JP2006090781A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for stabilizing an activated ester generated by activating a carboxylic acid. <P>SOLUTION: In this biosensor, a carboxyl group existing on a surface of a substrate is activated by a carbodiimide derivative or a salt thereof, followed with esterification by any of a nitrogen-containing hetero-aromatic compound having hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group, and a carboxylic amide is formed thereafter by reaction with an amine. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

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

  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).

  When creating the surface of a measurement chip (biosensor) as described above, a carboxylic acid amide is often formed in water (reaction between a polymer and a linker, and further binding with a substance to be detected such as a protein). In order to carry out this reaction, the carboxylic acid is usually activated with 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS), which are water-soluble carbodiimides, and then reacted with an amine. Thus, it is common practice to form carboxylic acid amides. Even when surface plasmon resonance analysis (SPR) or quartz crystal microbalance (QCM) is used to create a biosensor surface, it is reported that an amide bond is formed in water by combining EDC and NHS (patent) References 3 and 4). Similarly, SPR surface creation is available at http://www.biacore.co.jp/2_2_1.shtml#a (Biacore), and QCM surface creation is available at http://www.initium2000.com/technology.html ( (Initial).

  However, when EDC and NHS are mixed in water, there is a problem that “the stability of the obtained active ester is not sufficient and it is hydrolyzed with time”. This problem causes a “reduction in reaction yield” and requires the use of a large excess of activator relative to the carboxylic acid.

Japanese Patent No. 2815120 JP-A-9-264843 Japanese Patent Laid-Open No. 11-281568 JP 2000-39401 A

  The present invention has been made to solve the above-described problems of the prior art. That is, this invention made it the subject which should be solved to provide the technique which stabilizes the activated ester produced | generated by activation of carboxylic acid.

  As a result of intensive studies to solve the above problems, the present inventors have activated a carboxyl group present on the surface of a substrate with a carbodiimide derivative or a salt thereof, a nitrogen-containing heteroaromatic compound having a hydroxyl group, electron withdrawing It has been found that the activated ester can be stabilized by esterifying with either a phenol derivative having a functional group or an aromatic compound having a thiol group, and the present invention has been completed.

  That is, according to the present invention, the carboxyl group present on the surface of the substrate is activated with a carbodiimide derivative or a salt thereof, and has a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or a thiol group. Provided is a biosensor in which a carboxylic acid amide group is formed by reacting with an amine after esterification with any of the aromatic compounds.

Preferably, the carbodiimide derivative or salt thereof is water soluble.
Preferably, the carbodiimide derivative is any one of compounds 1-3.

  Preferably, the nitrogen-containing heteroaromatic compound having a hydroxyl group is any one of compounds 4 to 12.

Preferably, in the phenol derivative having an electron withdrawing group, the σ value of the electron withdrawing group is 0.3 or more.
Preferably, the phenol derivative having an electron-withdrawing group is any one of compounds 13 to 16.

  Preferably, the aromatic compound having a thiol group is any one of Compounds 17-19.

  Preferably, the substrate is coated with a hydrophobic polymer compound, and the carboxyl group of the hydrophobic polymer compound is activated with a carbodiimide derivative or a salt thereof, and a nitrogen-containing heteroaromatic compound having a hydroxyl group, electron withdrawing A carboxylic acid amide group is formed by reacting with an amine after being esterified with either a phenol derivative having a functional group or an aromatic compound having a thiol group.

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

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

  According to another aspect of the present invention, a substrate having a carboxyl group on its surface is activated with a carbodiimide derivative or a salt thereof, and has a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or a thiol group. There is provided a method for producing a biosensor of the present invention, which comprises a step of forming a carboxylic acid amide group by reacting with an amine after esterification with any compound of an aromatic compound.

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

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, more preferably detected or measured by surface plasmon resonance analysis.

  The present invention makes it possible to stabilize an activated ester obtained by activating a carboxylic acid in the production of a biosensor. By using the biosensor of the present invention, the amount of non-specific adsorption on the sensor surface can be suppressed, and at the same time, the amount of physiologically active substance bound can be improved.

