JP2005195488A - Method for manufacturing solid substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a solid substrate for inhibiting a nonspecific adsorption, in particular a method for manufacturing a sensor substrate used for a surface plasmon resonance analysis and controlling the nonspecific adsorption. <P>SOLUTION: The method for manufacturing the sensor solid substrate includes a process for bringing the solid substrate into contact with a mixed liquid of two or more organic solvents not containing a polymer after the solid substrate is brought into contact with the hydrophobic polymer solution. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for manufacturing a solid substrate. More particularly, the present invention relates to a method for manufacturing a solid substrate for a sensor.

  At present, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations. 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.

  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 measuring chip consists of a transparent substrate (for example, glass), a deposited metal film, a thin film that is inactive to a biological substance, and a functional group that can immobilize a physiologically active substance. The physiologically active substance is immobilized on the metal surface via the group. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the biological substance.

  In the above technique, a bioactive substance is detected by immobilizing a bioactive substance on the surface of a substrate or particle and causing a specific recognition reaction between the bioactive substance and the specimen substance. On the other hand, the sample substance is not necessarily a single component, and it is also required to measure the sample substance in a heterogeneous system such as in a cell extract. In that case, when various kinds of contaminants such as proteins and lipids cause nonspecific adsorption on the detection surface, the measurement and detection sensitivity is remarkably lowered. The above detection surface has a problem that non-specific adsorption is very likely to occur.

  In order to suppress nonspecific adsorption to the detection surface of the substrate, several methods have been studied. For example, a method of forming a film of self-assembled molecules on a metal surface and suppressing physical adsorption has been used (see Patent Document 1). However, this method has problems of pinhole generation in the film and desorption of self-assembled molecules.

  The non-specific adsorption can also be suppressed by forming a hydrophobic polymer thin film that is inert to the biological substance on the surface of the sensor substrate. As a method for forming a hydrophobic polymer thin film on a sensor substrate, conventionally, a method of applying a polymer solution on a substrate by spin coating, air knife coating, cast coating spray coating, etc., and drying and removing the solvent has been performed. There was a problem that pinholes and thickness non-uniformity were likely to occur in the thin film during drying. In addition, there is a restriction that the surface of the substrate is flat so as not to cause uneven coating. Although a metal film and a plasma polymerized film formed on the metal film have been reported, since the monomer raw material is applied, the same problem as described above remains (see Patent Document 2). Although a method of depositing a monomer raw material and polymerizing it on a substrate has also been reported, there is a problem that the type of monomer is limited (see Patent Document 3).

  There has been reported a method of adsorbing a hydrophobic polymer from a single solvent solution on the QCM sensor surface using the immersion adsorption method (see Non-Patent Document 1). However, with a single solvent, it is difficult to search for a solvent that achieves both dissolution of the polymer in the solution and adsorption onto the sensor surface, and it is difficult to adapt to various hydrophobic polymers. Furthermore, no method has been reported to date to further modify this surface and analyze interactions between biomolecules.

JP-A-8-193948 JP-A-9-264843 JP 2003-221974 A Langmuir 2001, 17, 5513-5519.

  The present invention has been made to solve the above-described problems of the prior art. That is, the present invention has a problem to be solved by providing a method for producing a solid substrate in which non-specific adsorption is suppressed, particularly a method for producing a sensor substrate for use in surface plasmon resonance analysis in which non-specific adsorption is controlled. did.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made a process in which a solid substrate is brought into contact with a hydrophobic polymer solution and then brought into contact with a mixture of two or more organic solvents not containing the polymer. The present invention has been completed by discovering that a hydrophobic substrate can be provided by adsorbing various hydrophobic polymers to the surface of the solid substrate by the surface forming method including the above.

That is, according to the present invention, there is provided a method for producing a solid substrate for a sensor, comprising the step of contacting a solid substrate with a hydrophobic polymer solution and then contacting with a mixed solution of two or more organic solvents not containing the polymer. Is done.
Preferably, there is provided a method for producing a solid substrate for a sensor, further comprising a step of surface modification of the obtained solid substrate.

