JP2008286775A - Manufacturing method of physiologically active substance-immobilized substrate and the substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method of physiologically active substance-immobilized substrate and the substrate capable of preventing the loss of activity on the surface of the substrate and/or immobilizing to high concentration with the physiologically active substance without utilizing the electrostatic attraction force. <P>SOLUTION: The manufacturing method of the physiologically active substance-immobilized substrate of this invention is composed of the process of coating the solution containing the physiologically active substance capable of forming covalent bonding with the molecule forming a layer for immobilizing the physiologically active substrate on the metal substrate provided with the layer for immobilizing the physiologically active substance, and the process of drying after that. Herein, the physiologically active substance is a kind of avidins, which is fixed on the layer for immobilizing the physiologically active substance with the concentration of ≥1 pmol/mm<SP>3</SP>to ≤2.5 pmol/mm<SP>3</SP>by the drying process. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a physiologically active substance-immobilized substrate and a substrate. More specifically, the present invention relates to a method for producing a physiologically active substance-immobilized substrate and a substrate for use in surface plasmon resonance analysis and the like.

  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 method of immobilizing a physiologically active substance on a substrate by covalent bonding, a physiologically active substance having a charge opposite to that of the substrate is provided and the physiologically active substance is localized at a high concentration near the substrate using electrostatic attraction. There has been used a method of bonding in the presence. According to this method, a physiologically active substance can be immobilized on the sensor substrate uniformly at a high concentration, but it is necessary to use a large amount of solution, and the pH of the solution is limited to a range where charge concentration is possible. There was a concern that the physiologically active substance would be deactivated.

Patent Document 1 and Non-Patent Document 1 describe fixation of a physiologically active substance on a substrate, but both are fixed in a solution. It is known to dry a physiologically active substance and apply a solution with a spin coater or a dispenser. However, until now, a physiologically active substance immobilized on a substrate by a covalent bond has been easily deactivated by the covalent bond. For this reason, there has been a concern that by applying a small amount, it may be easy to dry or spontaneously dry. In Patent Document 1, since the physiologically active substance is fixed in the flow path, it is not possible to dry or apply a small amount. In Non-Patent Document 1, since the method for immobilizing a physiologically active substance is not a covalent bond, there is a high possibility that the bond will be removed in the washing step even if it is dried or applied.
Further, when the physiologically active substance to be immobilized is not a low molecular protein, it may be difficult to adjust the amount immobilized on the substrate while suppressing inactivation.

US Pat. No. 5,436,161 Journal of Colloid and Interface Science 259 (2003) 13-26

  The present invention provides a method for producing a physiologically active substance-immobilized substrate capable of preventing the deactivation of the physiologically active substance on the substrate surface and / or immobilizing the physiologically active substance at a high concentration without using electrostatic attraction. And providing a substrate is a problem to be solved.

  The present inventors have found that a thin film of a solution containing a physiologically active substance can be formed on a metal substrate, and further dried and concentrated to fix the physiologically active substance at a high concentration on the substrate surface without using electrostatic attraction. I found it. Furthermore, the present inventors have found that the physiologically active substance can be uniformly fixed by forming a thin film of a solution containing the physiologically active substance and shortening the drying time. The present invention has been completed based on these findings.

That is, the present invention
<1> A solution containing a physiologically active substance capable of forming a covalent bond with a molecule constituting the layer for immobilizing the physiologically active substance is applied to a metal substrate having a layer for immobilizing the physiologically active substance. And the subsequent drying step, wherein the physiologically active substance is an avidin, and the layer for immobilizing the physiologically active substance in the drying step is 1 pmol / mm ³ or more and 2.5 pmol / A method for producing a physiologically active substance-immobilized substrate, wherein the substrate is immobilized at a density of mm ³ or less.

<2> The method further comprises a step of binding a biotinylated polymer protein that is an avidin-binding protein to the avidin immobilized on the layer for immobilizing the physiologically active substance, The method for producing a physiologically active substance-immobilized substrate according to <1>, wherein the molar ratio of the bound biotinylated polymer protein is 0.17 or more and 0.69 or less.

<3> The method for producing a physiologically active substance-immobilized substrate according to <2>, wherein the polymer protein has a weight average molecular weight of 100,000 or more and 200,000 or less.

<4> The avidin is immobilized at a density of 1.07 pmol / mm ³ or more and 2.48 pmol / mm ³ or less in the layer for immobilizing the physiologically active substance in the drying step. The method for producing a physiologically active substance-immobilized substrate according to any one of <1> to <3>.

<5> Any one of the above <2> to <4>, wherein the molar ratio of the avidin to the bound biotinylated polymer protein is 0.35 or more and 0.55 or less A method for producing a physiologically active substance-immobilized substrate according to any one of the above.

<6> The method for producing a physiologically active substance-immobilized substrate according to any one of <1> to <5> above, wherein the avidin is streptavidin.

<7> The physiologically active substance-immobilized substrate according to any one of <1> to <6> above, wherein a variation coefficient (CV value) of a fixed amount of the physiologically active substance is 15% or less. It is a manufacturing method.

<8> The above <1> to the above, wherein the layer for immobilizing the physiologically active substance is composed of at least one selected from a hydrophilic polymer layer, a hydrophobic polymer layer, and a self-oxidation film. <7> The method for producing a physiologically active substance-immobilized substrate according to any one of <7>.

<9> The method for producing a physiologically active substance-immobilized substrate according to <8>, wherein the layer for immobilizing the physiologically active substance includes at least a hydrophilic polymer layer.

<10> The method for producing a physiologically active substance-immobilized substrate according to <9>, wherein the hydrophilic polymer is covalently bonded to the physiologically active substance by activating the hydrophilic polymer.

<11> The physiologically active substance immobilization according to any one of <8> to <10> above, wherein the hydrophilic polymer layer is fixed to the metal substrate via a self-oxidizing film. A method for manufacturing a substrate.

<12> The method for producing a physiologically active substance-immobilized substrate according to <11>, wherein the hydrophilic polymer layer is made of a polysaccharide and is fixed to the metal substrate via a self-oxidation film. .

<13> The method for producing a physiologically active substance-immobilized substrate according to <11>, wherein the polysaccharide has a weight average molecular weight of 100,000 to 200,000.

<14> In the drying step, the solution containing the physiologically active substance applied in the applying step is dried at a temperature difference of 7 ° C. or higher between the dry bulb temperature and the wet bulb temperature < 1>-<13> The method for producing a physiologically active substance-immobilized substrate according to any one of <13>.

<15> In the application step, the solution containing the physiologically active substance is applied to the metal substrate in a dry environment where a temperature difference between a dry bulb temperature and a wet bulb temperature is 7 ° C. or more. 1> to the method for producing a physiologically active substance-immobilized substrate according to any one of <14>.

<16> The physiologically active substance according to any one of <1> to <15> above, wherein in the drying step, the drying time of the solution containing the physiologically active substance is 10 minutes or less. It is a manufacturing method of a fixed substrate.

<17> A substrate obtained by the method for producing a physiologically active substance-immobilized substrate according to any one of <1> to <16> above.

  According to the present invention, it is possible to prevent inactivation of a physiologically active substance on the substrate surface and / or to immobilize the physiologically active substance at a high concentration without using electrostatic attraction.

Hereinafter, embodiments of the present invention will be described in detail. In the method for producing a physiologically active substance-immobilized substrate according to the present invention, a covalent bond is formed with a molecule constituting the layer for immobilizing the physiologically active substance on a metal substrate having a layer for immobilizing the physiologically active substance. A step of applying a solution containing a physiologically active substance capable of forming, and a subsequent drying step, wherein the physiologically active substance is avidin, and the avidin is 1 pmol / mm on the metal substrate by the drying step. ^A method for producing a physiologically active substance-immobilized substrate, wherein the substrate is immobilized at a density of ³ or more and 2.5 pmol / mm ³ or less.