Hereinafter, embodiments of the present invention will be described.
The biosensor of the present invention activates a carboxyl group present on the surface of a substrate with a carbodiimide derivative or a salt thereof, a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic having a thiol group. A carboxylic acid amide group is formed by reacting with an amine after forming an ester with any one of the group compounds.

  The carbodiimide derivative or a salt thereof is preferably water-soluble, and specifically, the compounds 1 to 3 described above can be used in the present specification, but are not limited thereto.

  The kind of the nitrogen-containing heteroaromatic compound in the nitrogen-containing heteroaromatic compound having a hydroxyl group is not particularly limited, but 5- or 7-membered saturation containing at least one nitrogen atom (for example, 1, 2, or 3). Or an unsaturated monocyclic ring or condensed ring can be mentioned. Specific examples of the nitrogen-containing heteroaromatic compound having a hydroxyl group include, but are not limited to, compounds 4 to 12 described above in the present specification.

Specific examples of the electron-withdrawing group in the phenol derivative having an electron-withdrawing group include —NO ₂ , halogen (—F, —Cl, —Br, or —I), —S (CH ₃ ) ₂ X ⁻ (X Is a monovalent anion (for example, F ⁻ , Cl ⁻ , Br ⁻ , I ⁻ , At ⁻ , BF ₄ ⁻ , AsF ₆ ⁻ , PF ₆ ⁻ , SbF ₆ ⁻ , SbCl ₆ ⁻ , SnCl ₆ ²⁻ , FeCl _{4). ^{-, BiCl 5 2-, CF 3}} SO 2 -, ClO 4 -, FSO 2 -, F 2 PO 2 - shows the like)), - COOH, may be mentioned -CN, or -CHO and the like. The σ value of the electron withdrawing group is preferably 0.3 or more. Preferably, the σ value of the electron-withdrawing group is 0.5 or more, more preferably 0.7 or more. The σ values are summarized in, for example, the appendix of Naoki Inamoto's “Hammett Kaku” Maruzen (1983). Specific examples of the phenol derivative having an electron-withdrawing group include compounds 13 to 16 described above in the present specification, but are not limited thereto.

  The kind of aromatic compound in the aromatic compound having a thiol group (—SH) is not particularly limited, and may be an aryl compound (such as benzene or naphthalene) or a heterocyclic compound containing at least one or more heteroatoms. Heterocyclic compounds include those that are 5- or 7-membered saturated or unsaturated monocyclic or condensed rings containing one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur atoms. Quinoline, isoquinoline, pyrimidine, pyrazine, pyridazine, phthalazine, triazine, furan, thiophene, pyrrole, oxazole, benzoxazole, thiazole, benzothiazole, imidazole, benzimidazole, thiadiazole, triazole, benzotriazole, 7-azabenzotriazole, benzo Examples include triazine. Specific examples of the aromatic compound having a thiol group include, but are not limited to, compounds 17 to 19 described above in the present specification.

  Any compound of a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group, wherein a carboxyl group present on the surface of the substrate is activated with a carbodiimide derivative or a salt thereof There are no particular limitations on the method used to form an ester, and it can be carried out by a conventional method known to those skilled in the art. Specifically, any of a carbodiimide derivative or a salt thereof, a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group on a substrate having a carboxyl group on the surface. The esterification can be performed by contacting a solution containing the compound (for example, an aqueous solution).

The above-mentioned compounds 1 to 3 and compounds 4 to 19 are known compounds and can be synthesized by conventional methods, or commercially available products can also be used. Specifically, compounds 1 to 19 are commercially available from, for example, domestic chemistry, Aldrich, Sakai Kogyo, or Dojin Chemical, or literature (Bull. Chem. Soc. Jpn., 60 , 2409 (1987). , J. Polym. Sci., Polym. Chem. Ed., 17
2013 (1979). J. Polym. Sci., Polym. Chem. Ed., 16 , 475 (1978))
It can be synthesized by the method described in 1.