Preferably, the mixed solution of two or more organic solvents not containing the polymer is a mixed solution containing a good solvent and a poor solvent for the polymer.
Preferably, the liquid mixture of two or more organic solvents not containing the polymer has a lower liquid temperature that does not generate a hydrophobic polymer precipitate when the liquid mixture has the same hydrophobic polymer concentration as the hydrophobic polymer solution. On the other hand, it is used at a liquid temperature higher by 1 ° C. or more and 50 ° C. or less.
More preferably, the solvent of the hydrophobic polymer solution and the solvent of the liquid not containing the polymer are the same solvent. Preferably, the surface modification is a surface modification that introduces a functional group capable of generating a covalent bond. Preferably, the solid substrate contacted with the hydrophobic polymer solution is a solid substrate coated with a metal surface or metal film. Preferably, the sensor solid substrate is a substrate used for surface plasmon resonance analysis.

  According to another aspect of the present invention, the method includes the steps of producing a sensor solid substrate by the above-described method of the present invention, and contacting and immobilizing a physiologically active substance on the surface of the obtained sensor solid substrate. A method for producing a solid substrate for a sensor in which a physiologically active substance is bound to a surface is provided.

  According to still another aspect of the present invention, a step of producing a sensor solid substrate by the above-described method of the present invention, a step of contacting and immobilizing a physiologically active substance on the surface of the obtained sensor solid substrate, and Provided is a method for detecting or measuring a substance that interacts with a physiologically active substance, which comprises the step of bringing a solid substrate for a sensor having the obtained physiologically active substance bonded to the surface into contact with a test substance.

  The method for producing a solid substrate of the present invention makes it possible to provide a sensor substrate in which various hydrophobic polymers are adsorbed on the surface of the solid substrate and non-specific adsorption is suppressed. Further, it has become possible to provide a sensor substrate that suppresses non-specific adsorption regardless of the planar shape of the solid substrate.

Embodiments of the present invention will be described below.
The method for producing a solid substrate for a sensor according to the present invention is characterized in that a surface is formed by bringing a solid substrate into contact with a hydrophobic polymer solution and then contacting with a mixed solution of two or more organic solvents not containing the polymer. And

  The hydrophobic polymer used in the present invention is substantially insoluble in water, and specifically has a solubility in water of less than 0.1%. The hydrophobic polymer of the present invention preferably contains 30% by weight or more and 100% by weight or less of a monomer whose solubility in water at 25 ° C. is 0% by weight or more and 20% by weight or less.

  Hydrophobic monomers that form hydrophobic polymers include vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic diesters, maleic diesters, and fumaric diesters. , Allyl compounds, vinyl ethers, vinyl ketones and the like. The hydrophobic polymer may be a homopolymer composed of one kind of monomer or a copolymer composed of two or more kinds of monomers.

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

  The polymer dissolving solvent used in the present invention is not particularly limited, and any solvent can be used as long as it dissolves a part of the hydrophobic polymer. 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 hydrophobic polymer solution to be brought into contact with the solid substrate may be a polymer completely dissolved or a suspension containing a polymer insoluble component, but it is preferable that the polymer is completely dissolved. The liquid temperature is not particularly limited as long as a part of the hydrophobic polymer is dissolved, but is preferably higher than the liquid temperature at which the polymer generates a precipitate. The liquid temperature may be varied while the substrate is in contact with the hydrophobic polymer solution. Although there is no restriction | limiting in particular in the polymer 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 contacting the solid substrate with the hydrophobic polymer solution 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.

  In the method of the present invention, after the solid substrate is brought into contact with the hydrophobic polymer solution, the solid substrate is brought into contact with a mixed solution of two or more organic solvents not containing the polymer. The mixed solution of two or more organic solvents not containing a polymer in the present invention means an organic solvent not containing a polymer. A mixed solution of a good solvent and a poor solvent of the polymer is preferable. The preferable liquid temperature of the solvent containing no polymer is a liquid temperature higher by 1 ° C. or more and 50 ° C. or less than the lower limit liquid temperature at which the polymer aggregate is not generated. Further preferably, the solvent of the hydrophobic polymer solution and the mixed solution of two or more organic solvents not containing the polymer have the same solvent composition.