  In the method of the present invention, the physiologically active substance immobilized on the substrate by covalent bond is not inactivated even when dried, and the physiologically active substance is immobilized on the substrate surface by covalent bond. The physiologically active substance does not come off. Furthermore, when a physiologically active substance is immobilized in a solution, it cannot be immobilized unless the pH is controlled. However, in the method according to the present invention (drying method), the physiologically active substance can be immobilized at a preferred pH. Further, since it is not necessary to control the pH of the solution containing the physiologically active substance, the physiologically active substance is not deactivated. Furthermore, according to the method of the present invention, the physiologically active substance can be immobilized by applying a small amount, and the amount of the solution used can be reduced, so that the cost can be reduced. Conventionally, a physiologically active substance was thought to be deactivated when dried, but was not deactivated when dried. Further, in the method of the present invention, since the physiologically active substance film is uniform, errors in quantitative and kinetic evaluation can be minimized.

In addition, when avidin is used as a physiologically active substance and avidin is immobilized on the metal substrate by the coating and drying process, the avidin is immobilized on the metal substrate by covalent bond. By being fixed by the coating and drying step, the film made of avidin becomes a uniform film as described above. When immobilizing the avidin at a density of 1 pmol/mm ³ or 2.5 pmol / mm ³ or less, biotinylated polymeric proteins, avidin this avidin - is immobilized in a high concentration biotin binding . Specifically, the avidin is immobilized so that the molar ratio of the biotinylated polymer protein immobilized on the metal substrate via the avidin is 0.17 or more and 0.69 or less. The Furthermore, since the biotinylated polymer protein is immobilized on the metal substrate via avidins, inactivation is suppressed.

  The physiologically active substance-immobilized substrate produced by the method of the present invention can be used as a biosensor substrate. 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. Hereinafter, a method for producing a physiologically active substance-immobilized substrate according to the present invention will be specifically described.

  (1) In the present invention, a physiologically active substance is applied on a metal substrate having a layer for immobilizing a physiologically active substance, on the layer for immobilizing the physiologically active substance. In 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.

  The substrate is fixed to the dielectric block of the measurement unit and integrated to form a measurement chip, and the measurement chip may be formed to be replaceable. An example is shown below.

  FIG. 1 is an exploded perspective view of a sensor unit 10 used for measurement using SPR. The sensor unit 10 includes a total reflection prism (optical block) 20 that is a transparent dielectric, and a flow path member 30 that is mounted on the total reflection prism 20. The flow path member 30 has two types of flow paths: a first flow path 31 located on the back side in the figure and a second flow path 32 located on the near side in the figure. Although details will be described later, when measurement is performed using the sensor unit 10, one sample is measured using the two flow paths 31 and 32 as a set. The channel member 30 is provided with six channels 31 and 32 in the longitudinal direction, respectively, so that one sensor unit 10 can measure six samples. In addition, the number of each flow path 31 and 32 is not restricted to six, Five or less may be sufficient and seven or more may be sufficient.

  The total reflection prism 20 includes a prism main body 21 formed in a long trapezoidal column shape, a gripping portion 22 provided at one end of the prism main body 21, and a protrusion 23 provided at the other end of the prism main body 21. Become. The total reflection prism 20 is molded by, for example, an extrusion method, and the prism main body 21, the grip portion 22, and the protruding portion 23 are integrally formed.

  The prism main body 21 has a substantially trapezoidal vertical section whose upper base is longer than the lower base, and condenses the light irradiated from the bottom side surface on the upper surface 21a. A metal film (thin film layer) 25 for exciting SPR is provided on the upper surface 21 a of the prism body 21. The metal film 25 has a rectangular shape so as to face the flow paths 31 and 32 of the flow path member 30, and is formed by, for example, a vapor deposition method. As the metal film 25, for example, gold or silver is used, and the film thickness is, for example, 50 nm. The film thickness of the metal film 25 is appropriately selected according to the material of the metal film 25, the wavelength of light irradiated during measurement, and the like.

  On the metal film 25, a layer 26 for immobilizing a physiologically active substance is provided. The layer 26 for immobilizing the physiologically active substance has a binding group for immobilizing the physiologically active substance, and the physiological layer is placed on the metal film 25 via the layer 26 for immobilizing the physiologically active substance. The active substance is fixed.

(2) Layer for immobilizing physiologically active substance The metal substrate is provided with a layer for immobilizing the physiologically active substance. The layer for immobilizing the physiologically active substance is at least one layer selected from a self-assembled film, a hydrophilic polymer layer, and a hydrophobic polymer layer, and is a layer including at least a hydrophilic polymer layer. It is preferable. A particularly preferred embodiment of the layer for immobilizing the physiologically active substance is a combination of a self-assembled film and a hydrophilic polymer layer.

(2-1) Self-assembled film The self-assembled film referred to in the present invention is a structure having a certain order formed by the mechanism of the film material itself, without fine control from the outside. It refers to ultra-thin films such as monomolecular films and LB films. This self-organization forms ordered structures and patterns over long distances in a non-equilibrium situation.

The coating method of metal films using self-assembled films (SAMs) has been vigorously developed by Professor Whidesides of Harvard University, and the details are reported, for example, in Chemical Review, 105, 1103-1169 (2005). ing. When gold is used as the metal, an alkanethiol derivative represented by general formula A-1 (in general formula A-1, n represents an integer of 3 to 20 and X represents a functional group) is used as the organic layer forming compound. Thus, a monomolecular film having an orientation is formed in a self-organized manner based on the van der Waals force between the Au—S bond and the alkyl chains. 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. Specifically, for example, a gold surface can be covered with an organic layer having an amino group by forming a self-assembled film using a compound in which X = NH ₂ in the general formula A-1. .

The alkanethiol having an amino group at the terminal where X is NH ₂ in the general formula A-1 may be a compound in which a thiol group and an amino group are linked via an alkyl chain, and a carboxyl group at the terminal It may be a compound obtained by reacting alkanethiol (general formulas A-3, A-4) having a large excess with hydrazide or diamine. The reaction of the alkanethiol having a carboxyl group at the terminal with a large excess of hydrazide or diamine may be carried out in a solution state, or after binding the alkanethiol having a carboxyl group at the terminal to the substrate surface, Hydrazide or diamine may be reacted.

In the above general formula A-2, the repeating number n of the alkyl group represents an integer of 3 to 20, more preferably an integer of 3 to 16, and particularly preferably an integer of 4 to 8. In the general formula A-3, the repeating number n of the alkyl group represents an integer of 3 to 20, more preferably an integer of 3 to 16, and particularly preferably an integer of 4 to 8. In the general formula A-4, the repeating number n of the alkyl group independently represents an integer of 1 to 20, preferably an integer of 3 to 20, more preferably an integer of 3 to 16, and an integer of 4 to 8. Is most preferred.
If the alkyl chain of the alkanethiol derivative is short, it is difficult to form a self-assembled film, and if the alkyl chain is long, the water solubility decreases and handling becomes difficult.
Moreover, in the said general formula A-4, the repeating number m of an ethylene oxide group shows the integer of 1-20 each independently, the integer of 3-20 is preferable, the integer of 3-16 is further more preferable, and 4-8 An integer is most preferred.

