JP2006098261A - Method for manufacturing sensor chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a sensor chip in which the amount of immobilization of ligand is stabilized by stably producing a solid phase reaction for immobilizing the ligand on a sensor chip substrate. <P>SOLUTION: In the method for manufacturing a sensor chip, optical glass or an optical plastic substrate, a metal film, and a hydrophobic polymer film are laminated in this order, further a compound expressed by at least one type of general expression (1) X-L-Y (X and Y in the expression indicate each group that reacts with an organic molecule independently, and L indicates a divalent linkage group) is bonded. In the method for manufacturing a sensor chip, the ratio of a surface area that is not in contact with air to a surface area in contact with air in the droplet of a reaction liquid during reaction is larger than 1, when bringing the reaction liquid for bonding the compound expressed by the general expression (1) to the sensor chip into contact with the substrate surface of the sensor chip for reaction. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for manufacturing a sensor chip for a biosensor. In particular, the present invention relates to a method for manufacturing a sensor chip for a surface plasmon resonance measurement biosensor.

  Drugs often interact and function with proteins in vivo. For example, an inhibitor binds to an enzyme and inhibits the enzyme reaction, thereby changing a biochemical reaction in the living body and expressing a pharmacological action. For this reason, it is important to know the strength of binding between a protein and a drug candidate compound in drug search.

In recent years, the surface plasmon resonance method has attracted attention as a method of interaction between proteins and chemical substances. In this method, protein is solid-phased on a metal surface such as gold, and the interaction is quantified using a change in resonance when a chemical substance is donated here. The analyte molecule, which is a chemical substance, is adsorbed over time to molecules that interact with the analyte molecule, such as proteins immobilized on the metal surface. At this time, the amount of adsorption, that is, the change of the SPR signal changes according to the following equation.
dR / dt = ka ・ Cs ・ (Rmax-R)-kd ・ R Equation (1)
(In the formula, R is an SPR signal, Rmax is a maximum SPR signal, ka is a binding rate constant, kd is a dissociation rate constant, and Cs is a concentration of an analyte molecule near the metal surface.)

  Here, Cs is constant under ideal conditions in which the metal surface is constantly replaced with fresh liquid, and ka and kd can be obtained from the measurement results by solving a simple differential equation. The association constant (KD) indicates the ratio of association to dissociation and is expressed in kd / ka.

  In searching for low molecular weight drugs, screening of at least 10,000 low molecular weight compounds is usually performed for one target protein. The hit compound group obtained as a result is subjected to further detailed evaluation and narrowed down to one lead compound.

  The measurement of the interaction between the protein and the test compound by the surface plasmon resonance method is used in the drug search process as described in Non-Patent Document 1, but the number of measurements per day is about 100 samples. Because of its low processing capacity, it was not used for large-scale screening as described above.

  For large-scale screening, the fluorescent labeling method typified by FRET (fluorescense resonance energy transfer) method and the radiolabeling method typified by SPA (scintillation proximity assay) method are mainly used, but both need to be labeled with a ligand. It is. In the former case, the measurement system cannot often be constructed due to binding inhibition by the label, and in the latter case, there is a problem in handling radioactive substances. In recent years, a large number of target proteins with unknown ligands have been found due to advances in genomic science. Against this background, a label-free large-scale screening method has been strongly desired.

  The label-free large-scale screening can be performed, for example, by immobilizing a ligand protein on a disposable sensor chip in advance and performing parallel measurement of the interaction between the ligand protein and the test substance in the SPR measurement unit. Therefore, high processing capability can be realized. However, if the quality of the protein-immobilized membrane is not stable, the amount of ligand protein immobilized is not stable, and accurate measurement cannot be performed.

  In order to form a protein-immobilized film on a chip substrate, it is necessary to perform a multi-step solid-phase reaction, but how to perform these reactions stably and to reduce the loss of chemicals for mass production is a major issue. It was a challenge. In addition, there has been a demand for a control method for defects, thickness uniformity, and chemical surface modification uniformity of an ultrathin polymer film of several nm to several tens of nm.

