JP4372142B2 - Biosensor manufacturing method - Google Patents

Description

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

  Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations, etc., but conventional methods require complicated operations and labeling substances, so measurement without the need for labeling substances Several techniques that can detect a change in the amount of a substance bound with high sensitivity are used. For example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, and measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. SPR measurement technology detects adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a fixed wavelength. It is a method to do. The QCM measurement technique is a technique capable of detecting 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 chip is formed on the metal surface via the functional group. Immobilize physiologically active substances. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance.

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

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

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

Japanese Patent No. 2815120

  The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a biosensor capable of easily producing a hydrogel capable of immobilizing a physiologically active substance using a safe raw material and a method for producing the same. Furthermore, the present invention has an object to be solved by providing a biosensor having a large amount of physiologically active substance immobilized and a small amount of non-specific adsorption and a method for producing the same.

  As a result of intensive studies to solve the above problems, the present inventors have immobilized a physiologically active substance by reacting with a substrate having an amino group in a state where a hydrophilic polymer having a carboxyl group is activated. The inventors have found that the resulting hydrogel can be easily produced, and have completed the present invention.

  That is, according to the present invention, an activated carboxyl group-containing polymer is brought into contact with a substrate surface coated with an amino group-containing organic layer, and the polymer is bonded to the organic layer. A method of manufacturing a biosensor is provided.

Preferably, the activated carboxyl group is an active esterified carboxyl group.
Preferably, the substrate is a metal surface or a metal film.
Preferably, the metal is any of gold, silver, copper, platinum or aluminum.

Preferably, the substrate surface coated with the organic layer having an amino group is a substrate surface coated with an alkanethiol having an amino group.
Preferably, the alkanethiol having an amino group is any of a compound in which a thiol group and an amino group are linked via an alkyl chain, or a compound obtained by reacting an alkanethiol having a carboxyl group at the terminal with a hydrazide or diamine. It is.

Preferably, the substrate surface coated with an organic layer having an amino group is a substrate surface coated with a mixture of an alkanethiol having an amino group and an alkanethiol having a hydrophilic group.
Preferably, the hydrophilic group of the alkanethiol having a hydrophilic group is a hydroxyl group or an oligoethylene glycol group.
Preferably, the molar ratio in the mixture of alkanethiol having an amino group and alkanethiol having a hydrophilic group is in the range of 1/1 to 1 / 1,000,000.
Preferably, the molecular length of the alkanethiol having an amino group is longer than the molecular length of the alkanethiol having a hydrophilic group.

Preferably, the polymer containing carboxyl groups is a polysaccharide.
Preferably, the average molecular weight of the polymer containing a carboxyl group is 1000 to 5000000.

Preferably, the polymer containing the activated carboxyl group is reacted with the organic layer having an amino group in a state where a thin film is formed on the substrate.
Preferably, a thin film is formed on the substrate by spin coating or spray coating.

  Preferably, the polymer containing an activated carboxyl group is obtained by activating a polymer containing a carboxyl group with a carbodiimide derivative, a nitrogen-containing compound, or a phenol derivative.

Preferably, the carbodiimide derivative is water soluble.
Preferably, the carbodiimide derivative is any one of compounds 1 to 3 .

[式中、Ｒ₁及びＲ₂は、互いに独立して置換基を有しても良いカルボニル基、炭素原子、窒素原子を表し、Ｒ₁及びＲ₂は結合により５〜６員環を形成しても良く、Ａは置換基を有する炭素原子またはリン原子を表し、Ｍは（n-1）価の元素を表し、Ｘはハロゲン原子を表す]で表される化合物である。 Preferably, the nitrogen-containing compound has the following general formula (Ia) or (Ib):

[Wherein, R ₁ and R ₂ each independently represent a carbonyl group, a carbon atom or a nitrogen atom which may have a substituent, and R ₁ and R ₂ form a 5- to 6-membered ring by a bond. A represents a carbon atom or phosphorus atom having a substituent, M represents an (n-1) valent element, and X represents a halogen atom.

[式中、Ｙ及びＺは、互いに独立して、ＣＨまたは窒素原子を表す]で表される化合物である。 Preferably, the nitrogen-containing compound has the following general formula (II):

[Wherein Y and Z each independently represent CH or a nitrogen atom].

Preferably, the phenol derivative is a compound having an electron withdrawing group.
Preferably, the σ value of the electron-withdrawing group of the phenol derivative is 0.3 or more.

  Preferably, when activating a polymer containing a carboxyl group, a carbodiimide derivative and a nitrogen-containing compound, or a carbodiimide derivative and a phenol derivative are used in combination.

Preferably, when activating a polymer containing a carboxyl group, the following morpholine derivative is used.

Preferably, the mixing molar ratio of the carbodiimide derivative or the morpholine derivative to the carboxyl group in the polymer is 1 × 10 ⁻⁴ to 1.
Preferably, the mixing molar ratio of the nitrogen-containing compound to the carboxyl group in the polymer is 1 × 10 ⁻⁷ to 1.

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

  According to still another aspect of the present invention, a bioactive substance is immobilized on a biosensor comprising the step of bringing the biosensor of the present invention into contact with a bioactive substance and binding the bioactive substance to the biosensor. A method is provided.