  As a large excess of diamine to be reacted with alkanethiol having a carboxyl group at the terminal (general formulas A-3 and A-4), any compound can be used, but when used on the biosensor surface, Diamines are preferred. Specific examples of the water-soluble diamine include ethylenediamine, tetraethylenediamine, octamethylenediamine, decamethylenediamine, piperazine, triethylenediamine, diethylenetriamine, triethylenetetraamine, dihexamethylenetriamine, and 1,4-diaminocyclohexane. Diamine, paraphenylenediamine, metaphenylenediamine, paraxylylenediamine, metaxylylenediamine, 4,4'-diamonobiphenyl, 4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenyl ketone, 4,4 ' -Aromatic diamines such as diaminodiphenylsulfonic acid. From the viewpoint of improving the hydrophilicity of the biosensor surface, it is also possible to use a compound (general formula A-5) in which two amino groups are linked by an ethylene glycol unit. The diamine used in the present invention is preferably a compound represented by ethylenediamine or general formula A-5 (in general formula A-5, n and m each independently represents an integer of 1 to 20), and more Preferably, ethylenediamine or 1,2-bis (aminoethoxy) ethane (in formula A-5, n = 2, m = 1).

  The alkanethiol having an amino group can form a self-assembled film alone, or can be mixed with another alkanethiol 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 capable of suppressing the non-specific adsorption of physiologically active substances has been studied in detail by the above-mentioned professor Whitesides et al., And the self-assembled membrane formed from alkanethiol having a hydrophilic group is non-specifically adsorbed. It is 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 A-6) having a hydroxyl group or ethylene group is used 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 inhibiting ability and easy availability. Alkanethiol having a call unit (general formula A-7) (in formula A-6, n represents an integer of 3 to 20, and in formula A-7, n and m are each independently 1 to 20) It is preferable to use an integer. Preferably, in General Formula A-6, n is an integer of 5 or more, more preferably an integer of 10 or more, further preferably an integer of 10 to 20, and most preferably an integer of 6 to 16.

In addition, when the alkanethiol having an amino group is mixed with another alkanethiol to form a self-assembled film, the repeating number n of the alkyl groups of the general formulas A-2 to A-4 is 4 or more. An integer of 20 or less, preferably an integer of 4 to 16, and most preferably an integer of 4 to 10.
Similarly, when an alkanethiol having an amino group is mixed with another alkanethiol to form a self-assembled film, the repeating number n of the alkyl groups of the general formulas A-6 and A-7 is: It is an integer of 3 or more and 16 or less, preferably an integer of 3 or more and 12 or less, and most preferably an integer of 3 or more and 8 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, active esterification is performed. When the amount of the carboxyl group-containing polymer bound is reduced and the proportion of alkanethiol having a hydrophilic group is small, the nonspecific adsorption inhibiting ability is reduced. 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 in the range of 1 to 1/1000. It is more preferable that it is in the range of 1 to 1/10. 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 on the basis of a review by Prof. Grzybowski et al. Of Northwestern University (Curr. Org. Chem., 8, 1763-197 (2004)) and references cited therein. Commercially available compounds may also be used. These compounds are available from Dojindo Chemical Co., Ltd., Aldrich, SensoPath Technologies, Frontier Scientific Inc. It can be purchased from companies. In the present invention, the disulfide compound that is an oxidation product of alkanethiol can be used in the same manner as alkanethiol.

(2-2) Hydrophilic polymer As the hydrophilic polymer that can be used in the present invention, gelatin, agarose, chitosan, dextran, carrageenan, alginic acid, starch, cellulose, or a derivative thereof such as carboxymethyl derivative or water is used. Swellable organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyethylene glycol or derivatives thereof can be mentioned.

  As the hydrophilic polymer used in the present invention, a carboxyl group-containing synthetic polymer and a carboxyl group-containing polysaccharide can be further used. Examples of the carboxyl group-containing synthetic polymer include polyacrylic acid, polymethacrylic acid, and copolymers thereof, for example, JP-A 59-53836, page 3, upper right 2nd line to page 6, lower left 9th line; -71048, page 3, lower left line 1 to page 5, upper left line 3, methacrylic acid copolymer, acrylic acid copolymer, itaconic acid copolymer, crotonic acid copolymer, maleic acid copolymer Examples include polymers, partially esterified maleic acid copolymers, and polymers 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.

  A commercially available compound can be used as the carboxyl group-containing polysaccharide, and specifically, CMD (carboxymethyl dextran), CMD-L, CMD-D40 (with a weight average molecular weight in the range of 100,000 to 200,000 ( For example, sodium carboxymethylcellulose (manufactured by Wako Pure Chemical Industries, Ltd.), sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd.), and the like.

  The weight average molecular weight of the hydrophilic polymer used in the present invention is not particularly limited, but the weight average molecular weight is preferably 1,000 to 5,000,000, more preferably 10,000 to 2,000,000, and the weight average molecular weight is 100,000 to More preferably, it is 1000000.

  The hydrophilic polymer as described above may be fixed to the substrate through a self-assembled film or a hydrophobic polymer as described herein below, or formed directly on the substrate from a solution containing the monomer. It can also be made. Furthermore, the hydrophilic polymer described above can also be crosslinked. Crosslinking of the hydrophilic polymer is obvious to those skilled in the art.

  The hydrophilic polymer fixed to the sensor surface 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 for the analyte to diffuse into the film, and the distance from the detection surface to the interaction formation part is long especially when detecting the interaction from the opposite side of the hydrophilic polymer fixing surface of the sensor substrate. Thus, the detection sensitivity is lowered. The hydrophilic polymer layer thickness in the aqueous solution can be evaluated by AFM, ellipsometry or the like.

Further, in the present invention, a fixed amount of hydrophilic polymer immobilized to the sensor surface, 3 ng / mm ² or more 30 ng / mm ² or less ^{(3 × 10 -6 pmol / mm} 3 or more ^{30 × 10 -6 pmol / mm 3} ) is preferably less, 3 ng / mm ² or more 20 ng / mm ² or less ^{(3 × 10 -6 pmol / mm} 3 or more ^{20 × 10 -6 pmol / mm 3} ) are more preferred, 3 ng / mm ² or more 15 ng / mm ² or less (3 × 10 ⁻⁶ pmol / mm ³ or more and 15 × 10 ⁻⁶ pmol / mm ³ ) is most preferable. Alternatively, the thickness of the hydrophilic polymer is preferably 3 nm to 30 nm, more preferably 3 nm to 20 nm, and most preferably 3 nm to 15 nm.

  As the fixed amount of the hydrophilic polymer, values detected by various film thickness measurement methods / weight measurement methods can be used. Examples of film thickness measurement methods include atomic force microscope (AFM), scanning probe microscope (SPM) such as scanning tunneling microscope (STM), transmission electron microscope (TEM), scanning electron microscope (SEM), Examples thereof include an electron microscope such as a scanning transmission electron microscope (STEM) and an ellipsometry method.

  As a specific calculation method of the film thickness measurement method, for example, in SPM, the film thickness can be calculated from the difference in surface unevenness between the area where the hydrophilic polymer on the substrate is fixed and the area where the hydrophilic polymer is not fixed. In this case, the film thickness can be calculated from observation of a section of the substrate on which the hydrophilic polymer is fixed. When the thickness of the hydrophilic polymer layer is first determined using spectroscopic ellipsometry (manufactured by Fibrabo), the respective optical constants are calculated by measuring the substrate before and after fixing the hydrophilic polymer in a dry state. . When the substrate surface has a multilayer structure, the optical constants of each layer can be calculated sequentially by ellipsometry measurement each time each layer is installed, and it is possible to calculate up to about two layers as one layer. Using the calculated optical constant of each layer as a parameter, the film thicknesses of the substrate before hydrophilic polymer fixation and the dried substrate after hydrophilic polymer fixation were determined by monochromatic ellipsometry (He-Ne laser, manufactured by Fibrabo). The difference can be obtained as the film thickness of the hydrophilic polymer layer.

