JP4943888B2 - Biosensor manufacturing method - Google Patents

Description

  The present invention relates to a method for producing a biosensor and a method for analyzing an interaction between biomolecules using the biosensor. In particular, the present invention relates to a method for producing 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 detection surface having a functional group capable of immobilizing a physiologically active substance, for example, Patent Document 1 discloses a method for producing a hydrogel in detail. Specifically, a barrier layer is formed by bonding a 16-mercaptohexadecanol layer to a gold film. On this gold film, the hydroxyl group of the barrier layer is epoxy activated by treating with epichlorohydrin. In the next step, dextran is attached to the barrier layer via an ether linkage. Next, a carboxymethyl group is introduced by reacting the dextran matrix with bromoacetic acid.

  As a technique for immobilizing a physiologically active substance having an amino group (for example, a protein or amino acid) on the surface of a carboxymethyl-modified dextran produced based on this method, the following technique is disclosed. That is, for example, N-hydroxysuccinimide (NHS) and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) hydrochloric acid can be used to generate a reactive ester function in part of the carboxyl group in carboxymethyl-modified dextran. It is denatured by treating with an aqueous solution. Residual charges, i.e. unreacted carboxyl groups, will contribute to the concentration of the bioactive substance to the detection surface. By bringing an aqueous solution of a physiologically active substance (protein or amino acid) containing an amino group into contact with such a detection surface, the physiologically active substance containing an amino group can be covalently bound to the dextran matrix.

  The hydrogel produced by the method described above exhibits excellent performance as a detection surface of a biosensor because a physiologically active substance containing an amino group can be immobilized three-dimensionally. However, the hydrogel production method by the above-described method is complicated, has a long production time, and requires the use of a compound such as epichlorohydrin or bromoacetic acid.

  On the other hand, for immobilization of substances using photoreactive groups, a photoreactive group (such as an azidophenyl group) is introduced into a polymer and bound to a substrate together with protein (Patent Document 2), and the substrate is photoreactive. Introducing a group (benzophenone) and binding a hydrophobic polymer (polystyrene, polyethyloxazoline) (Non-patent Document 1), and immobilizing DNA fragments on a solid surface via a graft polymer to produce a DNA chip (Patent Document 3) has been reported.

J. Am. Chem. Soc., 121, 8766 (1999) Japanese Patent No. 2815120 JP 2004-125781 A JP 2003-130878 A

  The present invention has been made to solve the above-described problems of the prior art. That is, the present invention should solve the problem of providing a method for easily immobilizing a hydrogel capable of immobilizing a physiologically active substance on the surface of a biosensor using a safe raw material, and a biosensor produced by the above method. It was an issue.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors can form a covalent bond with the reactive group after binding a compound that generates a reactive group to the sensor substrate surface by an external stimulus. The inventors have found that a biosensor surface to which a hydrophilic polymer is bound can be easily produced by applying an external stimulus while the hydrophilic polymer is in contact with the substrate, and the present invention has been completed.

  That is, according to the present invention, after a compound that generates a reactive group by an external stimulus is bonded to the substrate surface, a hydrophilic polymer that can form a covalent bond with the reactive group is contacted with the substrate in an external state. Providing a stimulus provides a method for producing a biosensor comprising a substrate coated with a hydrophilic polymer, comprising binding the hydrophilic polymer to the substrate surface via a covalent bond with the reactive group. .

Preferably, the compound that generates a reactive group by an external stimulus is a photo radical generator.
Preferably, the hydrophilic polymer is a hydrophilic polymer having a double bond.
Preferably, the hydrophilic polymer is a hydrophilic polymer having a carboxyl group.
Preferably, the polymer containing carboxyl groups is a polysaccharide.
Preferably, the average molecular weight of the polymer containing a carboxyl group is 1000 to 5000000.

Preferably, the hydrophilic polymer is bonded to a reactive group generated by an external stimulus in a state where a thin film is formed on the substrate.
Preferably, a thin film is formed on the substrate by spin coating or spray coating.

Preferably, the substrate is a metal surface or a metal film.
Preferably, the metal is any of gold, silver, copper, platinum or aluminum.

According to another aspect of the present invention, there is provided a biosensor produced by the above-described method of the present invention.
Preferably, the biosensor of the present invention is used for non-electrochemical detection, more preferably for surface plasmon resonance analysis.

  According to still another aspect of the present invention, a bioactive substance is immobilized on a biosensor comprising the step of bringing the biosensor of the present invention into contact with a bioactive substance and binding the bioactive substance to the biosensor. A method 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, and more preferably, the substance that interacts with the physiologically active substance is detected or measured by surface plasmon resonance analysis.

  According to the present invention, a hydrogel capable of immobilizing a physiologically active substance can be simply immobilized on the surface of a biosensor using a safe raw material.

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

  In the biosensor of the present invention, a metal surface or a metal film can be used as a substrate. 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, the thickness is preferably 0.1 nm to 500 nm, particularly preferably 1 nm to 200 nm. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

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

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

  In the present invention, a compound that generates a reactive group by an external stimulus is bound to the biosensor surface, that is, the metal film. The compound that generates a reactive group by external stimulation may be a compound that generates radicals by heat (thermal radical generator) or a compound that generates radicals by light (photo radical generator). Further, it may be a compound known as a photoaffinity labeling agent.

  As the thermal radical generator, a known polymerization initiator or a compound having a bond with a small bond dissociation energy can be used. Examples of the thermal radical generator include organic halogenated compounds, carbonyl compounds, organic peroxide compounds, azo polymerization initiators, azide compounds, metallocene compounds, hexaarylbiimidazole compounds, organic boric acid compounds, disulfonic acid compounds, oxime esters. Compounds, onium salt compounds, and the like. These thermal radical generators may be used alone or in combination of two or more. Specific compound skeletons of the thermal radical generator are disclosed in [Chemical Formula 4] to [Chemical Formula 14] of JP-A-2005-335366.

  The photoradical generator has a single bond that is cleaved by light. As the single bond that is cleaved by light, carbonyl α-cleavage, β-cleavage reaction, photofleece rearrangement reaction, phenacyl ester cleavage reaction, sulfonimide cleavage reaction, sulfonyl ester cleavage reaction, N-hydroxysulfonyl ester cleavage reaction, benzyl Examples thereof include a single bond that can be cleaved using an imide cleavage reaction, a cleavage reaction of an active halogen compound, and the like. These reactions break a single bond that can be cleaved by light. Examples of the single bond that can be cleaved include a C—C bond, a C—N bond, a C—O bond, a C—Cl bond, a N—O bond, and a S—N bond. Examples of the group having a single bond that is cleaved by light include an aromatic ketone group, a phenacyl ester group, a sulfonimide group, a sulfonyl ester group, an N-hydroxysulfonyl ester group, a benzylimide group, a trichloromethyl group, and a benzyl chloride group. Is mentioned.