The good solvent referred to in the present invention is a solvent having a polymer solubility of 0.1% or more, and the poor solvent referred to in the present invention refers to a solvent that does not substantially dissolve the polymer. For example, when polymethyl methacrylate is used as a polymer, acetone, acetonitrile, benzene, 2-butanone, tetrahydrofuran, acetic acid, ethyl acetate, chloroform, chlorobenzene, methylene chloride, cyclohexanone, dioxane, 2-ethoxyethanol are used as good solvents. , Etc. Examples of the poor solvent include cyclohexane, dimethyl ether, ethylene glycol, formamide, hexane, methanol, ethanol, carbon tetrachloride, cresol, naphthalene, and the like. Examples of good and poor solvents for hydrophobic polymers are J. Brandrup, E.I. H. Immergut, E .; A. Grulk edition "POLYMER HANDBOOK FOURTH EDITION" VII
Examples include those described in Chapters 497 to 545, JOHN WILEY & SONS (1999).

  The liquid temperature of the mixed liquid of two or more organic solvents not containing a polymer in the present invention is not particularly limited, but when the mixed liquid has the same hydrophobic polymer concentration as the hydrophobic polymer solution used in the present invention, A liquid temperature that does not generate a conductive polymer precipitate is preferred. Specifically, a liquid temperature higher by 1 ° C. or more than the lower limit liquid temperature at which no polymer precipitate is generated is preferable. Furthermore, for the purpose of preventing desorption of the hydrophobic polymer from the solid substrate generated by increasing the liquid temperature, a liquid temperature as high as 50 ° C. or less with respect to the lower limit liquid temperature is preferable.

  The time for bringing the substrate into contact with the mixed solution of two or more organic solvents not containing the polymer is not particularly limited, but is preferably 1 second to 24 hours, and more preferably 3 seconds to 1 hour. 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 thickness of the hydrophobic polymer layer 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.

  In the method of the present invention, the solid substrate is brought into contact with a hydrophobic polymer solution and then brought into contact with a liquid not containing the polymer, and then the resulting solid substrate is surface-modified. The surface modification method can be appropriately selected from chemical treatments using chemicals, coupling agents, surfactants, surface deposition, etc., and physical treatments using heating, ultraviolet rays, radiation, plasma, ions, etc. .

Therefore, it is preferable to introduce a functional group capable of generating a covalent bond by surface modification into the hydrophobic polymer layer. Preferred functional groups include —OH, —SH, —COOH, —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group), —CHO, —NR ³ NR ^1. R ² (wherein R ¹ , R ² and R ³ each independently represents a hydrogen atom or a lower alkyl group), —NCO, —NCS, an epoxy group, or a vinyl group can be mentioned. 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.

As a method for introducing these functional groups on the surface, a hydrophobic polymer containing precursors of these functional groups is coated on a metal surface or a metal film, and then a precursor located on the outermost surface is subjected to chemical treatment. A method for generating these functional groups can be mentioned. For example, after coating polymethyl methacrylate, which is a hydrophobic polymer compound containing —COOCH ₃ groups, on a metal film, when the surface is brought into contact with an aqueous NaOH solution (1N) at 40 ° C. for 16 hours, —COOH groups are formed on the outermost surface. Produces. For example, when a polystyrene coating layer is treated with UV ozone, -COOH groups and -OH groups are generated on the outermost surface.

  The solid substrate referred to in the present invention is interpreted in the broadest sense and refers to a base that supports a material having a function, and includes not only a hard substrate but also a flexible substrate such as a film or a sheet.

  The solid substrate used in the present invention is preferably a metal surface or a metal film coated with a hydrophobic polymer. 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 sensor substrate 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 substances into a signal such as an electrical signal. The sensor substrate of the present invention can be used as a biosensor. 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 sensor substrate obtained as described above can immobilize the physiologically active substance 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 sensor substrate of the present invention is not particularly limited as long as it interacts with the measurement object. For example, immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-immune proteins And immunoglobulin binding proteins, sugar binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, polypeptides or oligopeptides having ligand binding ability, and the like.