  Specific calculation methods of the weight measurement method include an infrared spectroscopy method such as a quartz crystal microbalance method (QCM), a total reflection measurement method (ATR), and an external reflection method. As a specific calculation method of the weight measurement method, for example, in the infrared spectroscopy, the fixed amount (weight) of the hydrophilic polymer can be quantified from a calibration curve of absorption intensity attributed to the hydrophilic polymer. When determining the amount of hydrophilic polymer fixed using QCM (manufactured by USA System Co., Ltd.), the frequency of the substrate before and after fixing the hydrophilic polymer is measured in a dry state, and the difference is taken as the amount of hydrophilic polymer fixed. Can be sought.

Here, <dry state> means that the water content of the hydrophilic polymer is 30% by weight or less. Specifically, the above dry state can be obtained by standing at a temperature of 5 ° C. to 30 ° C. and a humidity of 0% RH to 90% RH, preferably a humidity of 0% RH to 70% RH for 5 minutes or more. it can.
In the surface plasmon resonance method (SPR), the amount of change in the resonance signal before and after the hydrophilic polymer is immobilized on the substrate in the solution can be calculated as the amount of the hydrophilic polymer immobilized.

(2-3) Activation of hydrophilic polymer When a polymer containing a carboxyl group is used as the hydrophilic polymer, it can be immobilized on a substrate coated with a self-assembled film by activating the carboxyl group. it can. 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 ethylcarboimide (EDC) and N-hydroxysuccinimide (NHS), Alternatively, a method of activation with EDC alone can be preferably used. By reacting a polymer containing a carboxyl group activated by this method with a substrate having an amino group, the biosensor of the present invention can be produced.

Further, as a method for activating a polymer containing a carboxyl group, there is a method using a nitrogen-containing compound. Specifically, the following general formula (Ia) or (Ib) [wherein R ¹ and R ² are Independently represents a carbonyl group which may have a substituent, a carbon atom, or a nitrogen atom, R ¹ and R ² may form a 5- or 6-membered ring by a bond, and A is a carbon atom having a substituent Alternatively, a nitrogen atom-containing compound represented by the following can be used: a phosphorus atom, M represents an (n-1) -valent element, and X represents a halogen atom.

Here, R ¹ and R ² each independently represent a carbonyl group, a carbon atom, or a nitrogen atom which may have a substituent. Preferably, R ¹ and R ² have a 5- to 6-membered ring by a bond. Form. Particularly preferably, hydroxysuccinic acid, hydroxyphthalic acid, 1-hydroxybenzotriazole, 3,4-dihydroxy-3-hydroxy-4-oxo-1,2,3-benzotriazine, and derivatives thereof are provided.

  Also preferably used are nitrogen-containing compounds represented by the following compounds Ic to Ie.

  Preferably, as the nitrogen-containing compound, a compound represented by the following general formula (II) [wherein Y and Z independently represent CH or a nitrogen atom] can also be used.

  As the nitrogen-containing compound represented by the general formula (II), specifically, the following compounds (II-1) to (II-3) can be used.

  Preferably, the following compound (II-4) can also be used as the nitrogen-containing compound.

  Preferably, the nitrogen-containing compound is represented by the following general formula (III) [wherein A represents a carbon atom or phosphorus atom having a substituent, and Y and Z each independently represent CH or a nitrogen atom. , M represents an (n-1) -valent element, and X represents a halogen atom].

  Here, as the substituent of the carbon atom or phosphorus atom represented by A, an amino group having a substituent is preferable, and a dialkylamino group such as a dimethylamino group or a pyrrolidino group is preferable. Examples of the (n-1) -valent element represented by M include a phosphorus atom, a boron atom, and an arsenic atom, and a phosphorus atom is preferable. Examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a fluorine atom is preferable.

  Specific examples of the nitrogen-containing compound represented by the general formula (III) include the following compounds (III-1) to (III-6).

  Preferably, the nitrogen-containing compound is represented by the following general formula (IV): wherein A represents a carbon atom or phosphorus atom having a substituent, M represents an (n-1) -valent element, and X represents a halogen atom. Can also be used.

  Specifically, the following compound (IV-1) and the like can be used.

Moreover, as a method for activating a polymer containing a carboxyl group, it is also preferable to use a phenol derivative having an electron-withdrawing group, and it is preferable that the σ value of the electron-withdrawing group is 0.3 or more. Specifically, the following compounds (V-1) to (V-4) can be used. In the following general formula (V-4), X ⁻ represents an anion.

  Furthermore, in the method of activating the polymer containing a carboxyl group, a carbodiimide derivative can be used together, preferably a water-soluble carbodiimide derivative can be used together, and more preferably the following compound (I-1 ), (1-Ethyl-3- (3-dimethylaminopropyl) carbohydrate, hydrochloride) to (I-3) can be used in combination.

  The above carbodiimide derivatives and nitrogen-containing compounds or phenol derivatives are not only used in combination, but can be used alone if desired. Preferably, a carbodiimide derivative and a nitrogen-containing compound are used in combination.

  Moreover, the following compound (V-6) can also be used as a method of activating the polymer containing a carboxyl group. Although this compound can also be used independently, you may use together with a carbodiimide derivative, a nitrogen-containing compound, and a phenol derivative.

  Furthermore, as a method for activating carboxylic acid in a polymer containing a carboxyl group, a method described in JP-A-2006-58071, paragraph numbers “0011” to “0022” (that is, a carboxyl group present on the surface of a substrate). In which a carboxylic acid amide group is formed by activating the compound with any one of a uronium salt, a phosphonium salt, or a triazine derivative having a specific structure, and paragraph number “0011” of JP-A-2006-90781 ”To“ 0019 ”(ie, a carboxyl group existing on the surface of a substrate is activated with a carbodiimide derivative or a salt thereof, a nitrogen-containing heteroaromatic compound having a hydroxyl group, a phenol derivative having an electron-withdrawing group, or Any of aromatic compounds having a thiol group and an ester Then, a method of forming a carboxylic acid amide group by reacting with an amine can be preferably used.

  The uronium salt, phosphonium salt, or triazine derivative having a specific structure in JP-A-2006-58071 described above is represented by the uronium salt represented by the following general formula (1) or the following general formula (2). Or a triazine derivative represented by the following general formula (3).

(In the general formulas (1) to (3), R ¹ and R ² each independently represent an alkyl group having 1 to 6 carbon atoms, or an N atom by forming an alkylene group having 2 to 6 carbon atoms by a bond. And R ³ represents an aromatic ring group having 6 to 20 carbon atoms or a heterocyclic group containing at least one heteroatom, and X ⁻ represents an anion In the general formula (2), R ⁴ and R ⁵ each independently represents an alkyl group having 1 to 6 carbon atoms, or an alkylene group having 2 to 6 carbon atoms is formed by a bond to form a ring together with an N atom, and R ⁶ has 6 to 20 carbon atoms An aromatic ring group or a heterocyclic group containing at least one or more hetero atoms, X ⁻ represents an anion, and in formula (3), R ⁷ represents an onium group, and R ⁸ and R ⁹ each independently represents an electron; Indicates a donor group.)
In the present invention, the amount of the hydrophilic polymer activated may be adjusted by adjusting the amount of addition to the hydrophilic polymer such as EDC or NHS used to activate the hydrophilic polymer. Specifically, the addition amount of an activator such as EDC or NHS with respect to 1 μmol of the hydrophilic polymer may be adjusted within a range of 2.4 mmol or less, preferably 2.0 mmol or less, more preferably 0.04 mmol to It may be adjusted to 1.5 mmol, most preferably in the range of 0.04 mmol to 0.3 mmol.
Moreover, what is necessary is just to adjust the solution density | concentration of the hydrophilic polymer in the polymer solution used in the said application | coating process for adjustment of the fixed amount to the board | substrate of a hydrophilic polymer. Specifically, the solution concentration of the hydrophilic polymer in the polymer solution is in the range of 0.005 wt% to 5 wt%, preferably in the range of 0.05 wt% to 3 wt%, particularly preferably 0.1%. What is necessary is just to adjust in the range of weight%-2 weight%.