  The photoradical generator preferably has a reactive group (base material binding group) that can react and bond with a functional group present on the surface of the substrate. Specific examples of the reactive group include the following: And groups as shown below.

  The group having a single bond that is cleaved by light and the substrate binding group may be directly bonded or may be bonded via a linking group. Examples of the linking group include a linking group containing an atom selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. Specifically, for example, a saturated carbon group, an aromatic group, an ester group, an amide group. Ureido group, ether group, amino group, sulfonamide group, and the like. The linking group may further have a substituent, and examples of the substituent that can be introduced include an alkyl group, an alkoxy group, and a halogen atom.

  These photo radical generators may be used alone or in combination of two or more. Specific compound skeletons of the photoradical generator include the following compounds described in [Chemical Formula 2] to [Chemical Formula 4] of JP-A-2005-277225, but are not limited thereto. Absent.

  As a compound that generates a reactive group by light irradiation, a compound used for photoaffinity labeling can also be preferably used. Examples of the active species generated from the photoaffinity label compound by light irradiation include carbon electrophiles such as nitrene, carbene, radical, ion radical, diradical, excited triplet, and carbocation. Examples of photoaffinity label compounds that generate these active species upon irradiation with light are described in, for example, S. Tetrahedron, Vol. 51, pages 12479-12520, published in 1995. A. It is described in a review by Flemig and a review by Yasumaru Hatanaka, Vol. 56, pages 581 to 590, published in 1998. That is, the compound generating nitrene is a compound having an azide group such as an aromatic azide, alkyl azide, and heterocyclic azide, and the compound generating carbene is a compound having a diazo group or a diazirine ring, Compounds that generate are conjugated ketones such as benzophenones and enones, aromatic halides, olefins, etc., and compounds that generate carbon nucleophiles include aromatic diazonium salts, nitrobenzenes, sulfonium salts, Examples thereof include phosphonium salts and ammonium salts. Since each of these photoreactive compounds has characteristics, it can be selected and used according to the use conditions, but a compound having a diazirine ring is preferable in terms of relatively high stability. In terms of stability and ease of synthesis, conjugated ketones, aromatic halides, olefins and the like are preferable. These photoaffinity label compounds may be used alone or in combination of two or more. Specific examples of the compound skeleton of the photoaffinity label compound include, but are not limited to, the following compounds described in [Chemical Formula 3] of JP-A No. 2004-301681.

  Since the patterning on the substrate surface is easy, the compound that generates a reactive group by external stimulation is preferably a photoradical generator or a photoaffinity label compound.

In the present invention, it is desirable that the compound that generates a reactive group by an external stimulus is bonded to a metal film covered with a self-assembled film. The coating method of metal films using self-assembled films (SAMs) has been vigorously developed by Prof. Whitesides et al. At Harvard University. The details are reported in, for example, Chemical Review, 105 , 1103-1169 (2005). ing. When gold is used as the metal, by using an alkanethiol derivative represented by general formula 1 (in general formula 1, n represents an integer of 3 to 20 and X represents a functional group) as an organic layer forming compound, Au Based on the van der Waals force between the -S bond and the alkyl chain, a monolayer with orientation is self-organized. The self-assembled film is prepared by an extremely simple technique in which a gold substrate is immersed in a solution of an alkanethiol derivative. In the present invention, it is preferable to use a self-assembled film having an amino group from the viewpoint that a reactive group can be easily bound by an external stimulus. By forming a self-assembled film using a compound where X = NH ₂ in the general formula 1, it is possible to cover the gold surface with an organic layer having an amino group.

The repeating number of the alkyl group of the general formula 1 is preferably 3 or more and 20 or less, more preferably 3 or more and 16 or less, and most preferably 4 or more and 8 or less. When the alkyl chain is short, it is difficult to form a self-assembled film, and when the alkyl chain is long, water solubility is lowered and handling becomes difficult.

  The alkanethiol having an amino group at the end may be a compound in which the thiol group and the amino group are linked via an alkyl chain (general formula 2) (in the general formula 2, n represents an integer of 3 to 20), Alkanethiol having a carboxyl group at the terminal (general formulas 3 and 4) (in general formula 3, n represents an integer of 3 to 20, and in general formula 4 n represents an integer of 1 to 20 each independently) and a large excess Or a compound obtained by reacting hydrazide or polyamine. The reaction between an alkanethiol having a carboxyl group at the terminal and a large excess of hydrazide or polyamine may be carried out in a solution state, or after binding an alkanethiol having a carboxyl group at the terminal to the substrate surface, a large excess of hydrazide Or you may make a polyamine react.

  Specific examples of the polyamine used in the present invention include fats such as ethylenediamine, tetraethylenediamine, octamethylenediamine, decamethylenediamine, piperazine, triethylenediamine, diethylenetriamine, triethylenetetraamine, dihexamethylenetriamine, and 1.4-diaminocyclohexane. Group diamine, paraphenylenediamine, metaphenylenediamine, paraxylylenediamine, metaxylylenediamine, 4,4'-diamonobiphenyl, 4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenyl ketone, 4,4 Aromatic diamines such as' -diaminodiphenylsulfonic acid, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, spermidine, sperm , It is possible to use preferably a polyamine such as polyethyleneimine. From the viewpoint of improving the hydrophilicity of the biosensor surface, it is also possible to use a compound (general formula 5) in which two amino groups are linked by an ethylene glycol unit.

The alkanethiol having an amino group can form a self-assembled film alone, or can be mixed with other alkanethiols to form a self-assembled film. When used on the biosensor surface, it is preferable to use a compound capable of suppressing nonspecific adsorption of a physiologically active substance as the other alkanethiol. The self-assembled membrane that can suppress non-specific adsorption of physiologically active substances has been studied in detail by the aforementioned Professor Whitesides et al. The self-assembled membrane formed from alkanethiol having a hydrophilic group is non-specifically adsorbed It has been reported to be effective for suppression (Langmuir, 17 , 2841-2850, 5605-5620, 6336-6343 (2001)). In the present invention, as the alkanethiol that forms a mixed monomolecular film with an alkanethiol having an amino group, the compounds described in the above paper can be preferably used. Alkane thiol (general formula 6) or ethylene glycol unit having a hydroxyl group as an alkane thiol that forms a mixed monomolecular film with an alkane thiol having an amino group, because of its excellent non-specific adsorption suppression ability and easy availability An alkanethiol having the following formula (general formula 7) (in general formula 6, n represents an integer of 3 to 20, and in general formula 7, n and m each independently represents an integer of 1 to 20). preferable.