BUNSEKI KAGAKU vol.51, No.6, pp.461-468 (2002)

  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 sensor chip manufacturing method that stably performs a solid-phase reaction for immobilizing a ligand on a sensor chip substrate and stabilizes the amount of ligand immobilized. did.

  As a result of intensive studies to solve the above problems, the present inventors have made a reaction solution for bonding a linker compound to the sensor chip to contact the substrate surface of the sensor chip to cause a reaction. The inventors have found that the above problem can be solved by increasing the ratio of (surface area not in contact with air) to (surface area in contact with air) of a droplet to be greater than 1, and the present invention has been completed.

That is, according to the present invention, an optical glass or optical plastic substrate, a metal film, and a hydrophobic polymer film are laminated in this order, and at least one compound represented by the general formula (1) is bonded. A method for producing a sensor chip, wherein a reaction liquid for binding a compound represented by the general formula (1) to the sensor chip is brought into contact with the substrate surface of the sensor chip to cause a reaction. A method of manufacturing a sensor chip is provided, wherein a ratio of a (surface area not in contact with air) to a (surface area in contact with air) of the droplet is greater than 1.
General formula (1): X-L-Y
(In the formula, X and Y each independently represent a group capable of reacting with an organic molecule, and L represents a divalent linking group.)

  Preferably, the reaction liquid for binding the compound represented by the general formula (1) to the sensor chip is a reaction liquid for activating a functional group present on the substrate and / or the general formula (1). It is the reaction liquid containing the represented compound.

Preferably, the hydrophobic polymer is applied on the metal film using an organic solvent having a water content of 100 ppm or less.
Preferably, the hydrophobic polymer is applied on the metal film and then vacuum-dried.
Preferably, a patterning reaction for patterning at least two types of surfaces on the surface of the sensor chip is performed, and after the patterning reaction, the surface of the sensor chip is washed with a liquid containing a carboxylic acid derivative.
Preferably, the sensor chip is a surface plasmon resonance measurement sensor chip.

  According to another aspect of the present invention, a sensor chip manufactured by the above-described method of the present invention is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the solid-phase reaction for fixing a ligand on a sensor chip board | substrate is performed stably, The manufacturing method of the sensor chip which stabilized the fixed amount of the ligand is provided.

Hereinafter, embodiments of the present invention will be described in detail.
In the method for producing a sensor chip of the present invention, an optical glass or optical plastic substrate, a metal film, and a hydrophobic polymer film are laminated in this order, and at least one compound represented by the general formula (1) is bonded. When the reaction liquid for binding the compound represented by the general formula (1) to the sensor chip is brought into contact with the substrate surface of the sensor chip to cause the reaction, In this method, the ratio of the (surface area not in contact with air) and (surface area in contact with air) droplets of the reaction solution is greater than 1.
General formula (1): X-L-Y
(In the formula, X and Y each independently represent a group capable of reacting with an organic molecule, and L represents a divalent linking group.)

  In the present invention, when the compound represented by the general formula (1) is reacted as a solution on the surface of the sensor chip substrate, the droplets of the compound solution during the reaction (surface area of the portion not in contact with air) and (air The ratio of the surface area of the part in contact with the surface is greater than 1. It is preferably 5 or more, and most preferably 10 or more.

  In order to make the ratio of (the surface area of the portion not in contact with air) and (the surface area of the portion in contact with air) of the droplet of the compound solution during the reaction larger than 1, cast the compound solution on the sensor chip. A film (for example, a PET film or the like) can be placed thereon to cover the surface while spreading the liquid. Thereby, the ratio of the surface area of the part which does not contact the air of the liquid at the time of reaction and the surface area of the part which contacts the air can be made larger than 1.

  The reaction liquid for binding the compound represented by the general formula (1) to the sensor chip is a reaction liquid for activating a functional group present on the substrate and / or the general formula (1). And a reaction solution containing the compound. Here, for example, when the functional group present on the substrate is a carboxyl group, examples of the reaction solution for activating the functional group include a Sulfo-NHS solution and an EDC solution.