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

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

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

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

  The biosensor on which a physiologically active substance is immobilized as described above can be used for detection and / or measurement of a substance that interacts with the physiologically active substance.

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

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

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

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

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

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

  In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the measured substance in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means. The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

  If the wave number of the surface plasmon is known from the incident angle at which this total reflection attenuation (ATR) occurs, that is, the total reflection attenuation angle (θSP), the dielectric constant of the substance to be measured can be obtained. In this type of surface plasmon measurement apparatus, as shown in Japanese Patent Application Laid-Open No. 11-326194, in order to measure the total reflection attenuation angle (θSP) with high accuracy and a large dynamic range, It is considered to use detection means. This light detection means is provided with a plurality of light receiving elements arranged in a predetermined direction, and arranged so that different light receiving elements receive light beam components totally reflected at various reflection angles at the interface. Is.

  In that case, there is provided differential means for differentiating the light detection signals output from the light receiving elements of the arrayed light detection means with respect to the arrangement direction of the light receiving elements, and based on the differential value output by the differential means. In many cases, the total reflection attenuation angle (θSP) is specified to obtain a characteristic related to the refractive index of the substance to be measured.

  Moreover, as a similar measuring device using total reflection attenuation (ATR), for example, “Spectroscopic Research” Vol. 47, No. 1, (1998), pages 21 to 23 and pages 26 to 27 are described. Devices are also known. This leakage mode measuring device is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be in contact with the sample liquid. Optical waveguide layer to be generated, a light source for generating a light beam, and the light beam to the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. The optical system includes an incident optical system and light detection means for detecting the excitation state of the waveguide mode, that is, the total reflection attenuation state by measuring the intensity of the light beam totally reflected at the interface.

  In the leakage mode measuring apparatus having the above-described configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle having a wave length propagates in the waveguide mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of guided light depends on the refractive index of the substance to be measured on the optical waveguide layer, knowing the specific incident angle at which total reflection attenuation occurs, the refractive index of the substance to be measured and the measurement object related thereto The properties of the substance can be analyzed.

  In this leakage mode measuring apparatus, the above-mentioned array-shaped light detecting means can be used to detect the position of the dark line generated in the reflected light due to the total reflection attenuation, and the above-described differentiating means is applied in conjunction therewith. Often done.

  In addition, the surface plasmon measurement device and the leakage mode measurement device described above may be used for random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research. In this case, the thin film layer A sensing substance is fixed on the sensing substance on the sensing substance (a metal film in the case of a surface plasmon measuring apparatus, a clad layer and an optical waveguide layer in the case of a leakage mode measuring apparatus), and various analytes are placed on the sensing substance. A sample solution dissolved in a solvent is added, and the total reflection attenuation angle (θSP) is measured every time a predetermined time elapses.

  If the analyte in the sample liquid binds to the sensing substance, the refractive index of the sensing substance changes with time due to this binding. Therefore, by measuring the total reflection attenuation angle (θSP) every predetermined time and measuring whether or not the total reflection attenuation angle (θSP) has changed, the binding state of the analyte and the sensing substance is determined. It is possible to determine whether or not the analyte is a specific substance that binds to the sensing substance based on the result. Examples of the combination of the specific substance and the sensing substance include an antigen and an antibody, or an antibody and an antibody. Specifically, a rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and a human IgG antibody can be used as the specific substance.

  Note that in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of total reflection attenuation (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

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

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

Example 1: PMMA / PSt block (1)
(1) Preparation of polymethyl methacrylate-polystyrene copolymer solution (0.1% PMMA / PSt (1)) Polymethyl methacrylate-polystyrene copolymer (number average molecular weight 60000, polymethyl methacrylate: polystyrene = 1: 1 (weight ratio)) 0.1 g was dissolved in a mixed solution of 2-butanone 60 mL and ethanol 40 mL to prepare 0.1% PMMA / PSt (1).
The lower limit liquid temperature at which no polymer precipitate was generated in this solution was 18 ° C.