According to still another aspect of the present invention, the bioactive substance interacts with the bioactive substance, comprising the step of bringing the biosensor of the present invention in which the bioactive substance is covalently bonded to the surface into contact with the test substance. A method for detecting or measuring a substance is provided.
Preferably, a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method, more preferably detected or measured by surface plasmon resonance analysis.

  According to the present invention, a biosensor with a large amount of physiologically active substance immobilized and a small amount of non-specific adsorption can be easily produced using safe raw materials.

  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 a chemical change generated therein into an electrical signal. In the living body, there are enzymes / substrates, enzymes / coenzymes, antigens / antibodies, hormones / receptors and the like as substances having affinity for each other. Biosensors use the principle that one of these substances having affinity with each other is immobilized on a substrate and used as a molecular recognition substance, thereby selectively measuring the other substance to be matched.

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

  Although the thickness of the metal film is arbitrary, for example, when considering use for a surface plasmon resonance biosensor, the thickness is preferably 0.1 nm to 500 nm, particularly preferably 1 nm to 200 nm. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.

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

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

  A polymer film 26 is provided on the metal film 25. The polymer film 26 has a binding group for immobilizing a physiologically active substance, and the physiologically active substance is immobilized on the metal film 25 via the polymer film 26.

  In the present invention, a metal film is coated with an organic layer having an amino group, and then a polymer containing an activated carboxyl group is reacted with the organic layer to produce a hydrogel capable of immobilizing a physiologically active substance. be able to.

In the present invention, a known method can be used as a method of coating a metal film with an organic layer having an amino group. However, a coating method using self-assembled films (SAMs) is preferable because the operation is simple. . The coating method of metal films using self-assembled films (SAMs) has been energetically developed by Prof. Whitesides et al. At Harvard University. The details are reported in, for example, Chemical Review, 105, 1103-1169 (2005). ing. When gold is used as the metal, 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. As a result, a monomolecular film having orientation is self-organized based on the van der Waals force between the Au—S bond and the alkyl chain. 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. By forming a self-assembled film using a compound where X = NH2 in the general formula A-1 , the gold surface can be covered with an organic layer having an amino group.

The alkanethiol having an amino group at the terminal is a compound (general formula A-2 ) in which the thiol group and the amino group are linked via an alkyl chain (in the general formula A-2 , n represents an integer of 3 to 20). The alkanethiol having a carboxyl group at the terminal (general formulas A-3 and A-4 ) (in the general formula A-3 , n represents an integer of 3 to 20, and in the general formula 4, each n is independently 1 Or a compound obtained by reacting a large excess of hydrazide or diamine. The reaction between the alkanethiol having a carboxyl group at the terminal and 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, a large excess of hydrazide. Or you may make diamine react.

  The number of repeating alkyl groups A-2 to A-4 is preferably 3 or more and 20 or less, more preferably 3 or more and 16 or less, and most preferably 4 or more and 8 or less. When the alkyl chain is short, it is difficult to form a self-assembled film, and when the alkyl chain is long, water solubility is lowered and handling becomes difficult.

As the diamine used in the present invention, any compound can be used, but when used on the biosensor surface, a water-soluble diamine is preferred. Specific examples of water-soluble diamines include aliphatic diamines such as ethylenediamine, tetraethylenediamine, octamethylenediamine, decamethylenediamine, piperazine, triethylenediamine, diethylenetriamine, triethylenetetraamine, dihexamethylenetriamine, and 1.4-diaminocyclohexane. , Paraphenylenediamine, metaphenylenediamine, paraxylylenediamine, metaxylylenediamine, 4,4'-diamonobiphenyl, 4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenyl ketone, 4,4'- Aromatic diamines such as diaminodiphenylsulfonic acid can be mentioned. From the viewpoint of improving the hydrophilicity of the biosensor surface, it is also possible to use a compound (general formula 5) in which two amino groups are linked by an ethylene glycol unit. The diamine used in the present invention is preferably ethylenediamine or a compound represented by the general formula A-5 (in the general formula A-5 , n and m each independently represents an integer of 1 to 20), and more Ethylenediamine or 1,2-bis (aminoethoxy) ethane (in formula A-5 , n = 2, m = 1) is preferable.

The alkanethiol having an amino group can form a self-assembled film alone, or can be mixed with other alkanethiols to form a self-assembled film. When used on the biosensor surface, it is preferable to use a compound capable of suppressing nonspecific adsorption of a physiologically active substance as the other alkanethiol. The self-assembled membrane that can suppress non-specific adsorption of physiologically active substances has been studied in detail by the aforementioned Professor Whitesides et al. The self-assembled membrane formed from alkanethiol having a hydrophilic group is non-specifically adsorbed It 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. As alkanethiol that forms a mixed monomolecular film with an alkanethiol having an amino group is excellent in non-specific adsorption inhibiting ability and is easily available, alkanethiol having a hydroxyl group (general formula A-6 ) or ethylene group in alkanethiol (general formula a-7) (in the formula a-6 with a call unit, n represents an integer of 3 to 20, in the general formula a-7, n and m are from 1 to 20 independently It is preferable to use an integer.

