JP2005283143A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection surface for a biosensor for stably immobilizing any physiologically active substances stably, namely by suppressing a decrease in the original activity. <P>SOLUTION: The biosensor is made of a substrate that is coated with a hydrophobic highly polymerized compound while a hydrophilic compound is combined with the surface, where there are 2-1000 groups combined with the physiologically active substances in one molecule of the hydrophilic compound. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

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

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

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

  As a 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).

In the above chip, the surface activity of the physiologically active substance may cause a decrease in the original activity, and only the physiologically active substance that is strong in immobilization has been used as a test material. In addition, it has been pointed out that the physiologically active substances are deactivated due to the influence of excess carboxylic acid on the surface. Among them, a surface capable of stably immobilizing any physiologically active substance was required in order to examine the correspondence with enzyme activity and the like.

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, an object of the present invention is to provide a detection surface for a biosensor that can be immobilized stably with any physiologically active substance, that is, while suppressing a decrease in the original activity.

  As a result of intensive studies to solve the above problems, the present inventors have found that a physiologically active substance can be stabilized stably (that is, active by binding a hydrophilic compound to the surface of a substrate coated with a hydrophobic polymer compound). The inventors have found that they can be immobilized (while suppressing the decrease) and have completed the present invention.

  That is, according to the present invention, the substrate consists of a substrate coated with a hydrophobic polymer compound, and a hydrophilic compound is bonded to the surface of the substrate. 1000 biosensors are provided.

Preferably, the molecular weight of the hydrophilic compound is 50 or more and 20000 or less.
Preferably, the hydrophilic compound is gelatin, alginic acid, chitosan, dextran, polyvinyl alcohol, polyethylene glycol or a derivative thereof, carrageenan, agarose, polyacrylic acid, or polyacrylamide.

Preferably, the substrate is a substrate made of a metal surface or a metal film.
Preferably, the metal surface or the metal film is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum, and aluminum.
Preferably, the coating thickness of the hydrophobic polymer compound is 1 angstrom or more and 5000 angstrom or less.

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

  According to another aspect of the present invention, the step of coating a substrate with a hydrophobic polymer compound, and the hydrophobic polymer compound coated on the substrate have 2 to 2 groups bonded to a physiologically active substance in one molecule. There is provided a method for producing the biosensor of the present invention as described above, which comprises a step of binding 100 hydrophilic compounds.

According to still another aspect of the present invention, there is provided the biosensor of the present invention as described above, wherein a physiologically active substance is covalently bonded to the surface.
According to still another aspect of the present invention, the biosensor includes a step of bringing the biosensor of the present invention into contact with a physiologically active substance and covalently bonding the physiologically active substance to the surface of the biosensor. A method for immobilizing a physiologically active substance is provided.

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

  According to the present invention, it is possible to provide a detection surface for a biosensor that can be immobilized stably with any physiologically active substance, that is, while suppressing a decrease in the original activity.

Embodiments of the present invention will be described below.
The biosensor of the present invention comprises a substrate coated with a hydrophobic polymer compound and having a hydrophilic compound bonded to the surface thereof, and a group that binds to a physiologically active substance in the molecule of the hydrophilic compound is 2 to 1000. It is characterized by the existence of a piece.

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

  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 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, the values specified by Fedors were used (R. F. Fedors, Polym. Eng. Sci., 14 (2), P147, P472 (1974)).
δ = (ΣEcoh / ΣV) ^1/2
By way of example, the SP values of hydrophobic polymer compounds and solvents are as follows: Polymethylmethacrylate-polystyrene copolymer (1: 1): for 21.0 Solvent 2-phenoxyethanol: 25.3, for Polymethylmethacrylate: 20.3 Solvent acetonitrile: 22.9, polystyrene: 21.6 with respect to polystyrene: 21.6.

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

  The coating thickness of the hydrophobic polymer compound is not particularly limited, but is preferably 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 3000 angstrom or less.

  The biosensor of the present invention is preferably a metal surface or metal film coated with a hydrophobic polymer compound. 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 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.

The hydrophilic compound used in the present invention will be described.
The term “hydrophilic” as used herein refers to a compound that is soluble in 5 g or more in 100 g of water. Examples thereof include gelatin, alginic acid, chitosan, dextran, polyvinyl alcohol, polyethylene glycol, carrageenan, agarose, polyacrylic acid, polyacrylamide and the like. A polyethylene glycol derivative is preferably used.

  The hydrophilic compound used in the present invention is preferably a compound having a molecular weight of 50 to 30,000. Further, a compound having a molecular weight of 100 to 1500 is more preferable.