  In the biosensor of the present invention, a carboxylic acid amide group is formed by reacting an amine with the active ester formed as described above. The method of reaction with amine is not particularly limited, and can be performed by a conventional method known to those skilled in the art. For example, the reaction may be performed by bringing an ethanolamine / HCl solution into contact with the substrate.

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

  According to a preferred embodiment of the present invention, the substrate is coated with a hydrophobic polymer compound, and the carboxyl group of the hydrophobic polymer compound can be activated by the method described above in this specification.

  The hydrophobic polymer compound that can be used in the present invention is a polymer compound that does not absorb water, and has a solubility in water (25 ° C.) of 10% or less, more preferably 1% or less, and most preferably 0.8%. 1% or less.

  Hydrophobic monomers that form hydrophobic polymer compounds include vinyl esters, acrylic acid esters, methacrylic acid esters, olefins, styrenes, crotonic acid esters, itaconic acid diesters, and maleic acid diesters. , Fumaric acid diesters, allyl compounds, vinyl ethers, vinyl ketones and the like. The hydrophobic polymer compound may be a homopolymer composed of one type of monomer or a copolymer composed of two or more types of monomers.

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

  Coating of the hydrophobic polymer compound onto the substrate can be performed by a conventional method, for example, spin coating (spin coating method), air knife coating, bar coating, blade coating, slide coating, curtain coating, spray method, vapor deposition. It can be performed by the method, the casting method, the dipping method or the like. Among these, the spin coating (spin coating method) and the dipping method will be described in more detail.

  The spin coating method is a method of manufacturing a thin film by dropping a solution onto a substrate such as a rotating disk, and controlling the film thickness by adjusting the concentration of the solution, the number of rotations of the disk, the vapor pressure of the solvent, and the like. is there.

  The concentration of the hydrophobic polymer compound in the solution used in the spin coating method is preferably 0.001 to 50% by weight, more preferably 0.01 to 10% by weight, and more preferably 0.1 to 5% by weight. Is more preferable.

  The rotational speed of the disk during spin coating is preferably 10 rpm or more and 10,000 rpm or less, more preferably 50 rpm or more and 7500 rpm or less, and further preferably 100 rpm or more and 5000 rpm or less.

  The solvent used in the spin coating method preferably has a vapor pressure of 0.1 kPa or more and 100 kPa or less, more preferably 0.5 kPa or more and 50 kPa or less, and further preferably 1 kPa or more and 30 kPa or less, at an environmental temperature during thin film production by spin coating. . Specific examples of the solvent that can be used include methanol, ethanol, i-propanol, n-butanol, t-butanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethyl acetate, butyl acetate, dimethyl sulfoxide, dimethylformamide, and the like.

  In the method for producing a biosensor of the present invention, it is preferable to rotate the disk after dropping the coating solution onto the surface of the substrate held on the disk.

  Further, when the coating solution is dropped onto the substrate surface, the coverage with respect to the substrate surface is preferably 80% or more and 100% or less, and more preferably 90% or more and 100% or less.

  In the dipping method, coating is performed by bringing the substrate into contact with a hydrophobic polymer solution and then bringing the substrate into contact with a liquid not containing the hydrophobic polymer solution. Preferably, the solvent of the hydrophobic polymer compound solution and the solvent of the liquid not containing the hydrophobic polymer compound are the same solvent.

In the dipping method, a hydrophobic polymer compound layer having a uniform coating thickness can be obtained on the surface of the substrate by appropriately selecting a solvent for coating the hydrophobic polymer compound, regardless of the unevenness, curvature, and shape of the substrate.

  The coating solvent for the dipping method is not particularly limited, and any solvent can be used as long as it dissolves a part of the hydrophobic polymer compound. For example, a formamide solvent such as N, N-dimethylformamide, a nitrile solvent such as acetonitrile, an alcohol solvent such as phenoxyethanol, a ketone solvent such as 2-butanone, and a benzene solvent such as toluene can be used. However, it is not limited to these.