(2-4) Application of hydrophilic polymer to substrate In the present invention, the polymer containing an active esterified carboxyl group may be reacted with the substrate as a solution, or by using a technique such as spin coating. You may make it react in the state which formed the upper thin film. Preferably, the reaction is in a state where a thin film is formed.

  As described above, the polymer containing a carboxyl group that has been esterified in the present invention 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. Regarding these thin film formation methods, “Progress in coating technology” by Yuji Harasaki, General Technical Center (1988), “Coating Technology” Technical Information Association (1999), “Water-borne 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.

  The dispensing method is a method in which a polymer solution is uniformly applied onto a substrate using a dispenser that dispenses a coating solution in a fixed amount. While discharging the polymer solution from a discharge device such as a syringe pump, the discharge port can be operated on the substrate at a constant speed, so that it can be uniformly applied to any place on the substrate. Further, application may be performed by fixing the discharge port and operating the substrate at a constant speed. It is possible to make the coating amount uniform by keeping the distance between the substrate and the discharge port constant, and further reducing the thickness of the coating solution by narrowing the gap as much as possible to reduce the amount of coating solution used and the drying speed. Improvement is possible and preferable.

(2-5) Hydrophobic polymer The hydrophobic polymer that can be used in the present invention is a polymer compound that does not absorb water, and has a solubility in water (25 ° C) of 10 mass% or less, more preferably 1 mass. % Or less, most preferably 0.1% by mass or less.

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

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

  Coating of the hydrophobic polymer on the substrate can be performed by a conventional method, for example, spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, spraying, vapor deposition, casting, dipping. Etc.

  The coating thickness of the hydrophobic polymer is not particularly limited, but is preferably 0.1 nm to 500 nm, and particularly preferably 1 nm to 300 nm. The weight average molecular weight of the hydrophobic polymer is not particularly limited, but a preferred range is 10,000 to 50 million.

(3) Immobilization of the physiologically active substance to the layer for immobilizing the physiologically active substance The layer for immobilizing the physiologically active substance described above has a functional group capable of immobilizing the physiologically active substance. preferable.
For example, when the layer for immobilizing a physiologically active substance is composed of a hydrophilic polymer having a carboxyl group, after activating the carboxyl group, the physiologically active substance having an amino group is reacted. The physiologically active substance can be immobilized on a hydrophilic polymer. The carboxyl group activation in immobilizing the physiologically active substance in the layer for immobilizing the physiologically active substance is the same as the method described in (2-3) Activation of hydrophilic polymer in the present specification. A similar method can be used.

  In the present invention, a layer for immobilizing the physiologically active substance is formed on the surface of the metal substrate having a layer for immobilizing the physiologically active substance (layer for immobilizing the physiologically active substance). A solution containing a physiologically active substance capable of forming a covalent bond with a molecule is applied and dried to form a uniform film on the substrate surface. At this time, the physiologically active substance is immobilized on the surface of the metal substrate by covalently bonding with a molecule constituting a layer for immobilizing the physiologically active substance.

  In the present invention, as a functional group capable of immobilizing a physiologically active substance, in addition to a functional group such as a carboxyl group or an amino group, by introducing an alkane, a physiologically active substance having a membrane structure such as a lipid is immobilized. It becomes possible to do. 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. In addition, if NTA (nitrotropic acid) or the like is introduced into the polymer as a fixing group, a physiologically active substance with a His-tag ligand can be fixed via a metal chelate.

(4) Bioactive substances that can be used in the present invention Bioactive substances that can be used in the present invention include protein A, protein G, avidins (avidin, streptavidin, neutravidin), antigens, antibodies (for example, And known tag antibodies such as anti-GST antibodies). When these physiologically active substances are used, proteins and the like can be further bound on these physiologically active substances. For example, when avidin is used as a physiologically active substance, a biotinylated protein can be immobilized by an avidin-biotin bond, and when an antibody is used as a physiologically active substance, an antigen can be immobilized. .
In the present invention, it is preferable to use avidin as the physiologically active substance, and it is more preferable to use streptavidin.

  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 used at 0.1 mg / mL to 10 mg / mL, more preferably 1 mg / mL to 10 mg / mL.

(5) Application and drying of physiologically active substance In the present invention, after applying a solution containing a physiologically active substance, it is dried by allowing to stand.

  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 step is preferably maintained at 0.9 cP or higher.

  In the present invention, the step of drying the solution containing the physiologically active substance means that after the solution containing the physiologically active substance is applied to the substrate, the solution is naturally dried by standing or heated while standing. A process of increasing the speed at which the solution is dried by blowing air or the like and intentionally drying it. Increasing the drying rate of a solution (coating solution) containing a physiologically active substance is effective in suppressing variation 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. An air knife method may be used as a drying method.

  The drying step is preferably performed under the condition that the temperature and humidity are kept constant after the coating step. In this drying step, the temperature difference between the dry bulb temperature and the wet bulb temperature is preferably dried in a dry environment of 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 0.1 second or more and 10 minutes or less, more preferably 1 second or more and 5 minutes or less, and particularly preferably 1 second or more and 1 minute or less.

  If the temperature difference between the dry bulb temperature and the wet bulb temperature in the drying step is less than 7 ° C., there may be a problem that the fixed amount of the physiologically active substance varies.

  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. The dispenser may store the solution in a syringe and discharge it, or may discharge using a pipette. In particular, the use of pipette is preferable because it can reduce the residual amount of the physiologically active substance in the dispenser. 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 during rotation. .

  In the coating step, it is preferable to stand under the same environmental conditions as in the drying step.

  In the step of applying the solution and the drying step, at least one of these two steps is performed in the dry environment (the difference between the dry bulb temperature and the wet bulb temperature is 7 ° C. or more). It is preferable to perform all these two steps in a dry environment because the amount of biotinylated protein immobilized on the physiologically active substance can be increased.

  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. When performing these measurements, it is preferable to measure in a dry state, depending on the characteristics of the apparatus. As used herein, <dry state> refers to the same conditions as in the dry state when measuring the amount of hydrophilic polymer fixed described in paragraph [0065] of this specification.

  A high molecular weight protein such as IgG (immunoglobulin) CRP (C-reactive protein) or the like having a molecular weight of 105 kDa or more and 150 kDa or less is used as the physiologically active substance to be immobilized on the layer for immobilizing the physiologically active substance by the coating and drying step In such a case, a method of binding a biotinylated polymer protein using the avidin-biotin bond interaction after immobilizing a physiologically active substance avidin by the coating and drying step, etc. can give. However, in this case, a suitable amount of the high molecular protein may not be immobilized as a result due to steric hindrance caused by avidins.

Therefore, in the present invention, the preparation the density of avidin is immobilized to the layer for immobilizing a physiologically active substance by the coating and drying process, so that the density 1 pmol/mm ³ or 2.5 pmol / mm ³ or less To do.

  In the present invention, as a method for adjusting the density of the avidin to be immobilized, the density of the avidin is adjusted by adjusting the amount of the hydrophilic polymer immobilized on the substrate according to the conditions described in paragraph [0098] above. Can be adjusted. In addition to this method, the density of avidins in the present invention can be adjusted by reducing the amount of hydrophilic polymer activated when immobilizing physiologically active substances (avidins).

