COOH surface chip. An aqueous solution of α-amino-polyethyleneoxy-ω-carboxylic acid (average molecular weight 5000) is immersed in 2 ml of a mixed solution of til-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) for 60 minutes. (10 mM) It was immersed in 2 ml for 16 hours, and finally washed with water 5 times.

(2) Immobilization of trypsin A mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added to the sensor chip prepared in (1) above, and the mixture was allowed to stand for 20 minutes. Then, the plate was washed with HBS-N buffer containing 10 mM CaCl2. 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, the ethanolamine / HCl solution (1M, pH 8.5) was added to a measurement chip, and then washed with an HBS-N buffer (Biacore).
Through the above operation, trypsin was covalently fixed to the surface of the sensor chip.

(2) Detection of Compound Binding After washing the trypsin-fixed sensor chip with HBS-N buffer, the leupeptin solution (2 μM), the nitrofurantoin solution (2 μM), and the chymostatin solution (2 μM) were added to the SPR leader part, respectively. After standing for 10 minutes, the amount of change in resonance signal (RU value) was defined as the amount of each compound bound to trypsin.

(3) Selection of compounds As shown in Table 1, leupeptin and nitrofurantoin in which binding was observed were selected.

Example 2: Selection of compound by biological activity Using the amount of sensor chip bound to trypsin, the enzyme activity of trypsin was measured in the presence of the above compound. 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.

(1) Reaction of Bz-Arg-MCA Using the sensor chip binding amount to which trypsin prepared in (2) of Example 1 was immobilized, leupeptin solution (4 μM) and 0.2 mM Bz-Arg-MCA were respectively 1: One mixed solution, a mixed solution of nitrofurantoin solution (4 μM) +0.2 mM Bz-Arg-MCA, and 0.1 mM Bz-Arg-MCA solution were added and reacted for 10, 30, 60, 120 minutes.

(2) Fluorescence measurement The fluorescence measurement of each time was performed using the BMGLab Technologies FLUOstar measuring apparatus (Excitation380nm, Emission460nm).

(3) Selection of compounds As shown in Table 1, leupeptin that inhibited the enzyme activity of trypsin was selected.

FIG. 1 shows an example of an SPR system for performing the screening method of the present invention.

Claims (8)

(1) a step of detecting a test substance that interacts with the bioactive substance by surface plasmon resonance analysis or local plasmon resonance analysis by bringing the test substance into contact with a biosensor in which the bioactive substance is immobilized on a substrate; 2) A step of measuring the biological activity of the physiologically active substance in the presence of the test substance:
A method for screening a test substance. The screening method according to claim 1, wherein 1 to 10 molecules of a test substance binds to 1 molecule of a physiologically active substance. The screening method according to claim 1 or 2, wherein detection of a plurality of test substances by surface plasmon resonance analysis or local plasmon resonance analysis is performed by parallel detection. The screening method according to any one of claims 1 to 3, wherein immobilization of a physiologically active substance on a biosensor and detection of a test substance that interacts with the physiologically active substance are performed at different positions. The test substance that interacts with the physiologically active substance is detected by surface plasmon resonance analysis or local plasmon resonance analysis based on any of the binding amount, the binding constant, the binding rate constant, or the dissociation rate constant. The screening method according to claim 1. The biological activity of the physiologically active substance is any one of receptor activity, enzyme activity, antibody activity, metabolic activity, membrane potential, ion release, or gene expression. A screening method according to 1. The screening method according to any one of claims 1 to 6, wherein a virtual screening is further performed on the test substance before the step (1) to reduce the number of test substances to be subjected to the screening. The screening method according to claim 1, wherein screening is automatically performed using a screening apparatus in which a reference value is input in advance.

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