JP2007132669A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor with a physiologically active substance fixed thereto in large amounts. <P>SOLUTION: In this biosensor comprising a substrate having polysaccharide on its surface capable of chemically fixing thereto the physiologically active substance, the polysaccharide is straight-chain polysaccharide. <P>COPYRIGHT: (C)2007,JPO&INPIT

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 a biosensor that detects or measures a substance that interacts with a physiologically active substance (usually a protein) immobilized on the sensor surface at a high speed, the amount of immobilized physiologically active substance is directly related to the sensitivity of the sensor. It is necessary to fix the physiologically active substance. In the case of surface plasmon resonance, conventionally, carboxymethylated dextran is used on the surface, and the pH is higher than the isoelectric point of the carboxylic acid and lower than the isoelectric point of the physiologically active substance. The physiologically active substance is fixed using the effect of attracting the substance to the surface. However, in the case of surface plasmon resonance, since the binding signal of a substance to a physiologically active substance depends on its molecular weight, it is desired that more physiologically active substance be immobilized in order to measure the binding of low molecular weight compounds. Yes.

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

  This invention made it the subject which should solve the above-mentioned problem to be solved. That is, an object of the present invention is to provide a biosensor with a large amount of physiologically active substance immobilized.

  As a result of intensive studies to solve the above problems, the present inventors have found that the amount of physiologically active substance immobilized can be increased by using a linear polysaccharide as the water-soluble polymer on the biosensor surface. The headline and the present invention were completed.

  That is, according to the present invention, there is provided a biosensor comprising a substrate having a polysaccharide on its surface capable of chemically immobilizing a physiologically active substance, wherein the polysaccharide is a linear polysaccharide. Provided.

  Preferably, the linear polysaccharide is cellulose, pullulan, amylose, agarose, chitin, chitosan, carrageenan, pectin, or derivatives thereof.

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

  Preferably, the biosensor according to the present invention uses a flow path system including cells formed on the substrate, and after the liquid in the flow path system is replaced, the bioactive substance is stopped in a state where the flow of the liquid is stopped. It is used in a method for detecting or measuring a substance that interacts with.

  According to another aspect of the present invention, there is provided a method for producing the biosensor of the present invention as described above, which comprises the step of bringing a linear polysaccharide into contact with a substrate.

  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, a substance that interacts with a physiologically active substance, comprising the step of bringing the biosensor of the present invention in which the physiologically active substance is covalently bonded to the surface with the test substance. A method of detecting or measuring is provided.

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

  In the biosensor of the present invention, a larger amount of physiologically active substance can be immobilized.

Hereinafter, embodiments of the present invention will be described.
The biosensor of the present invention is a biosensor comprising a substrate having a polysaccharide on its surface capable of chemically immobilizing a physiologically active substance, wherein the polysaccharide is a linear polysaccharide. is there. That is, in the biosensor of the present invention, the substrate is coated with a linear polysaccharide.

  The polysaccharide refers to a polymer compound obtained by binding monosaccharides or derivatives thereof via glycosidic bonds. Glycoside binding sites are between the 1,4 and 1,6 positions of monosaccharides (mainly glucose), that is, a bond can extend from one monosaccharide in the three directions of 1,4,6 positions. Here, a polysaccharide in which all monosaccharides other than the terminal are composed of two-way bonds is a linear polysaccharide, and a polysaccharide in which some monosaccharides are branched by three-way bonds is a branched polysaccharide. Call.

  Examples of linear polysaccharides that can be used in the present invention include cellulose, pullulan, amylose, agarose, chitin, chitosan, carrageenan, pectin, and derivatives thereof. Among these, cellulose, pullulan, amylose, agarose, And their derivatives are preferred. Derivatives mentioned here mainly include those in which a part of the hydroxyl group in the unit is alkylated or hydroxyalkylated. Examples thereof include hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and the like.

  The linear polysaccharide as described above may be fixed to the substrate via a self-assembled film or a hydrophobic polymer compound as described below in this specification. That is, in the present invention, a self-assembled film can be formed on a substrate, or a hydrophobic polymer can be coated, and then a linear polysaccharide can be coated thereon. Hereinafter, the self-assembled film and the hydrophobic polymer compound will be described.