  When an alkanethiol having an amino group is mixed with another alkanethiol to form a self-assembled film, the repeating number of the alkyl groups of A-2 to A-4 is preferably 4 or more and 20 or less, and more preferably 4 or more and 16 The following is preferable, and 4 or more and 10 or less are most preferable. The number of repeating alkyl groups A-6 and A-7 is preferably 3 or more and 16 or less, more preferably 3 or more and 12 or less, and most preferably 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, but activated when the ratio of the alkanethiol having an amino group is small. When the binding amount of the carboxyl group-containing polymer 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 preferably 1/4 to 1 / 10,000. Is more preferable, and the range of 1/10 to 1/1000 is more preferable. From the viewpoint of reducing steric hindrance when reacting with a polymer containing an activated 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, 1763-1797 (2004)) and its cited references. Commercially available compounds may also be used. These compounds can be purchased from Dojindo Co., Ltd., Aldrich, SensoPath Technologies, Frontier Scientific Inc., etc. In the present invention, the disulfide compound that is an oxidation product of alkanethiol can be used in the same manner as alkanethiol.

  As the polymer containing a carboxyl group used in the present invention, a carboxyl group-containing synthetic polymer and a carboxyl group-containing polysaccharide can be used. Examples of the carboxyl group-containing synthetic polymer include polyacrylic acid, polymethacrylic acid, and copolymers thereof, for example, JP 59-53836, page 3, line 20 to page 6, line 49, JP 59-71048. Methacrylic acid copolymer, acrylic acid copolymer, itaconic acid copolymer, crotonic acid copolymer, maleic acid copolymer as described in the specification, page 3, line 41 to page 7, line 54, Examples include partially esterified maleic acid copolymer and those obtained by adding an acid anhydride to a polymer having a hydroxyl group. The carboxyl group-containing polysaccharide may be any one of an extract from a natural plant, a product of microbial fermentation, an enzymatic synthesis, or a chemical synthesis. Specifically, hyaluronic acid, chondroitin sulfate, heparin, Examples include dermatanic acid sulfuric acid, carboxymethylcellulose, carboxyethylcellulose, ceurouronic acid, carboxymethylchitin, carboxymethyldextran, and carboxymethyl starch. As the carboxyl group-containing polysaccharide, a commercially available compound can be used. Specifically, CMD, CMD-L, CMD-D40 (manufactured by Meisho Sangyo Co., Ltd.), sodium carboxymethylcellulose (Japanese) Kogaku Pharmaceutical Co., Ltd.), sodium alginate (Wako Pure Chemicals Co., Ltd.), and the like.

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

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

  As a method of activating a polymer containing a carboxyl group, a known method, for example, a method of activating with water-soluble carbodiimide 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) A method of activating 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, R1 and R2 may form a 5- or 6-membered ring by a bond, and A represents a carbon atom or phosphorus having a substituent. It is also possible to use a nitrogen-containing compound represented by the formula: A represents an 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, carbon atom or nitrogen atom which may have a substituent, but preferably R ₁ and R ₂ form a 5- to 6-membered ring by a bond. . 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, a nitrogen-containing compound represented by the following compound 7 can be used.

  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] may be used.

  Specifically, the following compounds can be used.

  Preferably, the following compounds 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 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.

  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. Representing an atom] can also be used.

  Specifically, the following compounds can be used.

  Further, 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 the σ value of the electron withdrawing group is preferably 0.3 or more. Specifically, the following compound 16 or the like can be used.

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

  The polymer containing a carboxyl group activated in the present invention may be reacted with the substrate as a solution, or may be reacted in a state where a thin film on the substrate is formed using a technique such as spin coating. . Preferably, the activated carboxyl group is an active esterified carboxyl group. Preferably, the reaction is in a state where a thin film is formed.

  As described above, the polymer containing a carboxyl group activated 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. For these thin film formation methods, "Progress in coating technology" by Yuji Harasaki, Comprehensive Technology Center (1988), "Coating Technology" Technical Information Association (1999), "Aqueous Coating Technology" CMC (2001), "Evolution" Described in "Organic Thin Film Deposition", Sumibe Techno Research (2004), "Polymer Surface Processing" by Iwamori Satoshi, Gihodo Publishing (2005), etc. Since a coating film with a controlled film thickness can be easily produced, the method for forming a thin film on a substrate in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method.

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

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

  The polymer containing a carboxyl group is activated by a known method, for example, 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS), which are water-soluble carbodiimides, or EDC alone. A physiologically active substance having a group can be immobilized. As a method for activating carboxylic acid, the method described in Japanese Patent Application No. 2004-238396 (Japanese Patent Laid-Open No. 2006-58071) “0011” to “0022” (that is, a carboxyl group present on the surface of a substrate is specified). A method of forming a carboxylic acid amide group by activation using any one of a uronium salt, a phosphonium salt, and a triazine derivative having a structure), and Japanese Patent Application No. 2004-275012 (Japanese Patent Application Laid-Open No. 2006-90781) ) The method according to “0011” to “0019” (that is, the carboxyl group present on the surface of the substrate is activated with a carbodiimide derivative or a salt thereof, and has a nitrogen-containing heteroaromatic compound having a hydroxyl group or an electron-withdrawing group. An ester of either a phenol derivative or an aromatic compound having a thiol group Later, it may be preferably used a method) for forming a carboxylic acid amide group by reaction with an amine.

  The uronium salt, phosphonium salt, or triazine derivative having a specific structure in the above Japanese Patent Application No. 2004-238396 (Japanese Patent Laid-Open No. 2006-58071) is a uronium salt represented by the following general formula 1, A phosphonium salt represented by the formula 2 or a triazine derivative represented by the following general formula 3 is shown.