  The hydrophilic compound used in the present invention has 2 or more and 1000 or less groups that immobilize physiologically active substances, preferably 2 or more and 100 or less groups that immobilize physiologically active substances per molecule. ing. In addition to the binding group, it preferably has a group that reacts with the biosensor surface. However, a group that binds to one physiologically active substance after binding to the sensor surface may be introduced.

The group capable of reacting with the biosensor surface is preferably a halogen atom, an amino group, or an amino group protected by a protective group, a carboxyl group, or a carbonyl group having a leaving group, a hydroxyl group, or a protective group. A hydroxyl group, an aldehyde group, —NHNH ₂ , —N═C═O, —N═C═S, an 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. Nylmethyloxycarbonyl group (Fmoc), nitrophenylsulfenyl group (Nps), dithiasuccinyl group (Dts) and the like can be mentioned.

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 Examples include groups.

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.

The group that immobilizes the physiologically active substance represents a group that can immobilize the physiologically active substance or a group that can bind to the compound that binds to the physiologically active substance, and preferably protected by a halogen atom, an amino group, or a protecting group amino group, carboxyl group or carbonyl group having a leaving group, a hydroxyl group, hydroxyl group protected by a protecting group, an aldehyde _{group, -NHNH 2, -N = C =} O, -N = C = S, an epoxy group, or Vinyl group. As the protecting group and the leaving group, the same as those described above can be used.

  Specific examples of the hydrophilic compound that can be used in the present invention are shown below, but are not limited thereto.

  The biosensor of 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 bioactive substance is covalently bonded to the metal surface or metal film through a functional group capable of immobilizing the bioactive substance on the biosensor surface obtained as described above. Can be immobilized.

  The physiologically active substance immobilized on the biosensor surface of the present invention is not particularly limited as long as it interacts with the measurement target, and examples thereof include immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-molecular compounds, and the like. Examples include 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.

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

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

The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.
As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

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

  When the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme, the immobilization can be performed by covalently bonding to a functional group on the metal surface using the amino group, thiol group or the like of the physiologically active substance. .

  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.

That is, according to the present invention, the biosensor of the present invention on which a physiologically active substance is immobilized is brought into contact with a test substance to thereby interact with the physiologically active substance immobilized on the biosensor. A method of detecting and / or measuring a substance to be provided is provided.
As the test substance, for example, a sample containing a substance that interacts with the above physiologically active substance can be used.

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

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

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

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

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

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

  In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the measured substance in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon. 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 the total reflection attenuation angle (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

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

When the biosensor of the present invention is used for surface plasmon resonance analysis, it can be applied as a part of various surface plasmon measurement devices as described above.
The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1: Production of biosensor chip (1) Production of biosensor chip coated with polymethylmethacrylate A 1 cm (1 cm cover glass) with gold deposited so that the thickness of the gold film is 500 angstroms After processing with 208UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, set it on a spin coater (MODEL ASS-303, manufactured by ABLE) and rotate it at 1000rpm. (2 mg / ml) was dropped 50 μl, and the rotation was stopped after 2 minutes.The film thickness was measured by ellipsometry (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab). Was 200 angstroms, this sample is called PMMA surface chip.

(2) 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 three times with water. This sample is called a PMMA / COOH surface chip.

(3) Creation of surface with linker-1
After immersing the PMMA / COOH surface chip prepared in (2) above in 2 ml of a mixture of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) for 60 minutes, an aqueous ethylenediamine solution (10 mM) It was immersed in 2 ml for 16 hours. Finally, it was washed 5 times with water. This sample is called a PMMA / Eda surface chip.

(4) Creation of surface with linker-2
After immersing the PMMA / COOH surface chip prepared in (2) above in 2 ml of a mixture of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) for 60 minutes, the following structure Six-Arm Poly (ethylene oxide) Amino Terminated (average molecular weight: 12000; manufactured by Polymer Source) solution (10 mM) having 16 ml was immersed for 16 hours. Finally, it was washed 5 times with water. This sample is called a PMMA / PEO-A6 surface chip.

(5) Creation of surface with linker-3
After immersing the PMMA / COOH surface chip prepared in (2) above in 2 ml of a mixture of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) for 60 minutes, the following structure It was immersed in 2 ml of a solution (10 mM) of Four-Arm Poly (ethylene oxide) Amino Terminated (average molecular weight 10,000; manufactured by Polymer Source) having 16 hours. Finally, it was washed 5 times with water. This sample is called a PMMA / PEO-A4 surface chip.