  The solution of the hydrophobic polymer compound to be brought into contact with the substrate may be either a completely dissolved hydrophobic polymer compound or a suspension containing an insoluble component of the hydrophobic polymer compound. The liquid temperature is not particularly limited as long as a part of the hydrophobic polymer compound is dissolved, but is preferably −20 ° C. or higher and 100 ° C. or lower. The liquid temperature may be varied while the substrate is in contact with the hydrophobic polymer compound solution. Although there is no restriction | limiting in particular in the hydrophobic polymer compound density | concentration of a solution, Preferably it is 0.01% or more and 30% or less, More preferably, it is 0.1% or more and 10% or less.

  The time for bringing the solid substrate into contact with the hydrophobic polymer solution is not particularly limited, but is preferably 1 second to 24 hours, more preferably 3 seconds to 1 hour.

As the liquid not containing the hydrophobic polymer compound, the difference between the SP value of the solvent itself (unit: (J / cm ³ ) ^1/2 ) and the SP value of the hydrophobic polymer compound is 1 or more and 20 or less. Preferably, it is 3 or more and 15 or less. The SP value is represented by the square root of the cohesive energy density between molecules and is also called a solubility parameter. In the present invention, the SP value δ is calculated by the following formula. As the cohesive energy Ecoh and the molar volume V of each functional group, values defined by Fedors were used (R. F. Fedors, Polym. Eng. Sci., 14 (2), P147, P472 (1974)).
δ = (ΣEcoh / ΣV) ^1/2
By way of example, the SP values of hydrophobic polymer compounds and solvents are as follows: Polymethylmethacrylate-polystyrene copolymer (1: 1): for 21.0 Solvent 2-phenoxyethanol: 25.3, for Polymethylmethacrylate: 20.3 Solvent acetonitrile: 22.9, polystyrene: 21.6 with respect to polystyrene: 21.6.

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

  The coating thickness of the hydrophobic polymer compound is 0.1 nm or more and 500 nm or less, and particularly preferably 1 nm or more and 200 nm or less.

  The biosensor of the present invention is preferably a metal surface or metal film coated with a hydrophobic polymer compound. The metal constituting the metal surface or metal film is not particularly limited as long as surface plasmon resonance can occur when, for example, a surface plasmon resonance biosensor is considered. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal.

  Although the thickness of the metal film is arbitrary, for example, when considering use for a surface plasmon resonance biosensor, it is preferably 0.1 nm or more and 500 nm or less, particularly preferably 1 nm or more and 200 nm or less. 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, when considering use for a surface plasmon resonance biosensor, generally, optical glass such as BK7, or synthetic resin, specifically, polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  In a biosensor comprising a substrate coated with a hydrophobic polymer compound, it is preferable to have —COOH as a functional group capable of immobilizing a physiologically active substance on the outermost surface of the substrate. Here, the “outermost surface of the substrate” means “the side farthest from the substrate”, and more specifically, “the side farthest from the substrate in the hydrophobic polymer compound coated on the substrate”. Meaning.

As a method of introducing —COOH groups on the outermost surface, a hydrophobic polymer containing the precursor is coated on a metal surface or a metal film, and then the functional group of the precursor is located on the outermost surface by chemical treatment. The method of producing | generating is 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.

  In the biosensor surface obtained as described above, the —COOH group is activated according to the method of the present invention to form a carboxylic acid amide group, and through this activated carboxylic acid amide group, Thus, the physiologically active substance can be immobilized on the metal surface or metal film by covalently bonding the physiologically active substance.

  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, etc. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.
As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
Examples of the low-molecular 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, an apparatus using a system called Kretschmann arrangement can be cited (for example, Japanese Patent Laid-Open No. 6-167443). See the official gazette). A surface plasmon measuring apparatus using the above system basically includes a dielectric block formed in a prism shape, for example, and a metal film formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a sample liquid. A light source that generates a light beam; an optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the metal film; and It comprises light detecting means for detecting the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface.

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

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

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

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

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

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

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

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

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

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

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

When the sensor chip of the present invention is used for surface plasmon resonance analysis, it can be applied as a part of various surface plasmon measuring 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:
In this example, the lifetime of the active ester was examined when various activators were allowed to act on the carboxyl group present on the surface of the hydrophobic polymer applied to the gold surface.