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

  The physiologically active substance immobilized on the biosensor of the present invention is not particularly limited as long as it interacts with the measurement target, and examples thereof include immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-immune proteins, Examples include immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand-binding ability.

  Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, and 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 antigen 26, 86, 55, 111, 157 and the like can be used.

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

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

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

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

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

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

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

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

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

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

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

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

  In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the measured substance in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, 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.

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

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

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

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

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

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

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

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

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

Example 1:
This example relates to the production of a sensor chip for immobilizing proteins.

(1) Preparation of Sample 1 (Comparative Example 1) Biacore sensor chip CM-5 as the surface to which carboxymethyldextran was bound
(Research grade) was used as sample 1 as it was.

(2) Preparation of Sample 2 (Comparative Example 2) (2-1) Preparation of substrate having OH group Experiment was performed using Biacore sensor chip Au as the surface on which only the gold film was formed on the sensor chip. It was. The sensor chip Au was subjected to UV ozone treatment for 12 minutes, and then immersed in an ethanol / water (80/20) mixed solution in which 5.0 mM 16-hydroxyhexadecanethiol (manufactured by Frontier Scientific) was dissolved. After incubating for 20 minutes in a shaking incubator, it was washed 5 times with water, 5 times with 50 ml ethanol / water (80/20), and 5 times with 50 ml water.

(2-2) Treatment with epichlorohydrin
20ml 0.4M sodium hydroxide, 20ml diethylene glycol dimethyl ether, 2.0ml
The substrate was dipped in a mixed solution of epichlorohydrin, reacted in a shaking incubator at 25 ° C. for 4 hours, and then washed twice with 50 ml of ethanol and five times with 50 ml of water.

(2-3) Treatment with dextran The substrate was immersed in a mixed solution of 40.5 ml of water, 13.5 g of dextran (T500, Pharmacia) and 4.5 ml of 1M sodium hydroxide, and reacted for 20 hours in a shaking incubator at 25 ° C. Washed 15 times with 50 ml of 50 ° C. water.

(2-4) Treatment with bromoacetic acid The substrate was immersed in a mixed solution of 3.5 g of bromoacetic acid and 27 g of 2M sodium hydroxide solution, reacted for 16 hours in a shaking incubator at 28 ° C., and then washed with water. Again, a reaction with a bromoacetic acid solution for 16 hours and washing with water were performed to obtain Sample 2.

(3) Preparation of Sample 3 (Invention) (3-1) Preparation of Substrate Having Amino Group An experiment was conducted using Biacore sensor chip Au as the surface on which only a gold film was formed on the sensor chip. . Sensor chip Au was treated with UV ozone for 12 minutes, then 8mL ethanol and 2mL ultrapure water, 45μmol 11-Hydroxy-1-undecanethiol (Aldrich) and 4μmol 16-Mercaptohexadecanoic acid (Aldrich) ) In a solution in which the solution was dissolved at 40 ° C. for 1 hour, and washed once with ethanol and once with ultrapure water. EDC (0.4M) /
100 μl of NHS (0.1M) mixed solution was added dropwise, reacted at room temperature for 15 minutes to activate, and then washed once with ultrapure water. 50 μl of 1,2-bis (aminoethoxy) ethane was dropped onto the substrate, reacted at room temperature for 1 hour, and then washed once with ultrapure water.

(3-2) Active esterification of CMD After dissolving CMD (manufactured by Meisei Sangyo Co., Ltd .: molecular weight 1 million) in ultrapure water, 2% of the carboxyl groups are activated when the total amount is reacted. The calculated amount of EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide) (0.4M) / NHS (N-Hydroxysuccinimide) (0.1M) mixed solution was added and stirred at room temperature for 5 minutes.

(3-3) Bonding of Active Esterified CMD to Substrate 200 μl of the active esterified CMD solution prepared in (3-2) was dropped on the substrate prepared in (3-1), By spin coating at 7000 rpm for 45 seconds, an active esterified carboxymethyl dextran thin film was formed on a substrate having an amino group. After reacting at room temperature for 1 hour, Sample 3 was obtained by washing once with 0.1 N NaOH and once with ultrapure water.

Example 2
This example relates to protein immobilization on the sensor chip obtained in Example 1. As the protein, CA (Carbonic Anhydrase: manufactured by SIGMA) was used. The CA used was an electrophoresis experiment using AE-8150 from ATTO, and the isoelectric point was about 5.8 based on comparison with the marker (Broad pI Kit, pH 3.5-9.3: Amersham Biosciences) measured simultaneously. I confirmed that there was. 10 μl of a solution of 1 mg of CA dissolved in 1 ml of HBS-EP buffer (Biacore, 0.01M HEPES pH7.4, 0.15M NaCl, 0.005% Surfactant P20, 3 mM EDTA) was weighed, and 90 μl of acetate buffer (Biacore) By adding 0.1 mg / ml CA solution (pH 5.0,
0.1 mg / ml) was adjusted.

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

  The CA fixed amounts were 7757RU (sample 1), 16487RU (sample 2), and 35309RU (sample 3), respectively. It was proved by the present invention that a CMD binding surface having a larger amount of protein immobilized than a commercially available CMD binding surface or a CMD binding surface prepared by a conventional preparation method can be easily prepared by the present invention.