When an alkanethiol having an amino group is mixed with another alkanethiol to form a self-assembled film, the number of repeating alkyl groups of formulas 2 to 4 is preferably 4 or more and 20 or less, and more preferably 4 or more and 16 or less. It is preferably 4 or more and 10 or less. Further, the repeating number of the alkyl groups of the general formulas 6 and 7 is preferably 3 or more and 16 or less, more preferably 5 or more and 12 or less, and most preferably 5 or more and 10 or less.

  In the present invention, the alkanethiol having an amino group and the alkanethiol having a hydrophilic group can be mixed at an arbitrary ratio. However, when the ratio of the alkanethiol having an amino group is small, it is an active ester. In addition, the binding amount of the carboxyl group-containing polymer is decreased, and when the proportion of alkanethiol having a hydrophilic group is small, the nonspecific adsorption suppressing ability is decreased. Therefore, the mixing ratio of the alkanethiol having an amino group and the alkanethiol having a hydrophilic group is preferably in the range of 1/1 to 1 / 1,000,000, and preferably 1/4 to 1 / 10,000. Is more preferable, and the range of 1/10 to 1/1000 is more preferable. From the viewpoint of reducing steric hindrance when reacting with a polymer containing an active esterified carboxyl group, the molecular length of the alkanethiol having an amino group is preferably longer than the molecular length of the alkanethiol having a hydrophilic group. .

The alkanethiol used in the present invention may be a compound synthesized based on a review by Prof. Grzybowski et al. Of Northwestern University (Curr. Org. Chem., 8 , 1763-1797 (2004)) and the cited references. Commercially available compounds may also be used. These compounds can be purchased from Dojindo Co., Ltd., Aldrich, SensoPath Technologies, Frontier Scientific Inc., etc. In the present invention, the disulfide compound that is an oxidation product of alkanethiol can be used in the same manner as alkanethiol.

  As the hydrophilic polymer that can be used in the present invention, a known compound can be used. However, in consideration of the use as a sensor surface, a carboxyl group capable of forming a covalent bond with an amino group of a physiologically active substance. It is preferable that it is a polymer containing. These hydrophilic polymers need to be covalently bonded to reactive groups generated by external stimuli. Therefore, these hydrophilic polymers preferably have a functional group such as a C—H bond capable of extracting hydrogen, an unsaturated double bond, an epoxy group, or an oxetane group. From a synthetic viewpoint, a hydrophilic polymer into which a double bond is introduced is preferable. By utilizing a reaction with a functional group such as a carboxyl group, an amino group or a salt thereof, a hydroxyl group, and an epoxy group in the hydrophilic polymer, a double bond can be introduced into the hydrophilic polymer. Compounds used to introduce double bonds in this reaction include (meth) acrylic acid, glycidyl (meth) acrylate, allyl glycidyl ether, 2-isocyanatoethyl (meth) acrylate, 2-aminoethyl (meth) There are criates. In addition to these compounds, there is also a method of introducing a double bond by base treatment after introducing a functional group such as 2-methyl-2-bromopropionic acid.

  As the polymer containing a carboxyl group used in the present invention, a carboxyl group-containing synthetic polymer and a carboxyl group-containing polysaccharide can be used. Examples of the carboxyl group-containing synthetic polymer include polyacrylic acid, polymethacrylic acid, and copolymers thereof, for example, JP-A-59-44615, JP-B-54-34327, JP-B-58-12777, JP-B-54- No. 25957, JP-A-59-53836, JP-A-59-71048, methacrylic acid copolymer, acrylic acid copolymer, itaconic acid copolymer, crotonic acid copolymer , Maleic acid copolymers, partially esterified maleic acid copolymers, and those obtained by adding an acid anhydride to a polymer having a hydroxyl group. The carboxyl group-containing polysaccharide may be any one of an extract from a natural plant, a product of microbial fermentation, an enzymatic synthesis, or a chemical synthesis. Specifically, hyaluronic acid, chondroitin sulfate, heparin, Examples include dermatanic acid sulfuric acid, carboxymethylcellulose, carboxyethylcellulose, ceurouronic acid, carboxymethylchitin, carboxymethyldextran, and carboxymethyl starch. As the carboxyl group-containing polysaccharide, a commercially available compound can be used. Specifically, CMD, CMD-L, CMD-D40 (manufactured by Meisho Sangyo Co., Ltd.), sodium carboxymethylcellulose (Japanese) Kogaku Pharmaceutical Co., Ltd.), sodium alginate (Wako Pure Chemicals Co., Ltd.), and the like.

  The polymer containing a carboxyl group is preferably a polysaccharide containing a carboxyl group, more preferably carboxymethyl dextran.

  Although the molecular weight of the polymer containing a carboxyl group used in the present invention is not particularly limited, the average molecular weight is preferably 1,000 to 5,000,000, more preferably 10,000 to 2,000,000, and the average molecular weight is 100,000 to 1,000,000. More preferably it is. When the average molecular weight is smaller than this range, the fixed amount of the physiologically active substance becomes small, and when the average molecular weight is larger than this range, the handling becomes difficult due to the high solution viscosity.

  In the present invention, the hydrophilic polymer may be reacted with the substrate as a solution, or may be reacted in a state where a thin film on the substrate is formed using a technique such as spin coating. Preferably, the reaction is in a state where a thin film is formed.

  As described above, in the present invention, the hydrophilic polymer is preferably reacted with the substrate in a thin film state. As a method for forming a thin film on a substrate, known methods can be used. Specifically, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spin coating method, and a spray coating method. , Slide bead coating method, slit and spin method, slit coating method, die coating method, dip coating method, knife coating method, blade coating method, flow coating method, roll coating method, wire bar coating method, transfer printing method, etc. It is possible. For these thin film formation methods, "Progress in coating technology" by Yuji Harasaki, Comprehensive Technology Center (1988), "Coating Technology" Technical Information Association (1999), "Aqueous Coating Technology" CMC (2001), "Evolution" Described in "Organic Thin Film Deposition", Sumibe Techno Research (2004), "Polymer Surface Processing" by Iwamori Satoshi, Gihodo Publishing (2005), etc. Since a coating film with a controlled film thickness can be easily produced, the method for forming a thin film on a substrate in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method.

  The spray coating method is a method in which a polymer solution is uniformly coated on a substrate by moving the substrate in a state where a fine polymer solution is sprayed on the substrate. When the trigger of the spray gun is pulled, the air valve and the needle valve are simultaneously opened, the polymer solution is ejected from the nozzle in a mist state, and the atomized polymer solution is further refined by the air ejected from the air cap at the tip of the nozzle. A polymer film with a controlled film thickness is easily prepared by forming a coating film of a fine polymer solution on the substrate surface and then evaporating the solvent. The film thickness of the polymer thin film can be controlled by the polymer solution concentration, the moving speed of the substrate, and the like.