  The self-assembled film referred to in the present invention is a monomolecular film or a LB film having a structure with a certain order formed by a mechanism of the film material itself without fine control from the outside. It refers to ultra-thin films. This self-organization forms ordered structures and patterns over long distances in a non-equilibrium situation.

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

  The sulfur-containing compound is preferably represented by X—R—Y.

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

  R (and R ') are optionally interrupted by heteroatoms, preferably straight-chain (unbranched) for adequate packing, optionally containing double and / or triple bonds Is a chain. The chain length is usually 5 atoms or more, preferably 10 atoms or more, and more preferably 10 to 30 atoms. The carbon chain can optionally be perfluorinated. In the case of an asymmetric molecule, R ′ or R may be H.

  Y and Y ″ are groups for binding a hydrophilic compound. Y and Y ″ are preferably the same, and have the property that they can be bonded directly or after activation to a hydrophilic compound (eg, hydrogel). Have. Specifically, hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, epoxy, vinyl group, or the like can be used.

  The compound represented by X—R—Y in the form of a densely packed monolayer can be attached to the metal surface by bonding of the group represented by X to the metal.

  Specific examples of the compound represented by X—R—Y include 10-carboxy-1-decanethiol, 4,4′-dithiodibutylic acid, 11-hydroxy-1-undecanethiol, 11-amino-1- And undecanethiol, 16-hydroxy-1-hexadecathiol, and the like.

  The hydrophobic polymer compound referred to 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 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.

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

  The linear polysaccharide used in the present invention is a polysaccharide capable of chemically immobilizing a physiologically active substance, preferably a polysaccharide having a functional group for binding the physiologically active substance.

Specific examples of functional groups for binding a physiologically active substance include —COOH, —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group), —OH, — SH, —CHO, —NR ³ NR ¹ R ² (wherein R ¹ , R ² and R ³ independently represent a hydrogen atom or a lower alkyl group), —NCO, —NCS, epoxy group, or vinyl group Etc. Here, the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.

  The functional group for binding a physiologically active substance is preferably a carboxyl group, an amino group or a hydroxyl group.

  The functional group for binding the physiologically active substance in the present invention is selected depending on the method for immobilizing the physiologically active substance. That is, even if the functional group is the same (for example, a hydroxyl group, etc.), depending on the method of immobilizing the physiologically active substance, it may fall under the “functional group for binding the physiologically active substance” or not. There is also.

  For example, when the functional group for binding a physiologically active substance is a carboxyl group, a commonly used method is to generate an active ester using a combination of carbodiimide and N-hydroxysuccinimide, and the amino group of the physiologically active substance. A covalent bond can be generated. In this case, a hydroxyl group, an amino group, or a polyether is introduced as a functional group that cannot bind to the physiologically active substance on the surface that does not have a functional group for binding the physiologically active substance.

  In addition, when the functional group for binding the physiologically active substance is an amino group, a commonly used method is to generate a covalent bond with the amino group of the physiologically active substance after acting glutaraldehyde, There is a method in which a substance is oxidized with periodate and directly bonded to an amino group. In this case, a hydroxyl group, a carboxyl group, a polyether, or the like can be introduced as a functional group that cannot bind to the physiologically active substance on the surface that does not have a functional group for binding the physiologically active substance.

  In addition, when the functional group for binding a physiologically active substance is a hydroxyl group, a commonly used method is to form a covalent bond with the amino group of the physiologically active substance after the action of a polyepoxy compound or epichlorohydrin. There is a way to do it. Chemically, there is a direct ether bond forming reaction using alkyl halide, but when applied to a physiologically active substance, it may be difficult to maintain the physiological activity. In this case, on the surface that does not have a functional group for binding a physiologically active substance, reactive functional hydrogen (specifically, a hydroxyl group, an amino group, a carboxyl group, etc.) It is possible to introduce a water-soluble group having no hydrogen) (for example, a polyether such as polyethylene glycol).