Example 3
This example relates to nonspecific adsorption of low molecular weight compounds to the sensor chip obtained in Example 1. As low molecular weight compounds, CGP74514, which is an inhibitor of Cyclin-Dependent Kinases, and Tween 20, which is a surfactant, were selected, and the ability to suppress nonspecific adsorption to the sensor chip surface was examined. FIG. 3 shows a sensorgram obtained when CGP74514 (50 μM) was flowed to each sample for 2 minutes and then Tween 20 (0.005 wt%) was flowed for 2 minutes.

  CGP74514 and Tween20 are non-specifically adsorbed on sample 1, which is a commercially available CMD binding surface, whereas CGP74514 and Tween20 are hardly adsorbed on samples 2 and 3, respectively. From this, it was proved that a CMD binding surface superior in the ability to suppress nonspecific adsorption of low molecular weight compounds than a commercially available CMD binding surface can be easily prepared by this method.

Example 4
This embodiment relates to the creation of a sensor chip when the CMD concentration for spin coating is changed.

(1) Preparation of sample 4 (Example)
Sample 4 was obtained by performing the same operation as Sample 3 except that the CMD concentration was changed to 0.2%.

(2) Preparation of sample 5 (Example)
Sample 5 was obtained by performing the same operation as Sample 3 except that the CMD concentration was changed to 0.1%.

(3) Preparation of sample 6 (Example)
Sample 6 was obtained by performing the same operation as Sample 3 except that the CMD concentration was changed to 0.08%.

(4) Preparation of sample 7 (Example)
Sample 7 was obtained by performing the same operation as Sample 3 except that the CMD concentration was changed to 0.05%.

(5) Preparation of sample 8 (Example)
Sample 8 was obtained by performing the same operation as Sample 3 except that the CMD concentration was changed to 0.02%.

Example 5
This example relates to protein preconcentration with respect to the sensor chip obtained in Example 1 and Example 4. Proteins include BSA (Bovine
Serum Albumin: manufactured by SIGMA) was used. The BSA used was an electrophoretic experiment using ATEO's AE-8150. Compared with the marker (Broad pI Kit, pH 3.5-9.3: manufactured by Amersham Biosciences), the isoelectric point was about 6.1. I confirmed that there was. 10 μl of a solution of 1 mg of BSA dissolved in 1 ml of HBS-EP buffer (Biacore, pH 7.4) is weighed, and 90 mg of acetate buffer (Biacore, pH 5.0) is added to give 0.1 mg / ml A BSA solution (pH 5.0, 0.1 mg / ml) was prepared.

Samples 1 to 8 prepared in Example 1 and Example 4 were set in a Biacore 3000 which is a surface plasmon resonance device manufactured by Biacore, and pre-washed with a BSA solution (pH 5.0, 0.1 mg / ml) for 5 minutes. Concentration was examined. The results obtained are summarized in Table 1.

It was proved that the amount of protein preconcentration can be controlled by controlling the concentration of the active esterified CMD solution to be spin-coated. It is considered that the spin coat thin film obtained from the active esterified CMD solution having a higher concentration increases the amount of CMD immobilized on the substrate surface, and consequently increases the amount of protein preconcentration.

Example 6
This example relates to bonding of a carbonxyl group-containing polymer other than CMD to the substrate surface.

(1) Preparation of Sample 9 (Example) Sample 9 was obtained by performing the same operation as Sample 3 except that the polymer was changed from CMD to sodium alginate 500 to 600 (Wako Pure Chemical Industries, Ltd.).

Example 7
This example relates to protein preconcentration with respect to the sensor chip obtained in Example 1 and Example 6. Using CA as the protein, CA immobilization was examined in the same manner as in Example 2. The results are shown in FIG.

  Even when the active esterified sodium alginate is bonded to the bonded substrate surface, it has a protein fixing ability equal to or better than that of a commercially available CMD bonded surface. Proven to be effective for binding to

Example 8
This example relates to nonspecific adsorption of low molecular weight compounds to the sensor chips obtained in Example 1 and Example 6. In the same manner as in Example 3, the ability of CGP74514 and Tween20, which are low molecular compounds, to suppress nonspecific adsorption on the sensor chip surface was examined. The obtained sensorgram is shown in FIG.

Similar to Example 3, Sample 9 prepared according to the present invention is superior in non-specific adsorption suppression ability of low molecular weight compounds compared to Sample 1 which is a commercially available CMD binding surface. Even when the containing polymer was used, it was proved that a surface excellent in the ability to suppress non-specific adsorption of low molecules compared to a commercially available CMD-binding surface can be easily prepared.

Example 9
Hydrogel membranes that can immobilize proteins were prepared using SAM compounds with high water solubility, and the amount of immobilized proteins and nonspecific adsorption performance were evaluated.

(1) Creation of substrate
Mixed aqueous solution of 6-Hydroxy-1-undecanethiol (Aldrich) and 8-Amino-1-octanethiol, hydrochloride (Dojin Chemical) (6-Hydroxy-1-undecanethiol 4.995 mM / 8-Amino-1-octanethiol, hydrochloride 0.005 mM). This solution is called A solution.