  The spin coating method is a method in which a polymer solution is dropped on a horizontally placed substrate and then rotated at a high speed, and the polymer solution is uniformly applied to the entire substrate by centrifugal force. A polymer film with a controlled film thickness is easily produced with the scattering of the polymer solution by the centrifugal force and the evaporation of the solvent. The film thickness of the polymer thin film can be controlled by the rotation speed, polymer solution concentration, solvent vapor pressure, and the like. In the present invention, the rotation speed during spin coating is not particularly limited, but if the rotation speed is too low, the solution remains on the substrate, and if the rotation speed is too high, usable devices are limited. Therefore, in this study, the rotation speed during spin coating is preferably 500 rpm to 10000 rpm, and more preferably 1000 rpm to 7000 rpm.

  The hydrophilic polymer used in the present invention preferably has a film thickness in an aqueous solution of 1 nm to 300 nm. If the film thickness is thin, the amount of the physiologically active substance immobilized decreases, and the hydrated layer on the sensor surface becomes thin, so that the interaction with the analyte substance becomes difficult to detect due to the denaturation of the physiologically active substance itself. When the film thickness is thick, it becomes an obstacle that the analyte substance diffuses into the film, and the distance from the detection surface to the interaction forming part is particularly large when detecting the interaction from the opposite side of the hydrophilic polymer fixing surface of the sensor substrate. Longer and lower detection sensitivity. The hydrophilic polymer film thickness in the aqueous solution can be evaluated by AFM, ellipsometry, or the like.

  As a method of activating a polymer containing a carboxyl group, a known method, for example, a method of activating with water-soluble carbodiimide 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) , A method of activating by EDC alone, a method described in Japanese Patent Application No. 2004-238396 (Japanese Patent Laid-Open No. 2006-58071) (that is, any compound of uronium salt, phosphonium salt, or triazine derivative having a specific structure) A method for activating a carboxyl group using an acid), a method described in Japanese Patent Application No. 2004-275012 (Japanese Patent Application Laid-Open No. 2006-90781) (ie, a nitrogen-containing heteroaromatic having a hydroxyl group activated by a carbodiimide derivative or a salt thereof) Of aromatic compounds having thiol groups, phenol derivatives having electron withdrawing groups Things a carboxyl group can be preferably used a method) or the like to activate with. By reacting a polymer containing a carboxyl group activated by these methods with a substrate having an amino group, the biosensor of the present invention can be produced.

  The uronium salt, phosphonium salt, or triazine derivative having a specific structure in Japanese Patent Application No. 2004-238396 described above is a uronium salt represented by the following general formula 1, a phosphonium salt represented by the following general formula 2, Or the triazine derivative represented by the following general formula 3 is shown.

(In General Formula 1, R ₁ and R ₂ each independently represent an alkyl group having 1 to 6 carbon atoms, or together, form an alkylene group having 2 to 6 carbon atoms to form a ring together with an N atom. And R ₃ represents an aromatic ring group having 6 to 20 carbon atoms or a heterocyclic group containing at least one hetero atom, and X ⁻ represents an anion, wherein R ₄ and R ₅ are each independently Represents an alkyl group having 1 to 6 carbon atoms, or together with each other, an alkylene group having 2 to 6 carbon atoms is formed to form a ring together with an N atom, and R ₆ is an aromatic ring having 6 to 20 carbon atoms A group or a heterocyclic group containing at least one heteroatom, and X ⁻ represents an anion, wherein R ₇ represents an onium group, and R ₈ and R ₉ each independently represent an electron donating group. .)

  In the present invention, the reactive group introduced into the polymer as the immobilizing group for the physiologically active substance is, for example, a biotin-binding protein (eg, avidin, streptavidin, neutravidin) in addition to a functional group such as a carboxyl group or an amino group. , A biologically active substance such as protein A, protein G, antigen, antibody (for example, a known tag antibody such as anti-GST antibody) is immobilized in advance, and a physiologically active substance as shown below is further formed on the physiologically active substance. A mode of immobilizing can be possible. In addition, if an immobilization layer in which alkane is introduced into a polymer is used, a physiologically active substance having a membrane structure such as lipid can be immobilized. In addition, by adjusting the length of the polymer chain, the thickness of the polymer, the density of the polymer, or the amount of reactive groups introduced into the polymer, it is possible to deal with various proteins depending on the application. If NTA (nitrilotriacetic acid) or the like is introduced into the polymer as a fixing group, the His-tag ligand or the like can be fixed via a metal chelate.

  The physiologically active substance immobilized on the biosensor 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-immune proteins, Examples include 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. Also, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit a specific reaction with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

  The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.

  As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.

  Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

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

  These physiologically active substances are preferably immobilized by applying a solution containing the physiologically active substance onto a substrate on which a hydrophilic polymer is immobilized and drying.

  The concentration of the solution (coating solution) containing the physiologically active substance is preferably higher in the concentration of the physiologically active substance immobilized on the substrate surface. Although it varies depending on the physiologically active substance, it is preferably 0.1 mg / mL to 10 mg / mL, more preferably 1 mg / mL to 10 mg / mL.

  In the drying process of a solution containing a physiologically active substance, the physiologically active substance tends to be deposited on the outer periphery of the applied solution or on the portion where the liquid remains until just before the coating solution is dried. This undesirably causes a distribution in the amount of the physiologically active substance immobilized on the substrate surface. In order to make the amount of the physiologically active substance immobilized on the substrate surface uniform, it is preferable to increase the viscosity of the coating solution as long as the binding of the physiologically active substance to the substrate surface is not inhibited. By increasing the viscosity of the coating solution, the horizontal movement of the physiologically active substance in the coating solution in the drying process with respect to the substrate surface can be suppressed, and as a result, variations in the amount of physiologically active substance fixed can be suppressed. The viscosity of the coating solution in the drying process is preferably kept at 0.9 cP or higher.

  The drying step of the present invention means that after application of a solution containing a physiologically active substance, the solution is naturally dried by standing, or the solution is dried by heating or blowing, and the solution is intentionally dried. Refers to a process. Here, increasing the drying rate of a solution (coating solution) containing a physiologically active substance is effective in suppressing variations in the amount of physiologically active substance immobilized. By increasing the drying speed and finishing the drying sufficiently quickly with respect to the horizontal movement speed of the physiologically active substance, it is possible to finish the drying before the physiologically active substance substantially moves and to suppress variations. Become. The method of increasing the drying speed is not particularly limited, but the coating solution temperature and drying environment temperature are increased, the evaporation energy is applied by irradiation with infrared rays, laser, etc., the solvent vapor pressure during drying is lowered by blowing, the solution is applied in a thin layer And a method of increasing the evaporation area with respect to the coating amount. In particular, when the coating solution contains water, the drying speed is increased by drying in an environment having a large difference between the dry bulb temperature and the wet bulb temperature. It is preferable to dry in an environment where the temperature difference between the dry bulb temperature and the wet bulb temperature is 7 ° C or higher, more preferably 10 ° C or higher, and further preferably 13.5 ° C or higher. Further, in the production process, the drying time is preferably within 10 minutes, more preferably within 5 minutes, and particularly preferably within 1 minute.