  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 biosensor of the present invention is obtained by coating a metal surface or metal film with a hydrophilic 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 0.1 nm to 500 nm, more preferably 0.5 nm to 500 nm, particularly 1 nm to 200 nm. It is preferable that: If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

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

  The metal film is preferably disposed on the substrate. Here, “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not directly in contact with the substrate, but through other layers. This also includes the case where they are arranged. As a substrate that can be used in the present invention, for example, in the case of a surface plasmon resonance biosensor, generally, optical glass such as BK7, or synthetic resin, specifically polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  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. As used herein, “the outermost surface of the substrate” means “the side farthest from the substrate”, and more specifically, “the side farthest from the substrate in the hydrophilic compound coated on the substrate”. is there.

  On the biosensor surface obtained as described above, the physiologically active substance can be immobilized on the metal surface or metal film by covalently bonding the physiologically active substance via the functional group.

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

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

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

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

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

  The biosensor of the present invention includes a dielectric block, a metal film formed on one surface of the dielectric block, a light source that generates a light beam, and the light beam with respect to the dielectric block. An optical system that allows the total reflection condition to be obtained at the interface between the surface and the metal film and includes various incident angle components, and the intensity of the light beam totally reflected at the interface to measure surface plasmon resonance. A measurement chip for use in a surface plasmon resonance measurement apparatus comprising a light detection means for detecting a state is preferable, and is composed of the dielectric block and the metal film, and the dielectric block is formed of the light beam. It is formed as one block including all of the entrance surface, the exit surface, and one surface on which the metal film is formed, and the metal film is integrated with the dielectric block. Formed in the serial measurement chip, it can be used.

  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 biosensor of the present invention uses a flow path system including cells formed on a substrate of the biosensor, and after replacing the liquid in the flow path system, the flow of the liquid is stopped. It can be used in a method for detecting or measuring a substance that interacts with an active substance. For example, a surface plasmon comprising: a channel system including cells formed on a metal film; and a light detection means for detecting the state of surface plasmon resonance by measuring the intensity of a light beam totally reflected on the metal film surface. When measuring the change in surface plasmon resonance by exchanging the liquid in the flow path system using a resonance measuring device, the surface of the liquid flow is stopped after replacing the liquid in the flow path system. Changes in plasmon resonance can be measured.

  The time for stopping the flow of the liquid is not particularly limited, but is, for example, 1 second to 30 minutes, preferably 10 seconds to 20 minutes, and more preferably about 1 minute to 20 minutes.

  Preferably, the liquid in the flow path system is changed from the control liquid not containing the test substance to be measured to the sample liquid containing the test substance to be measured, and then the flow of the sample liquid is stopped. Changes in surface plasmon resonance can be measured.

  Preferably, a reference cell that does not bind a substance that interacts with the test substance and a detection cell that binds a substance that interacts with the test substance are connected in series and installed in the flow path system, and the reference cell By flowing a liquid through the detection cell, the change in surface plasmon resonance can be measured.

In addition, the ratio of the liquid exchange amount (Ve ml) per measurement to the volume (Vs ml) of the cell used for measurement (the total volume of those cells when the reference cell and the detection cell described above are used) ( Ve / Vs) is preferably 1 or more and 100 or less. Ve / Vs is more preferably 1 or more and 50 or less, and particularly preferably 1 or more and 20 or less. Although the volume of the cell used for measurement (Vs ml) is not particularly limited, it is preferably 1 × 10 ^-6 ~1.0ml, particularly preferably about ^{1 × 10 -5 ~1 × 10 -1} ml. Further, the time required for exchanging the liquid is preferably 0.01 seconds or more and 100 seconds or less, and more preferably 0.1 seconds or more and 10 seconds or less.

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

  The following experiment was performed using the apparatus shown in FIG. 22 of Japanese Patent Laid-Open No. 2001-330560 (hereinafter referred to as the surface plasmon resonance measuring apparatus of the present invention) (shown as FIG. 1 in this specification), and FIG. 1 (hereinafter referred to as the dielectric block of the present invention) (shown as FIG. 2 in this specification).