Example 2: Performance evaluation of biosensor chip:
(1) Immobilization of protein Each chip of samples 1-1 to 1-6 is placed on a cartridge block of a commercially available surface plasmon resonance biosensor (Biacore 3000, manufactured by Biacore), and trypsin (manufactured by Nacalai Tesque) is immobilized. Went. The fixed amount was used as a factor in the subsequent comparison of enzyme activity.
Sample 1-1: PMMA / COOH surface chip (prepared by method (2) of Example 1)
Sample 1-2: PMMA / Eda surface chip (prepared by method (3) of Example 1)
Sample 1-3: PMMA / PEO-A6 surface chip (prepared by method (4) of Example 1)
Sample 1-4: PMMA / PEO-A4 surface chip (prepared by method (5) of Example 1)

The fixing method was as follows.
150 μL of an aqueous solution (500 mM) of Compound A was passed through the measurement cell at a flow rate of 10 μL / min. Thereafter, 150 μL of trypsin (manufactured by Nacalai Tesque) 0.1 mg / ml solution (acetic acid buffer pH 5.5) was passed through the measurement cell at a flow rate of 10 μL / min. Thereafter, 150 μL of ethanolamine / HCl solution (1 M, pH 8.5) was passed through the measurement cell at a flow rate of 10 μL / min. Thereafter, in order to remove trypsin that was not covalently bonded, 10 μL of 10 mM NaOH aqueous solution was flowed twice through the measurement cell at a flow rate of 10 μL / min and washed. Before and after each injection, HBS-EP buffer (Biacore, pH 7.4) (HEPES 0.01 mol / l (pH 7.4); NaCl 0.15 mol / l; EDTA 0.003 mol / l; Surfactant P20 0.005 wt%) At a flow rate of 10 μL / min.

Compound _{A: CH 2 = CHSO 2 -CH} 2 -CONH-CH 2 CH 2 -NHCO-CH 2 SO 2 -CH = CH 2

  The enzyme activity value described later was calculated by setting the value before injection of the chemical to 0 and the value after washing with NaOH solution as the fixed amount of trypsin.

(2) Measurement of enzyme activity The following measurement was performed using the chip fixed in (1) as it was.
The chip surface was washed on the third floor with 0.05 M Tris HCl (pH 8.0, 0.02 M CaCl 2), and then 100 μl of a 0.1 mM solution of Bz-Arg-MCA (Peptide Institute, Inc.) was placed on the chip. After 120 minutes, the entire amount was transferred onto a measurement plate, and fluorescence measurement was performed at a detection wavelength of 460 nm (excitation wavelength of 380 nm) using FLUOstar (BMG Labtechnologies GmbH). The value obtained by dividing the measured value by the trypsin fixation amount (RU) was defined as the enzyme activity value.

(3) Results Table 1 shows the results of fixed amounts of trypsin and enzyme activity values.

  From the results shown in Table 1, it can be seen that the structure of the present invention can provide a surface having a large enzyme activity value.

Claims (14)

A biosensor comprising a substrate coated with a hydrophobic polymer compound and having a hydrophilic compound bonded to the surface thereof, wherein 2 to 1000 groups that bind to a physiologically active substance are present in one molecule of the hydrophilic compound. The biosensor according to claim 1, wherein the hydrophilic compound has a molecular weight of 50 or more and 20000 or less. The biosensor according to claim 1 or 2, wherein the hydrophilic compound is gelatin, alginic acid, chitosan, dextran, polyvinyl alcohol, polyethylene glycol or a derivative thereof, carrageenan, agarose, polyacrylic acid, or polyacrylamide. The biosensor according to any one of claims 1 to 3, wherein the substrate is a substrate made of a metal surface or a metal film. The biosensor according to claim 4, wherein the metal surface or metal film is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum, and aluminum. The biosensor according to any one of claims 1 to 5, wherein the coating thickness of the hydrophobic polymer compound is 1 angstrom or more and 5000 angstrom or less. The biosensor according to any one of claims 1 to 6, which is used for non-electrochemical detection. The biosensor according to any one of claims 1 to 7, which is used for surface plasmon resonance analysis. A step of coating a substrate with a hydrophobic polymer compound, and a hydrophilic compound having 2 to 100 groups that bind to a physiologically active substance in one molecule is bonded to the hydrophobic polymer compound coated on the substrate. The manufacturing method of the biosensor in any one of Claim 1 to 8 including a process. The biosensor according to any one of claims 1 to 8, wherein the physiologically active substance is bound to the surface by a covalent bond. A biosensor comprising the biosensor according to claim 1, comprising the step of bringing the biosensor and the bioactive substance into contact with each other and covalently binding the bioactive substance to the surface of the biosensor. How to immobilize. Detecting or measuring a substance that interacts with the bioactive substance, comprising the step of bringing the biosensor according to any one of claims 1 to 8 into contact with a test substance, wherein the bioactive substance is covalently bonded to the surface how to. The method according to claim 12, wherein a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method. The method according to claim 12 or 13, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.