  Examples of a method for applying a solution containing a physiologically active substance include a method using a dispenser that dispenses a coating solution in a fixed amount. By operating the discharge port of the dispenser on the substrate at a constant speed and a constant interval, it is possible to uniformly apply to any place on the substrate. In the case of applying with a dispenser, the distance between the substrate and the discharge port is made as narrow as possible, and the thickness of the physiologically active substance can be made uniform by reducing the thickness of the coating solution, and the drying speed can be increased, which is preferable. A preferred method for applying a solution containing a physiologically active substance is spin coating. This method is particularly preferable when the coating film thickness is reduced. In order to dry after forming a solution having a uniform thickness, the spin coater preferably prevents evaporation of the solvent during rotation. For this reason, it is particularly preferable because the thin film formation during the rotation and the drying speed after the thin film formation can be controlled by maintaining the environment in which the solvent concentration around the substrate is high by a method such as placing the substrate in a sealed container at the time of rotation. . After these coating steps, it is preferable to dry under conditions where the temperature and humidity are kept constant.

  When detecting the interaction between the physiologically active substance and the analyte, the variation in the amount of the physiologically active substance immobilized on the sensor surface causes an error in the quantitative and kinetic evaluation of the interaction. For the purpose of minimizing this error, it is preferable to make the fixed amount of the physiologically active substance uniform. The variation of the fixed amount of the physiologically active substance on the substrate surface used for the detection of the interaction is preferably 15% or less, more preferably 10% or less in terms of CV value (variation coefficient) (standard deviation / average value). The CV value can be calculated from a fixed amount of at least 2 points on the substrate surface, preferably 10 points or more, more preferably 100 points or more. Uniformity can also be evaluated by quantifying the amount of the substance on the sensor substrate before and after immobilizing the physiologically active substance, but this is done by fluorescently labeling a substance known to bind to the physiologically active substance. It is also possible to measure the fluorescence intensity using a fluorescence microscope after fixing the labeling substance to the sensor substrate. In addition, physiologically active substances can be quantified using an SPR imager, ellipsometer, TOF-SIMS, ATR-IR apparatus or the like.

  In the present invention, a compound having a residue capable of forming a hydrogen bond (hereinafter referred to as Compound S) may be used for the purpose of improving the storage stability of a physiologically active substance immobilized on a substrate. .

  In general, physiologically active substances such as proteins maintain a three-dimensional structure by coordinating water molecules in an aqueous solution. However, when dried, they cannot retain the three-dimensional structure and are deactivated. Moreover, when it is carried in the hydrophilic polymer on the surface of the substrate, the physiologically active substances come close to each other and aggregate due to drying. The compound S having a residue capable of forming a hydrogen bond according to the present invention suppresses inactivation by retaining the three-dimensional structure of the physiologically active substance instead of the water molecule, or covers the physiologically active substance to provide a steric effect. Can be used for the purpose of suppressing aggregation.

  In the present invention, the compound S having a residue capable of forming a hydrogen bond is preferably added to a layer in which a physiologically active substance on a substrate is fixed with an aqueous solution. As an addition method, it may be applied to the substrate surface as a mixed solution with a physiologically active substance, or may be added by overcoating after fixing the physiologically active substance on the substrate surface. When applied as a mixed solution of Compound S and a physiologically active substance, variation in the amount of the physiologically active substance immobilized can be suppressed. Compound S aqueous solution is preferably added to the substrate in a thin film state. As a method for forming a thin film on a substrate, known methods can be used. Specifically, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spin coating method, and a spray coating method. , Slide bead coating method, slit and spin method, slit coating method, die coating method, dip coating method, knife coating method, blade coating method, flow coating method, roll coating method, wire bar coating method, transfer printing method, etc. It is possible. Since a coating film with a controlled film thickness can be easily produced, the method for forming a thin film on a substrate in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method.

  The concentration of the coating solution of Compound S is not particularly limited as long as there is no problem of coating properties and penetration into a layer containing a physiologically active substance, but it is preferably 0.1% by weight or more and 5% by weight or less. In addition, a surfactant, a buffering agent, an organic solvent, a salt, and the like may be added to the coating solution from the viewpoints of coating properties and pH adjustment.

  The compound S having a residue capable of forming a hydrogen bond is preferably a non-volatile compound at normal temperature and normal pressure, preferably having an average molecular weight of more than 350 and less than 5 million, more preferably 1200 or more and 2 million or less. Most preferably, it is 1200 or more and 70,000 or less. The compound S containing a hydroxyl group in the molecule is preferably a saccharide, and the saccharide may be a monosaccharide or a polysaccharide. In the case of n-saccharides, n is preferably 4 or more and 1200 or less, and more preferably n is 20 or more and 600 or less.

  If the average molecular weight of Compound S is low, it will crystallize on the surface of the substrate, causing the destruction of the hydrophilic polymer layer to which the physiologically active substance is immobilized and the three-dimensional structure of the physiologically active substance. Problems such as an obstacle to fixing the active substance to the substrate, impregnation into a layer containing a physiologically active substance, and occurrence of layer separation occur.

  For the purpose of suppressing the deterioration of the physiologically active substance immobilized on the substrate, the compound S having a residue capable of forming a hydrogen bond preferably has a dextran skeleton or a polyethylene oxide skeleton, thereby achieving the object of the present invention. Any substituent may be used as long as it is within the range. Moreover, it is preferable that it is a nonionic compound which does not have a dissociable group for the purpose of suppressing deterioration of the bioactive substance fixed on the board | substrate. The compound S having a residue capable of forming a hydrogen bond is preferably a compound having a high affinity for water molecules, and the partition coefficient LogP value between water and n-octanol is preferably 1 or more. The LogP value can be measured by a method described in JIS standard Z7260-107 (2000) "Measurement of distribution coefficient (1-octanol / water)-shaking method".