Next, a gold thin film was formed on the upper surface of a plastic prism obtained by injection molding ZEONEX (manufactured by Nippon Zeon Co., Ltd.) by the following method. A prism is attached to the substrate holder of the sputtering device, and vacuum is applied (base pressure 1 × 10 ^-3 Pa or less), then Ar gas is introduced (1 Pa), and the substrate holder is rotated (20 rpm) while RF power is applied to the substrate holder. (0.5kW) is applied for about 9 minutes to plasma-treat the prism surface. Next, Ar gas was stopped and vacuum was drawn, Ar gas was introduced again (0.5 Pa), and DC power (0.2 kW) was applied to the 8-inch Cr target for about 30 seconds while rotating the substrate holder (10 to 40 rpm). This is applied to form a 2 nm Cr thin film. next. Stop Ar gas, evacuate again, introduce Ar gas again (0.5 Pa), rotate the substrate holder (20 rpm), and apply DC power (1 kW) to an 8-inch Au target for about 50 seconds to 50 nm A thin Au film is formed.

  The sensor stick on which the Au thin film obtained above was formed was immersed in solution A for 1 hour at 40 ° C. and washed 10 times with ultrapure water.

(2) Active esterification of CMD (carboxymethyl dextran)
After dissolving 4.95 ml of 0.1% by weight CMD (manufactured by Sangyo Sangyo Co., Ltd .: molecular weight 1 million) solution, 2% of the carboxyl groups are activated when the whole amount is reacted (1-Ethyl-3- [ 3-Dimethylaminopropyl] carbodiimide hydrochloride (0.4M) / NHS (N-hydroxysulfosuccinimide) (0.1M) mixed solution (50 μl) was added and stirred at room temperature.

(3) Binding reaction of CMD to substrate Drop 500 μl of the active esterified CMD solution prepared in (2) on the substrate prepared in (1) and spin coat at 1000 rpm for 45 seconds. Then, an active esterified carboxymethyl dextran thin film was formed on a substrate having an amino group. After reacting at room temperature for 1 hour, Sample 1 was obtained by washing 5 times with 0.1 N NaOH and 5 times with ultrapure water.

Example 10
Example 10 relates to protein immobilization on the sensor sample obtained in Example 9. CA (Carbonic Anhydrase: manufactured by SIGMA) was used as the protein.

  Sample 1 prepared in Example 9 was set in a surface plasmon resonance apparatus. After injecting PBS buffer and confirming the baseline (with the resonance angle at this time as a reference point), 0.2 M EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide) and 50 mM NHS (N-Hydroxysuccinimide) ) And then let stand for 7 minutes, then inject PBS buffer and let stand for 1 minute, then inject 0.1 mg / ml CA solution (acetate buffer pH 5.0) and let stand for 15 minutes, then Inject PBS buffer and leave for 1 minute, then inject 1M ethanolamine solution (Biacore) for 7 minutes, then inject PBS buffer for 1 minute, then inject 10 mM NaOH for 1 minute. The solution was placed three times, and finally PBS buffer was injected and allowed to stand for 1 minute. The difference between the resonance angle at this time and the resonance angle at the origin was defined as the CA fixed amount. Here, the resonance angle 1/10000 degree is expressed as 1RU.

  The CA fixed amount was 5200RU. It was found that a large amount of protein can be immobilized even on the surface prepared using a highly water-soluble SAM compound.

Example 11
Example 11 relates to nonspecific adsorption of a low molecular weight compound to the sensor sample obtained in Example 9. As low molecular weight compounds, CGP74514, which is an inhibitor of Cyclin-Dependent Kinases, and Tween 20, which is a surfactant, were selected, and the ability to suppress nonspecific adsorption to the sensor sample surface was examined.

  Sample 1 prepared in Example 9 was set in a surface plasmon resonance apparatus. After injecting PBS buffer and confirming the baseline (with the resonance angle at this time as a reference point), inject CGP74514 in PBS buffer solution (50 μM) or Tween20 in PBS buffer solution (0.005 wt%) for 2 minutes. The sample was allowed to stand, and finally PBS buffer was injected and left to stand for 2 minutes. The difference between the resonance angle at this time and the resonance angle at the origin was defined as the nonspecific adsorption amount.

  The amount of non-specific adsorption for Sample 1 was 3RU for CGP74514 and 5RU for Tween20. It was found that non-specific adsorption of low molecular weight compounds can be suppressed even on surfaces prepared using SAM compounds with high water solubility.

Example 12
A hydrogel membrane capable of immobilizing proteins was prepared using a SAM compound with high water solubility, and the amount of immobilized proteins was evaluated.

(1) Creation of substrate
A 1 mM aqueous solution of 6-Amino-1-Hexanethiol, hydrochloride (manufactured by Dojindo) was prepared. This solution is called A solution.

  Next, a gold thin film was formed on the upper surface of a plastic prism obtained by injection molding ZEONEX (manufactured by Nippon Zeon Co., Ltd.) by the following method.

  A prism is attached to the substrate holder of the sputtering device, and vacuum is applied to the base (base pressure 1 × 10-3-3Pa or less), then Ar gas is introduced (1 Pa), and the substrate holder is rotated (20 rpm) while RF is applied to the substrate holder. Applying power (0.5kW) for about 9 minutes to plasma treatment of the prism surface (Substrate Etching). Next, Ar gas was turned off and evacuated, Ar gas was introduced again (0.5Pa), and the substrate holder was rotated ( 10 to 40 rpm), DC power (0.2 kW) was applied to an 8 inch Cr target for about 30 seconds to form a 2 nm Cr thin film. It was introduced again (0.5 Pa), and while rotating the substrate holder (20 rpm), DC power (1 kW) was applied to an 8-inch Au target for about 50 seconds to form an Au thin film of about 50 nm.