(2) Creation of gold block Gold was vapor-deposited on the dielectric block shown in FIG. 23 of JP 2001-330560 A so that the thickness of the gold film would be 500 angstroms to obtain a gold block.

(3) Preparation of PMMA / PSt block (1) After processing the gold block with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, 0.1% PMMA / PSt (1) is applied on the gold deposition surface. The solution was dropped and allowed to stand for 15 minutes. Subsequently, 0.1% PMMA / PSt on the gold-deposited surface was immersed in a mixed solution of 30 mL of 2-butanone and 20 mL of ethanol by immersing in a mixed solution of 30 mL of 2-butanone and 20 mL of ethanol at 25 ° C. for 5 minutes. Replaced. After the replacement, the mixed solution on the block surface was removed by nitrogen blowing and dried under vacuum for 16 hours. When the film thickness was measured by ellipsometry (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab), the thickness of the PMMA / PSt film was 50 Å. This sample is called a PMMA / PSt block (1).

Example 2: PMMA / PSt block (2)
(1) Preparation of polymethyl methacrylate-polystyrene copolymer solution (0.1% PMMA / PSt (2)) Polymethyl methacrylate-polystyrene copolymer (number average molecular weight 60000, polymethyl methacrylate: polystyrene = 1: 1 (weight ratio)) 0.1 g was dissolved in a mixed solution of 45 mL of 2-butanone and 55 mL of acetonitrile to prepare 0.1% PMMA / PSt (2).
The lower limit liquid temperature at which no polymer precipitate was generated in this solution was 20 ° C.

(2) Preparation of PMMA / PSt block (2) Instead of 0.1% PMMA / PSt (1), 0.1% PMMA / PSt (2), 2 instead of a mixed solution of 2-butanone 30 mL and ethanol 20 mL A sample was prepared in the same manner as in Example 1 (3) except that a mixed solution of 45 mL of butanone and 55 mL of acetonitrile was used. When the film thickness was measured by an ellipsometry method (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab), the thickness of the PMMA / PSt film was 50 Å. This sample is called a PMMA / PSt block (2).

Example 3: PMMA / PSt block (3)
A sample was prepared by the operation of Example 1 (3) except that the temperature of the mixed solution was 60 ° C. The thickness of the PMMA / PSt film was 30 Å. This sample is called a PMMA / PSt block (3).

Example 4: PMMA / PSt / COOH block (1)
The PMMA / PSt block (1) was immersed in an aqueous NaOH solution (1N) at 40 ° C. for 16 hours, then washed with water three times, and water was removed by nitrogen blowing. The thickness of the PMMA / PSt / COOH film was 50 angstroms as measured by ellipsometry. This sample is called PMMA / PSt / COOH block (1).

Example 5: PMMA / PSt / COOH block (2)
The operation of Example 4 was performed on the PMMA / PSt block (2) to obtain the PMMA / PSt / COOH block (2). The thickness of the PMMA / PSt / COOH film was 50 angstroms as measured by ellipsometry.

Example 6: PMMA / PSt / COOH block (3)
The operation of Example 4 was performed on the PMMA / PSt block (3) to obtain a PMMA / PSt / COOH block (3). The thickness of the PMMA / PSt / COOH film was 10 angstroms as measured by ellipsometry.

Comparative Example 1: PMMA / PSt block (4)
(1) Preparation of polymethyl methacrylate-polystyrene copolymer solution (0.1% PMMA / PSt (4)) Polymethyl methacrylate-polystyrene copolymer (number average molecular weight 60000, polymethyl methacrylate: polystyrene = 1: 1 (weight ratio)) 0.1 g was dissolved in 100 mL of 2-phenoxyethanol to prepare 0.1% PMMA / PSt (4).