  Specific examples of the compound S having a residue capable of forming a hydrogen bond include polyhydric alcohols such as polyvinyl alcohol, proteins such as collagen, gelatin, and albumin, hyaluronic acid, chitin, chitosan, starch, cellulose, alginic acid, and dextran. Polysaccharides such as Polysaccharides, Polyethylene Glycol, Polyethylene Oxide, Polypropylene Glycol, Polypropylene Oxide, Pluronic and other Polyoxyxy-Polypropylene Oxide Condensates, Tween 20, Tween 40, Tween 60, Tween 80 and Two Residues Or a derivative and a polymer of these compounds. Of these, polysaccharides and polyethers are preferable, and polysaccharides are more preferable. Specifically, dextran, cellulose, tween 20, tween 40, tween 60, and tween 80 are preferably used. Furthermore, the non-volatile monomer and non-volatile water-soluble oligomer described in JP-A-2006-170832 can also be used. For example, the non-volatile monomer may be tetrose, pentose, heptose, hexose and its glycoxide whose hydroxyl group may be protected with a protecting group, or methyl glucoside or cyclitols whose hydroxyl group may be protected with a protecting group. In addition, the nonvolatile water-soluble oligomer is represented by the formula (1), (2) or (3)-[CH2-CH (CONH2)-] n- (1)-[CH2-CH2-O-] n- (2)- [CH2-CH (OH)-] n- (3) (in the formulas (1) to (3), n represents any integer of 10 to 200), and the hydroxyl group is a protecting group. It is also possible to use oligosaccharides of n sugars (where 2 ≦ n ≦ 10) which may be protected. Furthermore, saccharides described in US 2003/0175827, DE 20306476A1, for example, trehalose, sucrose, maltose, lactose, xylitol, fructose, mannitol, glucose, xylol, maltodextran, saccharose, polyvinylpyrrolidone, and the like may be used. These compounds S are preferably substantially the same as the basic skeleton of the hydrophilic polymer used in the present invention. Here, the basic skeleton refers to, for example, a sugar ring structure, and is substantially the same if the ring structures are the same even if the functional groups and length are different.

  The content of the compound S having a residue capable of forming a hydrogen bond on the substrate is a ratio of the average molecular weight, and the ratio of the average molecular weight of the compound S to the average molecular weight of the hydrophilic polymer is 0.005 or more and 0. .2 or less is preferable. When the ratio is lower than this, the compound S is easily crystallized, and when it is higher, the penetration into the hydrophilic polymer layer is difficult. By setting the ratio within this range, these problems can be solved, and the physiologically active substance The deactivation suppression effect and the aggregation suppression effect can be obtained higher.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  Note that in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of total reflection attenuation (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application Laid-Open No. 2003-106926 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

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

  When the biosensor of the present invention is used for surface plasmon resonance analysis, it can be applied as a part of various surface plasmon measurement devices as described above.

  The biosensor of the present invention can be used as a biosensor that detects a change in refractive index using a waveguide, for example, having a waveguide structure on the substrate surface. In this case, the waveguide structure on the surface of the substrate has a diffraction grating and possibly an additional layer. The waveguide structure consists of a planar waveguide made of a thin dielectric layer. Light rays collected in the waveguide are guided into this thin layer by total reflection. The propagation speed of the guided light wave (hereinafter referred to as mode) takes the value of C / N. Here, C is the speed of light in vacuum, and N is the effective refractive index of the mode guided in the waveguide. The effective refractive index N is determined by the configuration of the waveguide on one side and the refractive index of the medium adjacent to the thin waveguide layer on the other side. Light wave conduction is performed not only in a thin planar layer, but also by another waveguide structure, in particular a strip-shaped waveguide. In that case, the waveguide structure is in the form of a strip-like film. An important factor for a biosensor is that the change in the effective refractive index N is caused by a change in the medium adjacent to the waveguiding layer and a change in the refractive index and thickness of the waveguiding layer itself or an additional layer adjacent to the waveguiding layer. It is.

  Regarding the configuration of the biosensor of this system, for example, from Japanese Patent Publication No. 6-27703, page 4, line 48 to page 14, line 15 and FIG. 1 to FIG. 8, US Pat. No. 6,829,073, column 6, line 31 It is described in the 47th line of column7 and FIGS. 9A and B.

  For example, as one embodiment, there is a structure in which a waveguide layer having a flat thin layer is provided on a base material (for example, Pyrex (registered trademark) glass). The waveguide layer and the substrate together form a so-called waveguide. The waveguide layer can be, for example, an oxide layer (SiO2, SnO2, Ta2O5, TiO2, TiO2-SiO2, HfO2, ZrO2, Al2O3, Si3N4, HfON, SiON, scandium oxide or a mixture thereof), a plastic layer (for example, polystyrene, polyethylene, A multilayer laminate such as polycarbonate) is possible. In order for light rays to propagate through the waveguide layer by total reflection, the refractive index of the waveguide layer must be greater than the refractive index of an adjacent medium (for example, a base material or an additional layer described later). A diffraction grating is disposed on the surface of the waveguide layer or the volume of the waveguide layer facing the substrate or the measurement substance. The diffraction grating can be formed in the substrate by embossing, holography or other methods. A thin waveguide film having a higher refractive index is then coated on the upper surface of the diffraction grating. The diffraction grating focuses incident light to the waveguide layer, emits a mode already guided in the waveguide layer, transmits part of the mode in the traveling direction, and reflects part of the mode. Has function. The waveguide layer covers the grating region with an additional layer. The additional layer can be a multilayer film as required. The additional layer can have a function that enables selective detection of a substance contained in the measurement substance. As a preferred embodiment, a layer having a detection function can be provided on the outermost surface of the additional layer. As a layer having such a detection function, a layer capable of immobilizing a physiologically active substance can be used.

  As another embodiment, a configuration in which an array of diffraction grating waveguides is incorporated in a well of a microplate is also possible (Japanese Patent Publication No. 2007-501432). That is, if the diffraction grating waveguides are arranged in an array on the bottom surface of the well of the microplate, it is possible to screen a drug or chemical substance with high throughput.

  In order to enable detection of a physiologically active substance on the upper layer (detection region) of the diffraction grating waveguide, the diffraction grating waveguide detects incident light and reflected light to detect a change in refractive characteristics. For this purpose, one or more light sources (eg lasers, diodes) and one or more detectors (eg spectrometers, CCD cameras or other photodetectors) can be used. . There are two different modes of operation—spectroscopy and angle methods for measuring refractive index changes. In spectroscopy, a broadband beam is sent as incident light to a diffraction grating waveguide and the reflected light is collected and measured, for example, with a spectrometer. By observing the spectral position of the resonance wavelength (peak), the refractive index change, that is, the coupling at or near the surface of the diffraction grating waveguide can be measured. Also, in the angle method, nominally single wavelength light is focused to produce a range of illumination angles and directed into the diffraction grating waveguide. The reflected light is measured by a CCD camera or other photodetector. By measuring the position of the resonance angle reflected by the diffraction grating waveguide, it is possible to measure the refractive index change, ie, coupling, at or near the surface of the diffraction grating waveguide.