Specific examples of the linker used in the present invention include compounds represented by the following general formula (1).
General formula (1): XY
(In the formula, X and Y each independently represent a group capable of reacting with an organic molecule, and L represents a divalent linking group.)

In the general formula (1), X represents, for example, a group capable of reacting with a functional group of an organic molecule such as a hydrophobic polymer compound, preferably a halogen atom, an amino group, or an amino group protected by a protecting group, A carboxyl group, or a carbonyl group having a leaving group, a hydroxyl group, a hydroxyl group protected by a protecting group, an aldehyde group, —NHNH ₂ , —N═C═O, —N═C═S, an epoxy group, or a vinyl group is there.
The protecting group here is a group that can be deprotected in the reaction system to form a functional group. For example, the protecting group for amino group includes tertbutyloxycarbonyl group (Boc), 9-fluorene group. Nylmethyloxycarbonyl group (Fmoc), nitrophenylsulfenyl group (Nps), dithiasuccinyl group (Dts) and the like can be mentioned.

Moreover, an acyl group etc. are mentioned as a hydroxyl-protecting group.
The leaving group here is a halogen atom, alkoxy group, aryloxy group, alkylcarbonyloxy group, arylcarbonyloxy group, halogenated alkylcarbonyloxy group, alkylsulfonyloxy group, halogenated alkylsulfonyloxy group, arylsulfonyloxy Groups and the like.
As the leaving group, an ester group produced by combining a carboxylic acid, a known dehydration condensation reagent (for example, carbodiimides) and an N-hydroxy compound is also preferably used.

  In General formula (1), L represents a bivalent coupling group, and it is preferable that the total number of atoms of L is 2-1000. Further, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyleneoxy group, a substituted or unsubstituted aryleneoxy group, or X in the general formula (1) and Y of another molecule are bonded to form a continuous structure. A divalent linking group is preferred.

In the general formula (1), Y represents a group capable of immobilizing an organic molecule such as a physiologically active substance, and is preferably a halogen atom, an amino group, or an amino group protected by a protective group, a carboxyl group, or A carbonyl group having a leaving group, a hydroxyl group, a hydroxyl group protected by a protecting group, an aldehyde group, —NHNH ₂ , —N═C═O, —N═C═S, an epoxy group, or a vinyl group.
As the protecting group and the leaving group, the same as those described above can be used.

  Specific examples of the compound of the general formula (1) are shown below, but the compound of the general formula (1) that can be used in the present invention is not limited thereto. Another specific example of the compound of the general formula (1) is α-amino-ω-carboxyl-polyethylene glycol.

  In the present invention, the hydrophobic polymer is preferably applied using an organic solvent having a water content of 100 ppm or less, and more preferably 50 ppm or less.

  In the present invention, it is preferable to vacuum dry after applying the hydrophobic polymer film. The degree of vacuum is preferably 1 kPa or less, more preferably 10 Pa or less, and most preferably 0.1 Pa or less. The time for vacuum drying is preferably from 30 minutes to 100 hours, more preferably from 2 hours to 100 hours, and most preferably from 10 hours to 100 hours.

  In the sensor chip in which at least two kinds of surfaces of the present invention are patterned, it is preferable to wash with a liquid containing a carboxylic acid derivative after the patterning reaction. Specific examples of the carboxylic acid derivative include acetic acid, citric acid, oxalic acid, malonic acid, succinic acid, and the like, and citric acid is particularly preferable.

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

  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.

  The hydrophobic polymer compound used in the present invention is a polymer compound having no water absorption, and its solubility in water (25 ° C.) is 10% or less, more preferably 1% or less, most preferably 0.1% or less. It is.

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

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

  In the present invention, a hydrophobic polymer compound having an alkyl group substituted with a fluorine atom can also be used. Hydrophobic monomers that form hydrophobic polymer compounds having alkyl groups substituted with fluorine atoms include vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, and crotonic esters. Itaconic acid diesters, maleic acid diesters, fumaric acid diesters, allyl compounds, vinyl ethers, vinyl ketones and the like can be arbitrarily selected. 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.