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

(2) Active esterification of CMD (carboxymethyl dextran)
After dissolving 10 g (carboxyl group amount: 3.3 × 10 ⁻² mol) of a 1% by weight CMD (manufactured by Sangyo Sangyo Co., Ltd .: molecular weight 1 million, substitution degree 0.65) solution, it is a carbodiimide derivative in the amount shown in Table 2. EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbodiimide Hydrochloride) and a nitrogen-containing compound having a hydroxyl group of the type and amount described in Table 2 (NHS (N-Hydroxysuccinimide) or HOBt (1-Hydroxybenzotriazole)) 10 ml of the mixed aqueous solution was added and stirred at room temperature for 1 hour.

(3) Binding reaction of CMD to substrate Drop 1 ml of the active esterified CMD solution prepared in (2) on the substrate prepared in (1) and spin coat at 1000 rpm for 45 seconds. Then, an active esterified carboxymethyl dextran thin film was formed on a substrate having an amino group. After reacting at room temperature for 15 minutes, samples 1-A to 8-A were obtained by immersing in 1 N NaOH aqueous solution for 30 minutes and washing 5 times with ultrapure water.

  Also, 65 minutes (B), 70 minutes (C), 75 minutes (D), and 80 minutes (E) after the start of the active esterification reaction, spin coating similar to the above is performed and the same treatment is performed after the reaction. Thus, Samples 1-B to 8-B, Samples 1-C to 8-C, Samples 1-D to 8-D, and Samples 1-E to 8-E were obtained.

Comparative Example 3
(I) Creation of substrate having OH group
A 5 mM solution (ethanol / water = 8/2) of 16-Hydroxy-1-hexadecanethiol (manufactured by Frontier Scientific) was prepared. This solution is called B solution. Next, a 50 nm gold film was formed on the sensor stick in the same manner as in Example 12, immersed in solution B at 40 ° C. for 60 minutes, 5 times with ethanol, once with 50 ml of ethanol / water (80/20), Washed 5 times with 50 ml of ultrapure water.

(Ii) Treatment with epichlorohydrin
The substrate is immersed in a mixed solution of 20 ml of 0.4 M sodium hydroxide, 20 ml of diethylene glycol dimethyl ether and 2.0 ml of epichlorohydrin, reacted in a shaking incubator at 25 ° C. for 4 hours, and then twice with 50 ml of ethanol. And washed 5 times with 50 ml of water.

(Iii) Treatment with dextran The above substrate was immersed in a mixed solution of 40.5 ml of water, 13.5 g of dextran (T500, Pharmacia) and 4.5 ml of 1M sodium hydroxide, reacted for 20 hours in a shaking incubator at 25 ° C, Washed 15 times with water at 50 ° C.

(Iv) Treatment with bromoacetic acid The substrate was immersed in a mixed solution of 3.5 g of bromoacetic acid and 27 g of 2M sodium hydroxide solution, reacted for 16 hours in a shaking incubator at 28 ° C., and then washed with water. Again, reaction with a bromoacetic acid solution for 16 hours and washing with water were performed to obtain Sample 9.

Example 13
Example 13 relates to protein immobilization on the sensor samples obtained in Example 12 and Comparative Example 3. CA (Carbonic Anhydrase: manufactured by SIGMA) was used as the protein.

  Samples 1 to 9 prepared in Example 12 and Comparative Example 3 were set in a surface plasmon resonance apparatus. After injecting PBS buffer and confirming the baseline (with the resonance angle at this time as a reference point), 0.2 M EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide) and 50 mM NHS (N-Hydroxysuccinimide) ) And then let stand for 7 minutes, then inject PBS buffer and let stand for 1 minute, then inject 0.1 mg / ml CA solution (acetate buffer pH 5.0) and let stand for 15 minutes, then Inject PBS buffer and leave for 1 minute, then inject 1M ethanolamine solution (Biacore) for 7 minutes, then inject PBS buffer for 1 minute, then inject 10 mM NaOH for 1 minute. The solution was placed three times, and finally PBS buffer was injected and allowed to stand for 1 minute. The difference between the resonance angle at this time and the resonance angle at the origin was defined as the CA fixed amount. Here, the difference in resonance angle per DMSO 1% is expressed as 1500 RV.

  Table 2 shows average CA fixed amounts and CV values (variation coefficient:%) of Samples 1 to 8 and CA fixed amount of Sample 9. It was found that a large amount of protein can be immobilized on the surface of the present invention by a simple method. Here, the CV value is a coefficient of variation and represents a value obtained by dividing the standard deviation (square root of variance) by the average. The smaller the CV value, the smaller the degree of variation.

Example 14
Hydrogel membranes that can immobilize proteins were prepared using SAM compounds with high water solubility, and the amount of immobilized proteins and nonspecific adsorption performance were evaluated.

(1) Creation of substrate
A 1 mM aqueous solution of 6-Amino-1-octanethiol, hydrochloride (manufactured by Dojin Chemical Co., Ltd.) was prepared. This solution is called A solution.