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

Example 1: Synthesis of photo radical generator (compound A):
(1) Synthesis of Intermediate 1 29.33 g (0.150 mol) of 4-Cyano-4′-hydroxybiphenyl and 75 ml of N, N-dimethylacetamide were placed in a 300 ml three-necked flask equipped with a condenser and stirred. A homogeneous solution was obtained. 22.81 g (0.165 mol) of potassium carbonate was added to the solution, heated to 80 ° C. in an oil bath, and 38.78 g (0.166 mol) of 11-bromo-1-undecene was gradually added dropwise. After 1.5 hours, the temperature of the reaction solution was raised to 100 ° C. and further reacted for 1.5 hours. The disappearance of the raw materials was confirmed by TLC. After completion of the reaction, the reaction solution was poured into 230 ml of water, and the precipitated white solid was separated by filtration and washed with water. The obtained solid was recrystallized from acetonitrile to obtain 43.11 g of Intermediate 1 . The yield was 82.6%.

(2) Synthesis of Intermediate 2 20.00 g (57.56 mmol) of Intermediate 1 was placed in a 300 ml three-necked flask equipped with a calcium chloride tube and dissolved in 150 g (1.0 mol) of trichloroacetonitrile under ice cooling. A homogeneous solution. 1.54 g (5.76 mmol) of aluminum bromide was added to the solution, and hydrochloric acid gas was bubbled for 4 hours while stirring the solution. After further stirring for 4 hours, the mixture was allowed to stand overnight. The product was extracted with ethyl acetate, and intermediate 2 was isolated by column chromatography (hexane / ethyl acetate = 3/1). The yield was 36.44 g, and the yield was 99.5%.

(3) Synthesis of Compound A 2.01 g (3.159 mmol) of Intermediate (B) was placed in a 50 ml three-necked flask equipped with a calcium chloride tube, and dissolved in 3 ml of tetrahydrofuran. The solution was cooled to 0 ° C. and 6 ml of trichlorosilane was added and stirred. Further, Spire catalyst (platinum chloride (IV) acid hexahydrate / 2-propanol 0.1M) was added and stirring was continued at room temperature. After 5 hours, unreacted trichlorosilane was distilled off under reduced pressure to obtain Compound A.

Example 2: Compound 1 introducing a double bond into CMD
Carboxymethyldextran (abbreviation: CMD-D40, manufactured by Meisei Sangyo Co., Ltd., molecular weight 40,000) 5.0 g (0.02 mol / 1 unit, 0.02 mol / sodium carboxylate) and 2-aminoethyl methacrylate hydrochloride (90%) 3.7 g ( 0.02 mol) was dissolved in 40 g of distilled water. It was cooled in an ice bath and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: WSC, manufactured by Dojindo Laboratories) 4.2 g (0.022 mol) dissolved in 12 g of distilled water was added. . After stirring overnight at room temperature, the reaction solution was poured into 500 ml of acetone, and the precipitated solid was filtered out. The solid was reslurried with methanol and dried to give 4.1 g of a light brown powder. When the structure was confirmed by NMR, it was found that about 20 mol% of sodium carboxylate contained in CMD-D40 reacted with 2-aminoethyl methacrylate and amidated.

Compound 2
Carboxymethyldextran (abbreviation: CMD-D40, manufactured by Meisei Sangyo, molecular weight 40,000) 5.0g (0.02mol / 1 unit, 0.02mol / sodium carboxylate) and 2-aminoethyl methacrylate hydrochloride (90%) 1.8g ( 0.01 mol) was dissolved in 40 g of distilled water. It was cooled in an ice bath, and 2.1 g (0.011 mol) of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: WSC, manufactured by Dojindo Laboratories) dissolved in 12 g of distilled water was added. . After stirring overnight at room temperature, the reaction solution was poured into 500 ml of acetone, and the precipitated solid was filtered out. The solid was reslurried with methanol and dried to give a light brown powder. When the structure was confirmed by NMR, it was found that about 8 mol% of sodium carboxylate contained in CMD-D40 reacted with 2-aminoethyl methacrylate and amidated.

Compound 3
Carboxymethyldextran (abbreviation: CMD, manufactured by Meisei Sangyo Co., Ltd., molecular weight 1 million) 5.0 g (0.02 mol / 1 unit, 0.013 mol / sodium carboxylate) and 2-aminoethyl methacrylate hydrochloride (90%) 2.4 g (0.013 mol) ) Was dissolved in 320 g of distilled water. It was cooled in an ice bath, and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: WSC, manufactured by Dojindo Laboratories) dissolved in 12 g of distilled water was added to 2.7 g (0.0143 mol). . After stirring at room temperature overnight, the reaction solution was poured into 3000 ml of acetone, and the precipitated solid was filtered out. The solid was reslurried with methanol and dried to give 4.6 g of a light brown powder. When the structure was confirmed by NMR, it was found that 9.4 mol% of sodium carboxylate contained in CMD reacted with 2-aminoethyl methacrylate and amidated.

Compound 4
Carboxymethyldextran (abbreviation: CMD, manufactured by Meisei Sangyo Co., Ltd., molecular weight: 1 million) 1.0 g (4 × 10 ⁻³ mol / 1 unit, 2.5 × 10 ⁻³ mol / sodium carboxylate) and 2-aminoethyl methacrylate hydrochloride ( 90%) 0.92 g (5.0 × 10 ⁻³ mol) was dissolved in 100 g of distilled water. 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: WSC, manufactured by Dojindo Laboratories) 1.05 g (5.5 × 10 ^-3 mol) cooled in an ice bath and dissolved in 5 g of distilled water ) Was added. After stirring overnight at room temperature, the reaction solution was poured into 2000 ml of acetonitrile, and the precipitated solid was taken out by decantation. The solid was reslurried with methanol and dried to give 0.94 g of a light brown powder. When the structure was confirmed by NMR, it was found that 13.7 mol% of sodium carboxylate contained in CMD reacted with 2-aminoethyl methacrylate and amidated.

Compound 5
Carboxymethyldextran (abbreviation: CMD, manufactured by Meisei Sangyo Co., Ltd., molecular weight: 1 million) 1.0 g (4 × 10 ⁻³ mol / 1 unit, 2.5 × 10 ⁻³ mol / sodium carboxylate) and 2-aminoethyl methacrylate hydrochloride ( 90%) 1.84 g (10.0 × 10 ⁻³ mol) was dissolved in 100 g of distilled water. 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: WSC, manufactured by Dojindo Laboratories) 2.11g (11.0 × 10 ^-3 mol) cooled in an ice bath and dissolved in 5 g of distilled water ) Was added. The reaction solution was poured into 2000 ml of acetonitrile, and the precipitated solid was taken out by decantation. The solid was reslurried with methanol and dried to give 0.89 g of a light brown powder. When the structure was confirmed by NMR, it was found that 18.2 mol% of sodium carboxylate contained in CMD reacted with 2-aminoethyl methacrylate and amidated.