(2) Preparation of PMMA / PSt block After processing the gold block with Model-208UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, 0.1% PMMA / PSt was dropped on the gold deposition surface and allowed to stand for 15 minutes. I put it. Subsequently, 0.1% PMMA / PSt on the gold-deposited surface was replaced with 2-phenoxyethanol by immersing in 50 mL of 2-phenoxyethanol 5 times for 1 minute each. Furthermore, 2-phenoxyethanol on the gold-deposited surface was replaced with ethanol by immersing it in 50 mL of ethanol 5 minutes each for 1 minute. After the replacement, ethanol on the block surface was removed by nitrogen blowing and dried under vacuum for 16 hours. When the film thickness was measured by ellipsometry (In-Situ Ellipsometer MAUS-101, manufactured by Five Lab), the thickness of the PMMA / PSt film was 10 angstroms. This sample is called a PMMA / PSt block (4).

Comparative Example 2: PMMA / PSt / COOH block (4)
A sample was prepared by the same operation as in Example 4 using the PMMA / PSt block (4). The thickness of the PMMA / PSt / COOH film was 10 angstroms. This sample is called PMMA / PSt / COOH block (4).

Comparative Example 3: A gold block having a SAM block gold-deposited film was treated with an ozone cleaner for 30 minutes, and then immersed in an ethanol solution of 1 mM 7-carboxy-1-heptanethiol for 18 hours. Then, it was washed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water. By this operation, a SAM block coated with a SAM compound (7-carboxy-1-heptanethiol) was obtained.

Comparative Example 4: Gold Block A gold block produced by the method described in Example 1 was used as Comparative Example 4.

Evaluation 1: Nonspecific adsorption of protein Adsorption of nonspecific protein to the biosensor surface causes noise, so it is preferable that the amount be as small as possible. Nonspecific adsorption properties of BSA (manufactured by Sigma) and avidin (manufactured by Nacalai Tesque) were measured.

Ethanol block treatment The PMMA / PSt / COOH block and the SAM block are installed in the apparatus shown in FIG. 22 of Japanese Patent Application Laid-Open No. 2001-330560 (hereinafter referred to as the surface plasmon resonance measuring apparatus of the present invention) and blocked with ethanolamine Measurements were made after processing. In the block treatment with ethanolamine, a mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was dropped on the sensor surface of the block, and left standing for 60 minutes. Carried out. Further, after washing with water, an ethanolamine / HCl solution (1 M, pH 8.5) was added to each measurement block and allowed to stand for 20 minutes. Further, it was washed with HBS-EP buffer (Biacore, pH 7.4). The composition of the HBS-EP buffer used above is HEPES (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4), NaCl 0.15 mol / l, EDTA 0.003 mol / l. , Surfactant P20 0.005% by weight.

Measurement of non-specific adsorption Each block subjected to the PMMA / PSt block and ethanolamine block treatment was placed in the surface plasmon resonance measuring apparatus of the present invention and washed with an HBS-EP buffer. Thereafter, a BSA solution (2 mg / ml, HBS-EP buffer) or an avidin solution (2 mg / ml, HBS-EP buffer) was added and allowed to stand for 10 minutes. Thereafter, the plate was washed with HBS-EP buffer, and the amount of change in resonance signal after 3 minutes was measured. The amount of change was evaluated as a relative value to the amount of change in the gold block (Comparative Example 4).

Evaluation 2: Measurement of interaction between protein and analyte compound Neutral avidin (PIERCE, hereinafter referred to as N-avidin) was immobilized on each measurement block of Examples 4 to 6 and Comparative Examples 2 and 3, and D-biotin ( Measurement of interaction with Nacalai Tesque was performed by the following method.

  To the measurement block, a mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was added, and after standing for 20 minutes, HBS-N buffer (Biacore, Washed with pH 7.4). Next, an N-avidin solution (100 μg / ml, HBS-N buffer) was added, allowed to stand for 30 minutes, and then washed with HBS-N buffer. By the above operation, N-avidin was immobilized on the surface of each measurement chip by a covalent bond. The amount of change in resonance signal before addition of N-avidin and after washing was defined as the amount of N-avidin immobilized, and was evaluated as a relative value to the amount of change in the SAM block (Comparative Example 3). The evaluation results are shown in Table 2. The larger the relative value of the change amount, the greater the amount of immobilization, which is preferable. The composition of the HBS-N buffer used above is HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4) and NaCl 0.15 mol / l.