  Next, a gold thin film was formed on the upper surface of a plastic prism obtained by injection molding ZEONEX (manufactured by Nippon Zeon Co., Ltd.) by the following method. A prism is attached to the substrate holder of the sputtering device, and vacuum (base pressure 1 × 10-3-3Pa or less) is applied, Ar gas is introduced (1 Pa), and the substrate holder is rotated (20 rpm) while RF is applied to the substrate holder. Plasma is applied to the prism surface by applying power (0.5kW) for about 9 minutes. Next, Ar gas is stopped and vacuum is drawn, Ar gas is introduced again (0.5 Pa), and DC power (0.2 kW) is applied to an 8-inch Cr target for about 30 seconds while rotating the substrate holder (10 to 40 rpm). This is applied to form a 2 nm Cr thin film. next. Stop Ar gas, pull vacuum again, introduce Ar gas again (0.5 Pa), rotate the substrate holder (20 rpm), and apply DC power (1 kW) to an 8-inch Au target for about 50 seconds to 50 nm A thin Au film is formed.

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

(2) Activation of CMD (carboxymethyl dextran)
After preparing 10 g of a 1% by weight CMD (Made in Sangyo Industry: average molecular weight 1 million, carboxymethyl group substitution degree per sugar unit 0.65) solution, EDC (1-Ethyl-3- [3- Dimethylaminopropyl] carbodiimide Hydrochloride) 0.4M aqueous solution 0.5ml and ultrapure water 9.5ml were added and stirred at room temperature for 5 minutes.

(3) Binding reaction of CMD to substrate After 200 μl of the activated CMD solution prepared in (2) is dropped on the substrate prepared in (1), it is stretched with a blade and applied onto the substrate having amino groups. An active esterified carboxymethyl dextran membrane was formed. After reacting at 25 ° C. and 10% RH for 60 minutes, sample 1 was obtained by immersing in 1 N NaOH aqueous solution for 60 minutes and washing 15 times with ultrapure water.

Example 15
The sensor sample obtained in Example 14 was evaluated in the same manner as in Example 10. The CA fixed amount was 6300RU.

Example 16
The sensor sample obtained in Example 14 was evaluated in the same manner as in Example 11. The amount of non-specific adsorption was 2RU for CGP74514 and 4RU for Tween20.

It is a disassembled perspective view which shows schematic structure of a sensor unit. FIG. 2 is a sensorgram showing protein immobilization on the sensor chip obtained in Example 1. FIG. 3 is a sensorgram showing nonspecific adsorption of a low molecular weight compound to the sensor chip obtained in Example 1. FIG. 4 is a sensorgram showing protein preconcentration with respect to the sensor chip obtained in Example 1 and Example 6. FIG. 5 is a sensorgram showing nonspecific adsorption of a low molecular weight compound to the sensor chip obtained in Example 1 and Example 6.

Explanation of symbols

10 Sensor unit 20 Total reflection prism (optical block)
21 Prism body 21a Upper surface 22 Grasping part 22a Groove 23 Projection part 25 Metal film (thin film layer)
26 Polymer Film 27 System Joint 28 Engagement Section 28a System Joint Surface 29a Reference Plane 30 Channel Member 31 First Channel 32 Second Channel 33 Main Body 34 Mounting Portion 35 System Joint 36 Opening SS1 Sensor Surface SS2 Sensor Surface

Claims (11)

In a method for producing a biosensor, characterized in that an activated carboxyl group-containing polymer is brought into contact with a substrate surface coated with an organic layer having an amino group, and the polymer is bonded to the organic layer. The activated carboxyl group-containing polymer is obtained by activating a carboxyl group-containing polymer with a compound represented by the following general formula (II) or the following general formula (III). The above manufacturing method.

[Wherein Y and Z each independently represent CH or a nitrogen atom]

[In the formula, A represents a carbon atom or a phosphorus atom having a substituent, Y and Z each independently represent CH or a nitrogen atom, M represents an (n-1) -valent element, and X represents Represents a halogen atom] The method of claim 1, wherein the substrate is a metal surface or metal film. The method according to claim 1 or 2, wherein the substrate surface coated with an organic layer having an amino group is a substrate surface coated with an alkanethiol having an amino group. The alkanethiol having an amino group is either a compound in which the thiol group and the amino group are linked via an alkyl chain, or a compound obtained by reacting an alkanethiol having a carboxyl group at the terminal with hydrazide or diamine. The method according to claim 3. 5. The method according to claim 1, wherein the substrate surface coated with an organic layer having an amino group is a substrate surface coated with a mixture of an alkanethiol having an amino group and an alkanethiol having a hydrophilic group. . Hydrophilic group alkanethiol having a hydrophilic group is a hydroxyl group or an oligo ethylene grayed recall group, The method of claim 5. The method according to claim 5 or 6, wherein the molecular length of the alkanethiol having an amino group is longer than the molecular length of the alkanethiol having a hydrophilic group. The method according to any one of claims 1 to 7, wherein the polymer containing a carboxyl group is a polysaccharide. The method according to any one of claims 1 to 8, wherein the polymer containing an activated carboxyl group is reacted with an organic layer having an amino group in a state where a thin film is formed on a substrate. The method according to any one of claims 1 to 9, wherein the mixing molar ratio of the compound represented by the general formula (II) or the general formula (III) to the carboxyl group in the polymer is 1 x ^10-7 to 1. The method according to any one of claims 1 to 10, further comprising immobilizing a physiologically active substance via a carboxyl group in the polymer.

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