Example 3:
This example relates to the production of a sensor chip for immobilizing proteins. (1) Preparation of sample 1 (comparative example) Biacore sensor chip CM-5 (research grade) was used as sample 1 as it was as the surface to which carboxymethyldextran was bound.

(2) Preparation of sample 2 (present invention) (2-1) Preparation of substrate having amino group An experiment was conducted using Biacore sensor chip Au as a surface on which only a gold film was formed on the sensor chip. . Sensor chip Au was treated with UV ozone for 12 minutes, then 8mL ethanol and 2mL ultrapure water, 45μmol 11-Hydroxy-1-undecanethiol (Aldrich) and 4μmol 16-Mercaptohexadecanoic acid (Aldrich) ) In a solution in which the solution was dissolved at 40 ° C. for 1 hour, and washed once with ethanol and once with ultrapure water. 100 μl of a mixed solution of EDC (0.4M) / NHS (0.1M) was dropped on the substrate, reacted for 15 minutes at room temperature, activated, and then washed once with ultrapure water. 50 μl of 1,2-bis (aminoethoxy) ethane was dropped onto the substrate, reacted at room temperature for 1 hour, and then washed once with ultrapure water.

(2-2) Binding of photoradical generator (compound A) Compound A was dissolved in dehydrated MEK to make a 0.5 wt% solution. The gold chip prepared in (3-1) was spin-coated with a photoinitiator solution (1000 rpm, 20 sec) and dried (80 ° C., 5 minutes), and then the surface was washed with MEK.

(2-3) Photoimmobilization of Compound 2 on a Gold Substrate 0.4 g of Compound 2 was dissolved in 1.0 g of acetonitrile and 2.0 g of water to form a 12 wt% solution, and filtered through a syringe-type filter to remove insoluble matters. This solution was spin-coated (750 rpm, 20 sec) and dried (80 ° C., 2 minutes) on the surface of the gold chip on which the photoinitiator had been fixed to form a dry film. Sample 2 was obtained by exposing with UV exposure apparatus UVX-02516S1LP01 (high pressure mercury lamp, manufactured by USHIO) for 1 minute, and developing by immersing in water for 48 hours.

(3) Preparation of Sample 3 (Invention) Compound 3 0.05 g was dissolved in 5.0 g of water, 1 drop of saturated saline and 1.5 g of acetonitrile to obtain a 0.76 wt% solution. The solution was applied onto the gold chip surface prepared in (3-2) using a # 36 bar and dried (80 ° C., 2 minutes) to form a dry film. The film was exposed to UV exposure apparatus UVX-02516S1LP01 (high pressure mercury lamp, manufactured by USHIO) for 0.5 minutes and 1 minute, and immersed in water for 12 hours for development. Further, the polymer remaining on the surface was removed by washing with ultrasonic waves in water for 5 minutes.

Example 4
This example relates to protein immobilization on the sensor chip obtained in Example 3. As the protein, CA (Carbonic Anhydrase: manufactured by SIGMA) was used. The CA used was an electrophoresis experiment using AE-8150 from ATTO, and the isoelectric point was about 5.8 based on comparison with the marker (Broad pI Kit, pH 3.5-9.3: Amersham Biosciences) measured simultaneously. I confirmed that there was. 10 μl of a solution of 1 mg of CA dissolved in 1 ml of HBS-EP buffer (Biacore, 0.01M HEPES pH7.4, 0.15M NaCl, 0.005% Surfactant P20, 3 mM EDTA) was weighed, and 90 μl of acetate buffer (Biacore) Product, pH 5.0) was added to prepare a 0.1 mg / ml CA solution (pH 5.0, 0.1 mg / ml).

  Samples 1 to 3 prepared in Example 3 were set in a Biacore 3000 which is a surface plasmon resonance apparatus manufactured by Biacore, and an aqueous solution containing 0.4 M EDC and 0.1 M NHS, a CA solution (pH 5.0, 0.1 mg / ml). ), An ethanolamine solution (Biacore) was allowed to flow for 5 minutes each, and then immobilization was examined when 10 mM NaOH was flowed twice for 1 minute. As a running buffer, HBS-N buffer (Biacore, 0.01M HEPES pH7.4, 0.15M NaCl) was used. The obtained sensorgram is shown in FIG.

  The CA fixed amounts were 7757RU (sample 1: comparative example), 13983RU (sample 2: the present invention), and 34807RU (sample 3: the present invention), respectively. It has been proved that a CMD binding surface having a larger amount of protein immobilized than a commercially available CMD binding surface can be easily prepared by the present invention.

FIG. 1 is obtained by setting samples 1 to 3 prepared in Example 3 to a surface plasmon resonance apparatus, flowing EDC / NHS solution, CA solution, and ethanolamine solution for 5 minutes each, and then flowing NaOH. The sensorgram is shown.

Claims (16)

Hydrophilic capable of forming a covalent bond with the reactive group of the photoradical generator after the photoradical generator reacts with the self-assembled film on the substrate made of a metal film coated with the self-assembled film. From a substrate coated with a hydrophilic polymer comprising applying the light in contact with the substrate to attach the hydrophilic polymer to the substrate surface via a covalent bond with the reactive group. A method for producing a biosensor. The method according to claim 1, wherein the self-assembled film is an alkanethiol represented by the following general formula 1.
General formula 1 HS (CH ₂ ) _n X
(However, the number of repeating alkyl groups is 3 or more and 20 or less, and X = NH _2. ) The method according to claim 1 or 2, wherein the hydrophilic polymer is a hydrophilic polymer having a double bond. The method according to any one of claims 1 to 3, wherein the hydrophilic polymer is a polymer containing a carboxyl group. The method according to claim 4 , wherein the polymer containing a carboxyl group is a polysaccharide. The method according to claim 4 or 5 , wherein the polymer having a carboxyl group has an average molecular weight of 1,000 to 5,000,000. The method according to claim 1, wherein the hydrophilic polymer is bonded to the reactive group generated by light in a state where a thin film is formed on the substrate. The method according to claim 7, wherein a thin film is formed on the substrate by a spin coating method or a spray coating method. The method according to claim 1, wherein the metal is any one of gold, silver, copper, platinum or aluminum. A biosensor produced by the method according to claim 1. The biosensor according to claim 10, which is used for non-electrochemical detection. The biosensor according to claim 10 or 11, which is used for surface plasmon resonance analysis. A method for immobilizing a physiologically active substance on a biosensor comprising the step of bringing the biosensor according to any one of claims 10 to 12 into contact with the physiologically active substance and binding the physiologically active substance to the biosensor. A substance that interacts with the physiologically active substance is detected or measured, comprising the step of bringing the biosensor according to any one of claims 10 to 12 and the test substance into contact with the surface by a covalent bond. how to. The method according to claim 14, wherein a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method. The method according to claim 14 or 15, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.

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