  The hydrophobic polymer compound having an alkyl group substituted with a fluorine atom preferably has a fluorinated alkyl group as an ester in the molecule. Particularly preferred are acrylic acid esters and methacrylic acid esters.

The fluorinated alkyl group has 1 or more carbon atoms and may be linear, branched or cyclic (hereinafter, the alkyl group substituted with a fluorine atom is referred to as “Rf”).

  Rf is an alkyl group substituted with at least one fluorine atom having 1 or more carbon atoms, and Rf only needs to be substituted with at least one fluorine atom, and has a linear, branched or cyclic structure. There may be. Further, it may be further substituted with a substituent other than a fluorine atom, or may be substituted only with a fluorine atom.

  Examples of the substituent other than the fluorine atom of Rf include an alkenyl group, an aryl group, an alkoxyl group, a halogen atom other than fluorine, a carboxylic acid ester group, a carbonamido group, a carbamoyl group, an oxycarbonyl group, and a phosphoric acid ester group.

  Rf is preferably a fluorine-substituted alkyl group having 1 to 16 carbon atoms, more preferably 1 to 12 carbon atoms, and still more preferably 4 to 10 carbon atoms. Preferred examples of Rf are shown below.

Rf is more preferably an alkyl group having 4 to 10 carbon atoms, the terminal of which is substituted with a trifluoromethyl group, and particularly preferably a carbon number of 3 represented by — (CH ₂ ) _n1 — (CF ₂ ) _n2 F (An n ¹ represents an integer of ¹ to 6; n ² represents an integer of 3 to 8). Specifically, —CH ₂ — (CF ₂ ) ₂ F, — (CH ₂ ) ₆ — (CF ₂ ) ₄ F, — (CH ₂ ) ₃ — (CF ₂ ) ₄ F, —CH ₂ — (CF _{2) 3 F, - (CH} 2) 2 - (CF 2) 4 F, - (CH 2) 3 - (CF 2) 4 F, - (CH 2) 6 - (CF 2) 4 F, - (CH ₂ ) _2- (CF ₂ ) ₆ F,-(CH ₂ ) _3- (CF ₂ ) ₆ F,-(CH ₂ ) _2- (CF ₂ ) ₆ F, and the like. Among these, in _{particular, - (CH 2) 2 -} (CF 2) 4 F and _{- (CH 2) 2 - (} CF 2) 6 F is most preferred.

  The hydrophobic polymer compound having an alkyl group substituted with a fluorine atom may be a copolymer with another monomer. In that case, as an example of the copolymer, a methacrylic acid ester such as methyl methacrylate, an acrylic acid ester such as methyl acrylate, or styrene is preferable.

  Specific examples of the hydrophobic polymer compound having an alkyl group substituted with a fluorine atom include the following.

  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
Examples of hydrophobic polymer compounds and solvent SP values are: Polymethyl methacrylate-polystyrene copolymer (1: 1): solvent for 21.0, 2-phenoxyethanol: 25.3, solvent for polymethyl methacrylate: 20.3 Solvent toluene: 18.7 against acetonitrile: 22.9, polystyrene: 21.6.

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

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

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

  Although the thickness of the metal film is arbitrary, for example, when considering the use for a surface plasmon resonance biosensor, it is preferably 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 2000 angstrom or less. If it exceeds 5000 angstroms, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 1 angstrom or more and 100 angstrom or less.

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

  The metal film is preferably disposed on the substrate. Here, “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not directly in contact with the substrate, but through other layers. This also includes the case where they are arranged. As a substrate that can be used in the present invention, for example, 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.

  A part of the surface of the biosensor of the present invention may be modified with a linker having a terminal capable of binding to a physiologically active substance. The surface of the biosensor of the present invention preferably has at least two kinds of modifications. The linker having an end capable of binding to the physiologically active substance used in the present invention is preferably a hydrophilic linker, and examples thereof include a linker made of polyethylene glycol.

  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.