(1) Production of Gold Surface Substrate Chrome on a substrate using a parallel plate type 6-inch sputtering apparatus (SH-550 manufactured by ULVAC, Inc.) on a glass substrate having a length of 8 mm, a width of 80 mm, and a thickness of 0.5 mm. The thickness of 1 nm,
Further, a sputter film was formed on chromium so that the thickness of gold was 50 nm. This substrate was treated with Model-208UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes to produce a gold surface substrate.

(2) Preparation of coating solution Poly (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate) -poly (benzyl methacrylate) copolymer (monomer weight 1.5 g was dissolved in methyl isobutyl ketone, and methyl isobutyl ketone was added so that the liquid volume became 100 mL. The polymer A solution was filtered through a 0.45 μm filter to prepare a coating solution A. Methyl isobutyl ketone was dehydrated for 16 hours in advance with Molecular Sieves 4A 1/16.

(3) Production of Polymer A Coating Chip The substrate was set in an aluminum container having a sealed structure of 40 mm length × 120 mm width × 20 mm depth. This aluminum container was fixed on the inner cup of a spin coater (MODEL SC408 (special), manufactured by Nanotech Co., Ltd.) having a hermetically sealed inner cup so that the tangential direction of the arc is the major axis at a position 135 mm from the center. . Using a micropipette, 100 μL of coating solution A was dropped on this glass substrate, and the entire glass substrate was coated with coating solution A. The aluminum container was sealed, rotated at 200 rpm, and stopped for 60 seconds. The substrate was allowed to stand in a sealed container for 5 minutes, then removed from the sealed container and dried at room temperature overnight at normal pressure to obtain a polymer A coated chip.

(4) Preparation of biosensor chip The polymer A coating chip prepared as described above was immersed in a 1N NaOH aqueous solution kept at 60 ° C. for 2 hours, then washed in running pure water, and COOH was applied to the surface of the polymer A coating layer. A biosensor chip into which a group was introduced was produced. When the film thickness distribution with a width of 75 mm at intervals of 0.1 mm in the center of the substrate was measured by ellipsometry (In-Situ Ellipsometer MAUS-101, manufactured by Fibravo), the average film thickness was 20 nm. When the surface of the polymer A layer of the sensor chip was observed with a scanning electron microscope (S-5200, manufactured by Hitachi High-Technologies Corporation) at an acceleration voltage of 0.5 kV, no defects were detected in the polymer A layer. It was.

(5) Performance evaluation of activator (active ester lifetime)
In a laboratory maintained at 25 ° C., dissolve the hydrochloride salt of Compound 1 (Dojin Chemical) and the ester-forming compound (Compounds 4 to 19 or NHS) in ultrapure water (MilliQ) to 1 mM each. did. After immersing the sensor chip prepared in (4) in this solution, the sensor chip after 1 hour, 4 hours, and 16 hours was washed with ultrapure water 5 times at 25 ° C. Was immersed in an aqueous solution (0.50 mg / L) of Cy-5-Hydrazide (manufactured by Amersham) for 30 minutes. This sensor chip is washed with ultrapure water five times, and unreacted Cy-5-Hydrazide is removed from the sensor chip surface. Then, using a fluoro image analyzer (FLA8000, manufactured by Fuji Photo Film Co., Ltd.), The relative values of fluorescence intensity were compared (excitation wavelength: 635 nm, measurement wavelength: 675 nm). Since the fluorescence on the sensor chip surface is observed only when the active ester on the sensor chip surface reacts with Cy-5-Hydrazide, this method is an effective method for evaluating the lifetime of the active ester. The obtained results are shown in Table 1.

  The NHS ester (sample No. 17), which is a comparative example, completely lost activity 16 hours after the reaction, whereas in the present invention (samples No. 1 to 16) using compounds 4 to 19, It was shown that the activity was maintained even after 16 hours.