  By adjusting the density of avidins immobilized on the layer for immobilizing the physiologically active substance so as to be in the above range, the biotinylated polymer protein is immobilized at a high concentration. Moreover, since the biotinylated polymer protein is immobilized by binding to avidins, inactivation is suppressed.

By the coating and drying steps, the physiologically active material density of avidin immobilized to the surface of the layer for immobilizing a density 1 pmol/mm ³ or 2.5 pmol / mm ³ or less, as described above there but is preferably from density 1.07pmol / mm ³ or more 2.48pmol / mm ^3, it is more preferably less density 1.58pmol / mm ³ or more 2.28pmol / mm ^3.

If the density of avidin immobilized on the layer for immobilizing the physiologically active substance is less than 1 pmol / mm ³ , there may be a problem that the amount of biotinylated polymer protein immobilized decreases. If it exceeds 2.5 pmol / mm ³ , the immobilization of the biotinylated polymer protein may be inhibited by steric hindrance due to avidins, and a high concentration of immobilization may not be achieved.

  The density of the avidins immobilized on the surface of the layer for immobilizing the physiologically active substance may be obtained in the same manner as the above-described method for calculating the amount of immobilized physiologically active substance.

  In order to bind the biotinylated polymer protein to the avidin immobilized at the predetermined density to the layer for immobilizing the physiologically active substance, in the solution containing the biotinylated polymer protein, A substrate on which avidins are immobilized may be brought into contact. Moreover, when making it contact, it is preferable to set it as neutral conditions.

As described above, by adjusting the density of the avidin immobilized to the metal substrate such that 1 pmol/mm ³ or 2.5 pmol / mm ³ or less, to bind the biotinylated polymer protein this avidins As described above, a high-concentration biotinylated polymer protein is immobilized. As described above, the amount of the biotinylated polymer protein immobilized at this high concentration is specifically, It is 0.41 pmol / mm ³ or more and 0.87 pmol / mm ³ or less. At this time, the molar ratio between the avidin immobilized on the layer for immobilizing the physiologically active substance and the biotinylated polymer protein is in the range of 0.17 to 0.69. Inside.

  The molar ratio of the avidin immobilized on the layer for immobilizing the physiologically active substance and the biotinylated polymer protein is 0.17 or more and 0.69 or less as described above, and 0 .35 or more and 0.55 or less is preferable.

If the molar ratio of avidin to biotinylated polymer protein is less than 0.17, there may be a problem that the amount of biotinylated polymer protein immobilized is small.
It should be noted that the molar ratio of the avidin immobilized on the layer for immobilizing this physiologically active substance and the biotinylated polymer protein is adjusted to be 0.17 or more and 0.69 or less. in order, the density of the avidin immobilized, may be prepared such that the above 1 pmol/mm ³ or 2.5 pmol / mm ³ or less.

In addition, the molar ratio of the avidin immobilized in the layer for immobilizing the physiologically active substance and the biotinylated polymer protein is 0.35 to 0.55, which is the above preferred range. In order to prepare in such a manner, the density of the avidin to be immobilized may be adjusted to be in the range of 1.58 pmol / mm 3 or more and 2.28 pmol / mm 3 or less.

  The molar ratio between the avidin and the biotinylated polymer protein can be obtained by the following formula (1).

  (Biotinylated polymer protein immobilized amount (RU) / immobilized biotinylated polymer protein weight average molecular weight) / (Avidin immobilized amount (RU) / immobilized avidin Weight average molecular weight) (1)

In Formula (1), RU (Resonance Unit) measures 120 points on the substrate at intervals of 0.25 mm using an SPR imager (SPR imager manufactured by CWG), and fixes the measurement object at each measurement point. It is obtained by measuring the amount of change in the resonance signal obtained after immobilizing the measurement object with respect to the reference point indicating the resonance signal in an unconverted state as a fixed amount (RU) and obtaining the average value. 1 RU corresponds to a fixed amount of 1 pg / mm ² .

(6) Biotinylated polymer protein In the present invention, the biotinylated polymer protein provides biotin capable of binding to avidins among proteins and nucleic acids shown in the following <1> to <5>. Furthermore, the weight average molecular weight is 100,000 or more and 200,000 or less. For example, immune proteins, enzymes, microorganisms, nucleic acids, non-immune proteins, immunoglobulin binding proteins, sugar binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides with ligand binding ability Among them, those having a weight average molecular weight of 100,000 or more and 200,000 or less and further biotinylated can be mentioned. Here, a known method can be used for biotinylation.

  <1> Examples of immunity proteins include antibodies and haptens that use a 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.

  <2> The enzyme is not particularly limited as long as it exhibits activity with respect to a measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, An isomerase, a desorption enzyme, a synthetic enzyme, etc. can be used. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase, dopamine esterase, and CRP (C-reactive protein) can be used.

  <3> The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.

  <4> As the nucleic acid, a nucleic acid that hybridizes complementarily to 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.

  <5> 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.

  Among the biotinylated macromolecular proteins, immune proteins or enzymes are preferably used, and IgG, IgM, IgA, IgE, IgD, and CRP are more preferably used.

(7) Method of using the substrate of the present invention The substrate of the present invention on which the 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. it can. In this detection and / or measurement, the substance that interacts with the physiologically active substance is not particularly limited, and detection and / or measurement from a low molecular compound to a high molecular compound such as a protein is possible. Here, “low molecular compound” refers to a compound having a weight average molecular weight of less than 1000, and “polymer compound” refers to a compound having a weight average molecular weight of 1000 or more.

  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 substrate produced by the method of the present invention is used as a biosensor for surface plasmon resonance, for example, comprising a metal film disposed on a transparent substrate. it can.

  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 apparatus for analyzing the characteristics of a substance to be measured by utilizing a phenomenon in which surface plasmons are excited by light waves, an apparatus using a system called a 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 there may be components that enter the light beam at various angles. As included, a relatively thick light beam may be incident on the interface in a convergent or divergent 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, so that the entire energy is transferred to the interface between the dielectric block and the metal film. 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.

  Further, as a similar measuring device using total reflection attenuation (ATR), for example, “Spectroscopy” Vol. 47, No. 1 (1998), pages 21 to 23 and pages 26 to 27 are disclosed. Mode measuring 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, rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and human IgG antibody can be used as the specific substance.

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

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

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

  The 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 formed into 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 waveguide layer and a change in the refractive index and thickness of the waveguide layer itself or an additional layer adjacent to the waveguide layer. It is.

  Regarding the structure of the biosensor of this system, for example, Japanese Patent Publication No. 6-27703, page 4, line 48 to page 14, line 15 and FIG. 1 to FIG. 8, column 6 of US Pat. No. 6,829,073. It is described from the 31st line to the 47th line of column 7 and FIGS. 9A and 9B.

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 may be, for example, an oxide layer (SiO ₂ , SnO ₂ , Ta ₂ O ₅ , TiO ₂ , TiO ₂ —SiO ₂ , HfO ₂ , ZrO ₂ , Al ₂ O ₃ , Si ₃ N ₄ , HfON, SiON, Multilayer laminates such as scandium oxide or mixtures thereof, plastic layers (eg polystyrene, polyethylene, polycarbonate, etc.) are 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 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 method for detecting and / or measuring a substance that interacts with a physiologically active substance using the substrate of the present invention is used as a fragment assay (a method for detecting a low molecular weight compound as a substance that interacts with a physiologically active substance). Is possible. Fragment assays can contribute to the efficiency of drug discovery techniques, and by using the above detection and / or detection method of the present invention as a search for compounds that serve as drug bases, it is possible to determine the structure of a physiologically active substance. The efficiency of drug discovery can be improved by detecting low molecular weight compounds with activity correlation and designing more active compounds. In a fragment assay using the substrate of the present invention and a method for detecting and / or measuring a substance that interacts with a physiologically active substance, a low molecular weight compound having a weight average molecular weight of about 100 to 500 can be measured.