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

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

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

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

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

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

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

  Next, the surface coated with 11-hydroxy-1-undecanthiol was brought into contact with a 10% by mass epichlorohydrin solution (solvent: 1: 1 mixture of 0.4M sodium hydroxide and diethylene glycol dimethyl ether), The reaction was allowed to proceed for 4 hours in a shaking incubator. The surface was washed twice with ethanol and five times with water.

  Next, 4.5 ml of 1M sodium hydroxide was added to 40.5 ml of a 25% by weight polysaccharide aqueous solution of the type listed in Table 1, and the solution was brought into contact with the epichlorohydrin treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. The surface was washed 10 times with 50 ° C. water.

  Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated in a shaking incubator at 35 ° C. for 3 hours. The surface was washed with water and then the above procedure was repeated three times.

(2) Preparation of protein-immobilized chip After removing the solution in the measurement chip, the dielectric block was covered with silicon rubber to prepare a cell having an internal volume of 15 μl. In addition, two 1 mmφ holes were made in the silicon rubber of the lid, and a Teflon (registered trademark) tube having an inner diameter of 0.5 mm and an outer diameter of 1 mm was passed through to create a flow path system. Each chip of this flow path system was installed in the surface plasmon resonance measuring apparatus of the present invention. Hereinafter, the liquid replacement was performed by sending 500 μl of the liquid in 5 seconds.

  The flow path was filled with 70 μl of a mixed solution of 200 mM EDC (N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 50 mM NHS (N-hydroxysuccinimide) and left for 10 minutes. After substituting twice with 100 μl of the buffer described in Table 1 (manufactured by BIAcore), the protein was immobilized by replacing it with an acetate buffer solution of the protein at the concentration and pH described in Table 1, and allowing to stand for 30 minutes. The inside of the chip was replaced with 1M ethanolamine solution and left for 10 minutes. The inside of the chip was replaced twice with 100 μl of the buffer described in Table 1 to prepare a protein-immobilized chip. The amount of immobilization was determined from signal changes before and after protein immobilization. The results are shown in Table 1.

(3) Hydrochlorothiazide binding performance evaluation Each CA fixed chip flow channel system in which a fixed amount of about 3000 RU was obtained was filled with 1 × PBS (pH 7.4) buffer. The signal change with reference to the time before liquid exchange was measured at 0.5 second intervals. The inside of the flow path system was replaced with a Hydrochlorothiazide (manufactured by SIGMA) solution (dissolved in 1 × PBS (pH 7.4) to 50 μM). The signal change 2 minutes after the replacement was calculated from 5 seconds before the start of the replacement.

  From the results in Table 1, it can be seen that more proteins can be immobilized in the biosensor of the present invention using a linear polysaccharide when proteins are immobilized. It can also be seen from levels 1 and 5 that a higher signal can be obtained for the binding of the physiologically active substance.

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

Explanation of symbols

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

Claims (10)

A biosensor comprising a substrate having on its surface a polysaccharide capable of chemically immobilizing a physiologically active substance, wherein the polysaccharide is a linear polysaccharide. The biosensor according to claim 1, wherein the linear polysaccharide is cellulose, pullulan, amylose, agarose, chitin, chitosan, carrageenan, pectin, or a derivative thereof. The biosensor according to claim 1 or 2, which is used for non-electrochemical detection. The biosensor according to any one of claims 1 to 3, which is used for surface plasmon resonance analysis. A flow path system including cells formed on the substrate is used to detect or measure a substance that interacts with the physiologically active substance in a state where the flow of the liquid is stopped after the liquid in the flow path system is replaced. The biosensor according to any one of claims 1 to 4, which is used in a method. The method for producing a biosensor according to claim 1, comprising a step of bringing a linear polysaccharide into contact with a substrate. The biosensor according to any one of claims 1 to 5, wherein the physiologically active substance is bound to the surface by a covalent bond. 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 5 and a test substance into contact with each other, wherein the bioactive substance is covalently bonded to the surface. how to. The method according to claim 8, wherein a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method. The method according to claim 8 or 9, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.

Images