Example 2
In this example, the performance as a biosensor chip when various activators are allowed to act on the carboxyl group present on the surface of the hydrophobic polymer applied to the gold surface is examined.

(1) Non-specific adsorption preventing performance A sensor chip was prepared according to the operations of (1) to (4) of Example 1. The prepared sensor chip is brought into contact with an aqueous solution containing 1 mM of Compound 1 and 1 mM of an ester-forming compound (compounds 4 to 19 or NHS) for 30 minutes, and then 50 mM acetate buffer (pH 4.5, Biacore). Washed by the company). Next, after contacting with ethanolamine / HCl solution (1M, pH 8.5) for 30 minutes, the surface COOH groups were blocked by washing with 50 mM acetate buffer (pH 4.5). Each of the sensor chips prepared by the above procedure was set on a surface plasmon resonance measuring apparatus (Applied Spectroscopy, SPR resonance apparatus described in FIG. 5 of 42 (8), 1375-1379 (1988)). The sensor chip was set at a center position where the laser light hits in the center in the vertical direction and 40 mm from the end in the horizontal direction. By covering the chip with a polypropylene member, a cell having a width (vertical direction) of 1 mm, a length (horizontal method) of 7.5 mm, and a depth of 1 mm was produced. The measurement cell was filled with HBS-EP buffer and measurement was started. The composition of the HBS-EP buffer is as follows: 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. The inside of the cell was replaced with a BSA solution (2 mg / ml, HBS-EP buffer (Biacore, pH 7.4)) or an avidin solution (2 mg / ml, HBS-EP buffer) and allowed to stand for 10 minutes. Thereafter, the plate was washed with HBS-EP buffer, and the amount of change in resonance signal (RU value) after 3 minutes was measured. The amount of change after 3 minutes of buffer washing before the addition of each protein was defined as the amount of non-specific adsorption of each protein, and evaluated by a relative value with respect to the amount of change on the unmodified gold surface substrate (sample No. 35).

(2) Interaction between protein and analyte compound Neutral avidin (PIERCE) was immobilized on a sensor chip, and interaction with D-biotin (Nacalai Tesque) was measured by the following method.

  A sensor chip was prepared according to the operations of (1) to (4) of Example 1. The prepared sensor chip is contacted with an aqueous solution containing 0.1 mM of Compound 1 and 0.1 mM of an ester-forming compound (Compounds 4 to 19 or NHS) for 30 minutes, and then HBS-N buffer (Biacore) And pH 7.4). The composition of the HBS-N buffer is HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4) and NaCl 0.15 mol / l. Next, the sensor chip was installed in the surface plasmon resonance apparatus of the present invention. The sensor chip was set at a center position where the laser light hits in the center in the vertical direction and 40 mm from the end in the horizontal direction. By covering the chip with a polypropylene member, a cell having a width (longitudinal direction) of 1 mm, a length (horizontal method) of 7.5 mm, and a depth of 1 mm was produced. The inside of the cell was replaced with a neutral avidin solution (100 μg / ml, HBS-N buffer), allowed to stand for 30 minutes, and then replaced with HBS-N buffer. Through the above operation, N-avidin was immobilized on the sensor chip surface by covalent bond.

  For the NHS esterified sensor chip (sample No. 34), the compound was determined using the amount of change in resonance signal (RU value) before and after neutral avidin addition and 3 minutes after the completion of HBS-N buffer substitution as a reference for the amount of binding. Resonance signal (RU value) change amount (ie, neutral avidin) after addition of neutral avidin of sensor chip (sample Nos. 18 to 33) esterified by 4 to 19 and 3 minutes after completion of HBS-N buffer substitution after addition The amount of binding) was evaluated as a relative value.

  Thereafter, the inside of the cell was replaced with an ethanolamine / HCl solution (1M, pH 8.5), and then replaced with an HBS-N buffer to block COOH groups remaining without reacting with neutral avidin. Next, the inside of the cell was replaced with D-biotin (1 μg / ml, HBS-N buffer), allowed to stand for 10 minutes, and then replaced with HBS-N buffer.