  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. The substance to be detected can be detected and / or measured.
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
The sensor chip of the present invention was produced by the following method.
(1) Gold film on plastic prism ZEONEX (manufactured by Nippon Zeon Co., Ltd.) was injection molded onto a plastic prism (FIG. 1) to form a gold thin film by the following method.
(1-1) Gold film A prism is attached to the substrate holder of the sputtering equipment, vacuum is applied (base pressure 1 × 10 ^-3 Pa or less), Ar gas is introduced (1 Pa), and the substrate holder is rotated (20 rpm) Then, RF power (0.5 kW) is applied to the substrate holder for about 9 minutes to plasma process the prism surface. Next, Ar gas was stopped and vacuum was drawn, Ar gas was introduced again (0.5 Pa), and DC power (0.2 kW) was applied to the 8-inch Cr target for about 30 seconds while rotating the substrate holder (10 to 40 rpm). This is applied to form a 2 nm Cr thin film. next. Stop Ar gas, pull vacuum again, introduce Ar gas again (0.5 Pa), rotate the substrate holder (20 rpm), and apply DC power (1 kW) to an 8-inch Au target for about 50 seconds to 50 nm A thin Au film is formed. The obtained sample is called chip A.

(2) Polymer application A polymer thin film was formed on the gold thin film of chip A by the following method.
(2-1) Preparation of polymer solution A 1.5 g of polymer (F-1) is dissolved in 100 mL of dehydrated MiBK (methyl isobutyl ketone) and filtered through a microfilter having a pore size of 0.45 μm. The water content of dehydrated MiBK is 20ppm.

(2-2) Spin coating Chip A is set on a spin coater (SC-408S sample sealed spin coater, manufactured by Oshikelle Co., Ltd.). The chip A is fixed at a position of 135 mm from the center of the spin coater as shown in FIG. 200 μL of the polymer solution A is cast so as to cover the entire gold film of the chip A. Next, a windshield cover is set so as to completely cover the chip A. Then spin at 200 rpm for 60 seconds. After the rotation stops, leave it for 5 minutes.

(2-3) Vacuum drying Chip A spin-coated with polymer is vacuum-dried for 16 hours. The obtained sample is called chip B.

(3) Hydrolysis of polymer surface By the following method, the polymer thin film surface of chip B was hydrolyzed to generate COOH groups on the outermost surface.
(3-1) Hydrolysis
Dip Chip B in 1N NaOH solution and store in a constant temperature bath at 60 ° C for 16 hours.
(3-2) Cleaning
After taking out from the thermostat at 60 ° C, it is naturally cooled for 15 minutes and then washed with ultrapure water. The obtained sample is called chip C.

(4) 5-aminovaleric acid bond The 5-aminovaleric acid reaction was covalently bonded to the COOH group present on the surface of chip C by the following method.
(4-1) Preparation of activation solution and 5-aminovaleric acid solution
0.1M NHS solution: Dissolve 1.16 g NHS (N-hydroxysulfosuccinimide) in ultrapure water to make 100 mL.
0.4M EDC solution: Dissolve 7.7 g of EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbodiimide Hydrochloride) in ultrapure water to make 100 mL.
1M 5-aminovaleric acid solution: 11.7 g of 5-aminovaleric acid is dissolved in 80 mL of ultrapure water and adjusted to pH 8.5 using 1N NaOH. Add ultrapure water to make 100 mL.

(4-2) Activation Chip C is drained with an air gun. Place Chip C in a wet box (tight box with wet cloth, keep the humidity above 90% RH in a sealed state), and cast 200 μL of a mixture of 100 μL of 0.1 M NHS solution and 100 μL of 0.4 M EDC solution. A PET film with a thickness of 175 μm cut into 120 mm × 8.5 mm is placed on it to cover the surface while spreading the liquid. In this reaction, the ratio of the surface area of the portion not in contact with air to the surface area of the portion in contact with air is 26. Seal the wet box and let stand at 25 ° C for 60 minutes.

(4-3) Washing Remove the PET film from the sample taken out of the wet box and wash it with pure water. The obtained sample is called chip D.