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

  Hereinafter, in this example, the ability of biotinylated polymer protein to be immobilized on the surface of a metal substrate having a layer having avidins immobilized on the surface was evaluated.

Example 1
(1) Preparation of substrate A 1 mM aqueous solution of 6-Amino-1-octanethiol, hydrochloride (manufactured by Dojindo) was prepared. This solution is called A liquid.

Next, pellets of ZEONEX (manufactured by Nippon Zeon Co., Ltd.) were melted at 240 ° C., and this melt was molded into a plastic prism substrate having a length of 8 mm × width of 120 mm × 1.5 mm using an injection molding machine. A prism is attached to a substrate holder of a parallel plate type 6-inch sputtering device (SH-550, ULVAC, Inc.) on this substrate, and after drawing a vacuum (base pressure 1 × 10 ⁻³ Pa or less), Ar gas is supplied. Introducing (1 Pa), while rotating the substrate holder (20 rpm), RF power (high frequency electrical signal) (0.5 kW) is applied to the substrate holder for about 9 minutes to plasma-treat the plastic prism surface. Next, the Ar gas was stopped and vacuum was drawn, and the Ar gas was reintroduced (0.5 Pa), and while rotating the substrate holder (10 rpm to 40 rpm), the DC power (DC voltage) (0. 2 kW) is applied for about 30 seconds to form a 2 nm Cr thin film. next. Stop Ar gas, evacuate again, introduce Ar gas again (0.5 Pa), apply DC power (1 kW) to the 8-inch Au target for about 50 seconds while rotating the substrate holder (20 rpm). An Au thin film having a thickness of about 50 nm was formed.

  The plastic prism on which the Au thin film obtained above was formed was immersed in the liquid A at 40 ° C. for 1 hour and washed 5 times with ultrapure water.

(2) Active esterification of CMD (carboxymethyl dextran) After preparing 20 g of 1% by weight of CMD (manufactured by Meitsu Sangyo: weight average molecular weight 1 million, carboxymethyl group substitution degree per sugar unit 0.67) 20 ml of an aqueous solution having a concentration of 15 mM was added to EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate), and the mixture was stirred at room temperature for 1 hour.

(3) Binding reaction of CMD to substrate On the substrate (plastic prism formed with an Au thin film) prepared in (1) above, the mixed solution of CMD and EDC prepared in (2) above is 1. 5 ml was dropped, spin-coated at 1500 rpm for 45 seconds, and allowed to stand at room temperature for 15 minutes to react, thereby fixing the carboxymethyldextran thin film on the substrate having amino groups. Next, a CMD film was formed by immersing in a 1N NaOH aqueous solution for 30 minutes and washing 5 times with ultrapure water.

(4) Production of sensor stick (4-1)
1 ml of a mixed aqueous solution of 0.2 M EDC / 50 mM NHS (N-Hydroxysuccinimide) was dropped on the CMD film prepared in (3) above and allowed to stand for 7 minutes. The same surface was washed 5 times with pure water. After thoroughly removing the cleaning solution from the surface using a nitrogen gun, it was set on a spin coat in an environment of 23 ° C. and 10%, and 100 ul cast of 5 mg / ml streptavidin (manufactured by Wako Pure Chemical Industries) Borate buffer (pH 8.5) Then, after being allowed to stand for 1 minute and then spin coating at 500 rpm for 45 seconds) 3 times, drying was performed by standing for 15 minutes.
(4-2) Blocking treatment After standing for 15 minutes, cast 1M 2-aminoethanol (manufactured by Wako Pure Chemical Industries, Ltd.), pH 8.5, let stand for 7 minutes, wash with Borate buffer, and soak in 1N NaOH aqueous solution for 15 minutes The avidin-modified substrate 2 on which streptavidin was immobilized was obtained by sequentially washing with the Borate buffer.

(4-3)
The avidin-modified substrate prepared by the above processes (4-1) and (4-2) was set on a surface plasmon resonance apparatus.
After injecting PBS buffer and confirming the baseline (with the resonance angle at this time as a reference point), a 0.5 mg / ml biotinylated IgG solution (manufactured by Fitzgerald, CRP monoclonal antibody) (PBS buffer pH 7.4) Then, a biotinylated protein having a molecular weight of 150 kDa) was injected and stirred for 30 minutes.
Next, inject PBS buffer and let stand for 1 minute, then inject 0.2 mg / ml D-Biotin solution and let stand for 1 minute, and finally inject PBS buffer and let stand for 1 minute twice. Then, a sensor stick 2 in which biotinylated IgG was immobilized via adibin was obtained.

(Example 2)
The biotinylated IgG was obtained after obtaining the avidin-modified substrate 3 on which avidin was immobilized in the same manner as in Example 1 except that the concentration of EDC was changed to 10 mM in the step (2) of Example 1 above. A sensor stick 3 was obtained, which was immobilized via ajbin.

(Example 3)
In the step (2) of Example 1, except that the concentration of EDC was changed to 6 mM, biotinylated IgG was obtained after obtaining the avidin-modified substrate 4 on which avidin was immobilized, in the same manner as in Example 1 above. The sensor stick 4 in which was immobilized via adivine was obtained.

Example 4
In the step (2) of Example 1, except that the concentration of EDC was changed to 3 mM, biotinylated IgG was obtained after obtaining the avidin-modified substrate 5 on which avidin was immobilized, in the same manner as in Example 1 above. A sensor stick 5 was obtained, which was immobilized via ajbin.

(Example 5)
In the step (2) of Example 1 above, except that the concentration of EDC was 1 mM, in the same manner as in Example 1 above, after obtaining the avidin-modified substrate 6 on which avidin was immobilized, biotinylated IgG Obtained a sensor stick 6 immobilized through ajbin.

(Example 6)
In the step (2) of Example 1, the avidin-modified substrate 7 on which avidin was immobilized was obtained in the same manner as in Example 1 except that the EDC concentration was 0.4 mM. Thus, a sensor stick 7 in which IgG was immobilized via adivine was obtained.

(Comparative Example 1)
In the step (2) of Example 1, except that the concentration of EDC was 25 mM, the biotinylated IgG was obtained after obtaining the avidin-modified substrate 1 on which avidin was immobilized in the same manner as in Example 1 above. A sensor stick 1 was obtained, which was immobilized via adivine.

(Comparative Example 2)
In the step (2) of Example 1 described above, the surface plasmon resonance apparatus was applied to the CMD film obtained by setting the EDC concentration to 10 mM and not fixing the avidin obtained in the step (3) of Example 1. After activating for 7 minutes using the above 0.2M EDC / 50 mM NHS mixed aqueous solution, 5 mg / ml Streptavidin (manufactured by Wako Pure Chemical Industries) Borate buffer (pH 8.5) was added and stirred for 30 minutes. After obtaining the avidin-modified substrate 8 on which avidin was immobilized by performing the steps (4-2) to (4-3) in Example 1, the biotinylated IgG was immobilized via adivine. The obtained sensor stick 8 was obtained.

(Comparative Example 3)
In the step (2) of Example 1 above, the surface plasmon resonance apparatus was applied to the CMD film obtained by setting the EDC concentration to 3 mM and not fixing the avidin obtained in the step (3) of Example 1 After activating for 7 minutes using the above 0.2M EDC / 50 mM NHS mixed aqueous solution, 5 mg / ml Streptavidin (manufactured by Wako Pure Chemical Industries) Borate buffer (pH 8.5) was added and stirred for 30 minutes, By performing the steps (4-2) to (4-3) in Example 1 above, the biotinylated IgG was immobilized via adivine after obtaining the avidin-modified substrate 9 on which avidin was immobilized. A sensor stick 9 was obtained.