  NHS esterified sensor chip (sample No. 34) was esterified with compounds 4-19 using the amount of change in resonance signal (RU value) before addition of D-biotin and 3 minutes after washing as a reference for the amount of binding. The amount of change in the resonance signal (RU value) (ie, the amount of D-biotin binding) before addition of D-biotin and 3 minutes after the washing of the sensor chip (sample Nos. 18 to 33) was expressed as a relative value. evaluated. The obtained results are shown in Table 2.

  When the NHS esterified sensor chip, which is a comparative example, is reacted with ethanolamine (sample No. 34), it has a non-specific adsorption preventing ability compared with the unmodified gold surface (sample No. 35). However, the degree is not enough. On the other hand, when the sensor chip activated by the compounds 4 to 19, which is the present invention, is reacted with ethanolamine (sample No. 18 to 33), it has sufficient non-specific adsorption preventing ability. It was shown that.

On the other hand, in the experiment of activating the carboxylic acid on the surface of the sensor chip and binding neutral avidin via an amide bond, the sensor chip activated by EDC / NHS as a comparative example was reacted with neutral avidin (sample No. .34), when the sensor chip esterified with compounds 4 to 19 according to the present invention is reacted with neutral avidin (sample No. 18 to 33), more neutral avidin is bonded with an amide bond. As a result, it was shown that more D-biotin can be detected.

Claims (19)

Any compound of a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group, wherein a carboxyl group present on the surface of the substrate is activated with a carbodiimide derivative or a salt thereof A biosensor in which a carboxylic acid amide group is formed by reacting with an amine after being converted to an ester. The biosensor according to claim 1, wherein the carbodiimide derivative or a salt thereof is water-soluble. The biosensor according to claim 2, wherein the carbodiimide derivative is any one of compounds 1 to 3.

The biosensor according to any one of claims 1 to 3, wherein the nitrogen-containing heteroaromatic compound having a hydroxyl group is any one of compounds 4 to 12.

The biosensor according to any one of claims 1 to 3, wherein in the phenol derivative having an electron-withdrawing group, the σ value of the electron-withdrawing group is 0.3 or more. The biosensor according to claim 5, wherein the phenol derivative having an electron-withdrawing group is any one of compounds 13 to 16.

The biosensor according to any one of claims 1 to 3, wherein the aromatic compound having a thiol group is any one of compounds 17 to 19.

A substrate is coated with a hydrophobic polymer compound, and the carboxyl group of the hydrophobic polymer compound is activated with a carbodiimide derivative or a salt thereof to form a nitrogen-containing heteroaromatic compound having a hydroxyl group, an electron-withdrawing group. The carboxylic acid amide group is formed in any one of the phenol derivative which has it, or the aromatic compound which has a thiol group, Then, the carboxylic acid amide group is formed by making it react with an amine. Biosensor. The biosensor according to any one of claims 1 to 8, wherein the coating thickness of the hydrophobic polymer compound is 1 angstrom or more and 5000 angstrom or less. The biosensor according to any one of claims 1 to 9, wherein the substrate is a metal surface or a metal film. The biosensor according to claim 10, wherein the metal surface or metal film is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum, and aluminum. The biosensor according to any one of claims 1 to 11, which is used for non-electrochemical detection. The biosensor according to any one of claims 1 to 12, which is used for surface plasmon resonance analysis. A substrate having a carboxyl group on the surface is activated with a carbodiimide derivative or a salt thereof, and esterified with a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or an aromatic compound having a thiol group The method for producing a biosensor according to claim 1, further comprising a step of forming a carboxylic acid amide group by reacting with an amine. The biosensor according to any one of claims 1 to 13, wherein the physiologically active substance is bound to the surface by a covalent bond. The biosensor according to claim 1, comprising the step of bringing the biosensor and the bioactive substance into contact with each other, and covalently bonding the bioactive substance to the surface of the biosensor. How to immobilize. A substance that interacts with the bioactive substance is detected or measured, comprising the step of bringing the biosensor according to any one of claims 1 to 13 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 17, wherein a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method. The method according to claim 17 or 18, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.