(4-4) 5-aminovaleric acid reaction
The 5-aminovaleric acid reaction starts within 1 hour after the activation reaction. First, the tip D is drained with an air gun. Place chip D in a wet box and cast 200 μL of 1M 5 aminovaleric acid solution. A PET film with a thickness of 175 μm cut into 120 mm × 8.5 mm is placed on it to cover the surface while spreading the liquid. In this reaction, the ratio of the surface area of the part not in contact with air to the surface area of the part in contact with air is 24. Seal the wet box and let stand at 25 ° C for 90 minutes.

(4-5) Washing Remove the PET film from the sample taken out of the wet box and wash with pure water. The obtained sample is called chip E.

(5) Pattern formation of non-ligand binding part The pattern of the part which cannot bind a ligand was formed on the surface of chip E by the following method. Specifically, PEG5000 (α-amino-ω-methoxy-polyethylene glycol) was covalently bonded to the COOH group of 5-aminovaleric acid at a specific location on chip E. Since the end of PEG5000 is a methoxy group, it cannot form a covalent bond with the ligand. This part is measured as a reference part at the time of analyte binding measurement.

(5-1) Preparation of reaction solution
20% PEG5000 solution: Dissolve 4.5 g of PEG5000 in 18.5 mL of ultrapure water and 4 mL of 1N NaOH.
0.2M NHS solution: Dissolve 2.32 g NHS in ultrapure water to make 100 mL.
0.4M EDC solution: Dissolve 7.7 g of EDC in ultrapure water to make 100 mL.
(5-2) Patterning reaction The chip E is drained with an air gun and fixed to a pedestal of a dispenser manufactured by Musashi Engineering. Next, a mixture of 1 mL of 20% PEG5000 solution, 1 mL of 0.2M NHS solution and 2 mL of 0.4M EDC solution is put into a syringe. The mixed solution is spotted on chip E at 15 points in intervals of 18 mm at 6 points. The diameter of the droplet is about 4 mm. Place spotted chip E in a wet box, seal the wet box, and leave it at 25 ° C. for 60 minutes.
(5-3) The sample taken out from the washing wet box is washed with a 1N aqueous citric acid solution and further washed with pure water. The obtained sample is called chip F.

(6) PEG Linker Binding A PEG linker (α-amino-ω-carboxyl-polyethylene glycol) was covalently bonded to the COOH group of 5-aminovaleric acid present on the surface of chip F by the following method. The PEG linker does not bind to the patterned surface.

(6-1) Preparation of activation solution and PEG linker solution
0.1M Sulfo-NHS solution: Dissolve 2.04 g NHS in ultrapure water to make 100 mL.
0.4M EDC solution: Dissolve 7.7 g of EDC in ultrapure water to make 100 mL.
10% PEG linker solution: 10 g PEG linker is dissolved in 80 mL of ultrapure water and adjusted to pH 8.5 using 1N NaOH. Add ultrapure water to make 100 mL.

(6-2) Activation Drain the tip F with an air gun. Chip F is set in a wet box, and 200 μL of a mixture of 100 μL of 0.1 M Sulfo-NHS solution and 100 μL of 0.4 M EDC solution is cast. A PET film with a thickness of 175 μm cut into 120 mm × 8.5 mm is placed on it to cover the surface while spreading the liquid. In this reaction, the ratio of the surface area of the portion not in contact with air to the surface area of the portion in contact with air is 26. Seal the wet box and let stand at 25 ° C for 60 minutes.

(6-3) Washing Remove the PET film from the sample taken out of the wet box and wash it with pure water. The obtained sample is called chip G.

(6-4) PEG linker reaction
The PEG linker reaction starts within 1 hour after completion of the activation reaction. First, the chip G is drained with an air gun and fixed to the pedestal of the dispenser made by Musashi Engineering. Next, 5 mL of 10% PEG linker solution is put into a syringe. 6 points of 10% PEG linker solution are spotted on chip G at 8 mm intervals at 18 mm intervals. A PET film with a thickness of 175 μm cut into 120 mm × 8.5 mm is placed on it to cover the surface while spreading the liquid. In this reaction, the ratio of the surface area of the part not in contact with air to the surface area of the part in contact with air is 90. Set this sample in a wet box, seal the wet box, and leave it at 25 ° C. for 16 hours.