  Further, an ethanolamine / HCl solution (1M, pH 8.5) was added to the measurement block, and then washed with HBS-N buffer to block the remaining COOH groups without reacting with N-avidin.

  Next, the measurement block is placed in the surface plasmon resonance measurement apparatus of the present invention, D-biotin (0.5 μg / ml, HBS-N buffer)) is added to the measurement block, and after standing for 10 minutes, HBS-N Washed with buffer. The amount of change in resonance signal before addition of D-biotin and after washing was defined as the amount of D-biotin bound to N-avidin, and was evaluated as a relative value to the amount of change in the SAM block (Comparative Example 3). The evaluation results are shown in Table 2. A larger change amount relative value indicates higher detection sensitivity, which is preferable.

  From the results in Table 1, it can be seen that the surface forming method of the present invention can provide a surface plasmon resonance substrate with very little nonspecific adsorption of protein. Each sample surface was immersed in a fluorescently labeled substrate FITC-avidin solution (1 mg / mL, HBS-EP buffer) for 15 minutes, then washed with water and observed with a fluorescence microscope, whereas the SAM block showed fluorescence derived from FITC. No fluorescence was observed in the inventive sample, and it was found that the surface forming method of the present invention provides a surface with very little nonspecific adsorption.

  From the results shown in Table 2, the sensor substrate produced by the surface forming method of the present invention was able to immobilize proteins and detect analyte compounds.

  In addition, the measurement block of Comparative Example 1 prepared using a single solvent forms a surface that suppresses nonspecific adsorption, so that the solubility of the polymer in about 50 types of solvents, the nonspecific adsorption of the prepared measurement block, and Evaluation was necessary, and a great deal of work was required to develop the measurement block. On the other hand, the measurement block creation of the present invention can be developed by mixing an arbitrary good solvent and poor solvent of the polymer, and the development time and work are greatly reduced.

Claims (10)

A method for producing a solid substrate for a sensor, comprising the step of bringing a solid substrate into contact with a hydrophobic polymer solution and then bringing the solid substrate into contact with a mixed solution of two or more organic solvents not containing the polymer. The method according to claim 1, further comprising the step of surface modifying the obtained solid substrate. The method of Claim 1 or 2 that the liquid mixture of the 2 or more types of organic solvent which does not contain the said polymer is a liquid mixture containing the good solvent and poor solvent of the said polymer. The liquid temperature of a mixed liquid of two or more organic solvents not containing the polymer is set to a lower limit liquid temperature that does not generate a hydrophobic polymer precipitate when the mixed liquid has the same hydrophobic polymer concentration as the hydrophobic polymer solution. On the other hand, the method in any one of Claim 1 to 3 which is 1 to 50 degreeC high liquid temperature. The method according to any one of claims 1 to 4, wherein the solvent of the hydrophobic polymer solution and the solvent of the mixed liquid of two or more organic solvents not containing a polymer have the same organic solvent composition. The method according to claim 1, wherein the surface modification is a surface modification that introduces a functional group capable of generating a covalent bond. The method according to any one of claims 1 to 6, wherein the solid substrate brought into contact with the hydrophobic polymer solution is a solid substrate coated with a metal surface or a metal film. The method according to claim 1, wherein the sensor solid substrate is a substrate used for surface plasmon resonance analysis. A physiologically active substance comprising the steps of: producing a sensor solid substrate by the method according to any one of claims 1 to 8; and immobilizing the physiologically active substance in contact with the surface of the obtained sensor solid substrate. Of a solid substrate for a sensor in which is bonded to the surface. A step of producing a sensor solid substrate by the method according to any one of claims 1 to 8, a step of bringing a physiologically active substance into contact with the surface of the obtained sensor solid substrate, and a physiological activity obtained. A method for detecting or measuring a substance that interacts with a physiologically active substance, comprising a step of bringing a test substance into contact with a solid substrate for sensors having a substance bonded to the surface.