(Comparative Example 4)
In the step (2) of Example 1 above, the surface plasmon resonance apparatus was applied to the CMD film obtained by setting the EDC concentration to 3 mM and not fixing the avidin obtained in the step (3) of Example 1 And activated for 7 minutes using the above 0.2 M EDC / 50 mM NHS mixed aqueous solution, 0.5 mg / ml of Streptavidin (manufactured by Wako Pure Chemical Industries), acetate buffer (pH 4.5) was added, and the mixture was stirred for 30 minutes. Thereafter, the steps (4-2) to (4-3) of Example 1 are performed to obtain an avidin-modified substrate 10 on which avidin is immobilized, and then biotinylated IgG is immobilized via adivine. The obtained sensor stick 10 was obtained.

The density of avidin (pmol / mm ³ ) immobilized on each of the avidin-modified substrates 1 to 10 on which avidins were immobilized, which were prepared in Examples 1 to 6 and Comparative Examples 1 to 4. ) Was measured. The measurement results are shown in Table 1.

Furthermore, each of the sensor sticks 1 to 10 immobilized with the biotinylated polymer protein via avidin prepared in Examples 1 to 6 and Comparative Examples 1 to 6 was immobilized. The amount of immobilized biotinylated polymer protein (pmol / mm ³ ) was measured. The measurement results are shown in Table 1.

It should be noted that the density of the immobilized avidin and the amount of the biotinylated polymer protein immobilized are determined by the resonance angle and the origin resonance angle in the sensor stick adjustment step (step (4-3) above). The difference was measured as the amount of immobilized biotinylated polymer protein. The resonance angle was calibrated with a DMSO solution, and the change in resonance angle between the PBS buffer and the PBS buffer containing 10% DMSO was proportionally calculated as a fixed amount of 1500 pg / mm ² biotinylated polymer protein.
Similarly, the fixed amount of avidin was calculated from the change in resonance angle before and after fixing the prepared avidin modified substrate.

  Further, an avidin immobilized on each of the sensor sticks 1 to 10 prepared in Examples 1 to 6 and Comparative Examples 1 to 4 and a biotinylated polymer protein, The molar ratio was determined. The results are shown in Table 1.

  In addition, this molar ratio was calculated | required by following formula (2).

  (Biotinylated polymer protein immobilized amount (RU) / immobilized biotinylated polymer protein weight average molecular weight) / (Avidin immobilized amount (RU) / immobilized avidin weight) Average molecular weight) Formula (2)

As shown in Table 1, the coating and the avidin immobilized on the substrate by drying, and the avidin when immobilized to a density 1 Pmol/mm ³ or 2.5 pmol / mm ³ or less of the density In the sensor stick obtained by binding biotinylated polymer protein, the molar ratio of avidin to biotinylated polymer protein is within the range of 0.17 to 0.69. There also a fixed amount of biotinylated polymeric protein became 0.41pmol / mm ³ or more 0.87pmol / mm ³ within the following range. For this reason, in Examples 1 to 6, a suitable amount of biotinylated polymer protein was immobilized.
On the other hand, in the sensor sticks 1 and 8 to 10 prepared in Comparative Examples 1 to 4, the amount of biotinylated polymer protein immobilized was smaller than that of the Examples, and a good amount of immobilization was not obtained. It can be said that.

Therefore, the immobilization on the substrate by coating and drying the avidin, by adjusting the amount of immobilized avidin such that the density 1 pmol/mm ³ or 2.5 pmol / mm ³ or less, the avidins Thus, it can be said that a suitable amount of the biotinylated polymer protein could be immobilized.

It is a disassembled perspective view which shows schematic structure of a sensor unit.

Explanation of symbols

25 Metal film (thin film layer)
26 Layer for immobilizing physiologically active substances

Claims (17)

Applying a solution containing a physiologically active substance capable of forming a covalent bond with a molecule constituting the layer for immobilizing the physiologically active substance on a metal substrate having a layer for immobilizing the physiologically active substance; And subsequent drying step,
The biologically active substance is avidin or the like, to be immobilized at a density of 1 pmol/mm ³ or 2.5 pmol / mm ³ or less on a layer for immobilizing the physiologically active substance by said step of drying A method for producing a physiologically active substance-immobilized substrate. Further comprising the step of binding a biotinylated macromolecular protein that is an avidin-binding protein to the avidin immobilized on the layer for immobilizing the physiologically active substance,
2. The bioactive substance-immobilized substrate according to claim 1, wherein a molar ratio of the avidin and the bound biotinylated polymer protein is 0.17 or more and 0.69 or less. Method.   The method for producing a physiologically active substance-immobilized substrate according to claim 2, wherein the high molecular weight protein has a weight average molecular weight of 100,000 or more and 200,000 or less. The avidin is immobilized at a density of 1.07 pmol / mm ³ or more and 2.48 pmol / mm ³ or less in a layer for immobilizing the physiologically active substance in the drying step. The method for producing a physiologically active substance-immobilized substrate according to any one of claims 1 to 3.   The molar ratio between the avidin and the bound biotinylated polymer protein is 0.35 or more and 0.55 or less, according to any one of claims 2 to 4. The manufacturing method of the bioactive substance fixed board | substrate of description.   The method for producing a physiologically active substance-immobilized substrate according to any one of claims 1 to 5, wherein the avidin is streptavidin.   The method for producing a physiologically active substance-immobilized substrate according to any one of claims 1 to 6, wherein a variation coefficient (CV value) of a fixed amount of the physiologically active substance is 15% or less.   The layer for immobilizing the physiologically active substance comprises at least one selected from a hydrophilic polymer layer, a hydrophobic polymer layer, and a self-oxidation film. A method for producing a physiologically active substance-immobilized substrate according to claim 1.   The method for producing a physiologically active substance-immobilized substrate according to claim 8, wherein the layer for immobilizing the physiologically active substance includes at least a hydrophilic polymer layer.   The method for producing a physiologically active substance-immobilized substrate according to claim 9, wherein the hydrophilic polymer is covalently bonded to the physiologically active substance by activating the hydrophilic polymer.   The method for producing a physiologically active substance-immobilized substrate according to any one of claims 8 to 10, wherein the hydrophilic polymer layer is fixed to the metal substrate via a self-oxidizing film.   12. The method for producing a physiologically active substance-immobilized substrate according to claim 11, wherein the hydrophilic polymer layer is made of a polysaccharide and is fixed to the metal substrate via an auto-oxidation film.   The method for producing a physiologically active substance-immobilized substrate according to claim 11, wherein the polysaccharide has a weight average molecular weight of 100,000 to 200,000.   The said drying process WHEREIN: The solution containing the said bioactive substance apply | coated by the said application | coating process is dried by the temperature difference of dry-bulb temperature and wet-bulb temperature being 7 degreeC or more. 14. The method for producing a physiologically active substance-immobilized substrate according to any one of items 13.   The said application | coating process apply | coats the solution containing the said bioactive substance to the said metal substrate in the dry environment whose temperature difference of dry bulb temperature and wet bulb temperature is 7 degreeC or more. Item 15. The method for producing a physiologically active substance-immobilized substrate according to any one of Items 14 to 14.   The method for producing a physiologically active substance-immobilized substrate according to any one of claims 1 to 15, wherein, in the drying step, the drying time of the solution containing the physiologically active substance is 10 minutes or less. .   The board | substrate obtained by the manufacturing method of the bioactive substance fixed board | substrate of any one of Claims 1-16.

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