(6-5) Washing The sample taken out from the wet box is washed with pure water. The obtained sample is called chip H.
(6-6) Storage Chip H is drained with an air gun and stored.

Example 2
In the sensor chip production method of Example 1, the following chip (chip P is a comparative example) in which only the following process was changed was produced.
Chip M: In (6-2), the reaction is performed without using a PET film. The ratio of the surface area of the portion not in contact with air to the surface area of the portion in contact with air is 0.7.
Chip N: In (6-4), 200 μL of 10% PEG linker solution is cast and reacted without using PET film. The ratio of the surface area of the portion not in contact with air to the surface area of the portion in contact with air is 0.7.
Chip P: In (6-2), the reaction is carried out without using a PET film. The ratio of the surface area of the portion not in contact with air to the surface area of the portion in contact with air is 0.7. In (6-4), 200 μL of 10% PEG linker solution is cast and reacted without using PET film. The ratio of the surface area of the portion not in contact with air to the surface area of the portion in contact with air is 0.7.

Example 3
The ligand protein was immobilized on each of 48 chips H, M and N of the present invention and 48 chips P of the comparative example by the following method, and the variation of the immobilized amount was evaluated. For protein fixation, the SPR device shown in FIG. 3 was used.

(1) Preparation of ligand solution: 0.5 mg of trypsin was dissolved in 1 ml of acetate buffer (pH 5.5).
(2) Activation of sensor chip surface: NHS (0.1M) / EDC (0.4M) = 1/1, 25 ° C., 30 minutes (3) Ligand fixation: the above ligand solution, 25 ° C., 30 minutes (4 ) Blocking: 1M ethanolamine solution (pH8.5), 25 ° C, 30 minutes

  The results are shown in Table 1 with the variation in the fixed amount as the CV value. If the CV value is 15% or more, it is not practical. The CV value is preferably 10% or less.

  From the results in Table 1, it can be seen that the chip of the present invention exhibits extremely excellent reproducibility of the amount of protein immobilized.

FIG. 1 shows a plastic prism used in the example. FIG. 2 shows a position when performing the spin coating in the embodiment. FIG. 3 shows an example of an SPR system for performing the screening method of the present invention.

Claims (7)

An optical glass or optical plastic substrate, a metal film, and a hydrophobic polymer film are laminated in this order, and further a method for producing a sensor chip in which at least one compound represented by the general formula (1) is bonded. When the reaction liquid for binding the compound represented by the general formula (1) to the sensor chip is brought into contact with the substrate surface of the sensor chip and reacted, the droplets of the reaction liquid during the reaction (in contact with air) The ratio of (surface area not in contact) and (surface area in contact with air) is greater than 1.
General formula (1): X-L-Y
(In the formula, X and Y each independently represent a group capable of reacting with an organic molecule, and L represents a divalent linking group.) The reaction liquid for binding the compound represented by the general formula (1) to the sensor chip is represented by the reaction liquid for activating the functional group present on the substrate and / or the general formula (1). The method for producing a sensor chip according to claim 1, which is a reaction solution containing a compound. The method for producing a sensor chip according to claim 1, comprising applying a hydrophobic polymer on the metal film using an organic solvent having a water content of 100 ppm or less. The method for producing a sensor chip according to claim 1, comprising applying a hydrophobic polymer onto the metal film and then vacuum drying. 5. The method includes performing a patterning reaction for patterning at least two types of surfaces on the surface of the sensor chip, and washing the surface of the sensor chip with a liquid containing a carboxylic acid derivative after the patterning reaction. A method for producing a sensor chip according to any one of the above. The sensor chip manufacturing method according to claim 1, wherein the sensor chip is a surface plasmon resonance measurement sensor chip. A sensor chip manufactured by the method according to claim 1.

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