JP4908385B2 - Biosensor chip, manufacturing method thereof, and sensor for surface plasmon resonance analysis - Google Patents

Description

  The present invention relates to a biosensor chip useful for immobilizing a physiologically active substance, a method for producing the same, and a sensor for surface plasmon resonance analysis using the biosensor chip.

  At present, many measurements using intermolecular interactions such as immune reactions are performed in clinical tests, etc., but it is possible to detect changes in the binding amount of the measurement substance with high sensitivity without requiring complicated operations and labeling substances. There are several techniques that can be used. Examples include 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.

  A measurement chip used for measurement using surface plasmon resonance sequentially has a thin film having a functional group capable of immobilizing a biologically active substance such as a deposited metal film or protein on a transparent substrate (for example, glass). A physiologically active substance is immobilized on the metal surface via a functional group. The interaction between biomolecules can be analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance.

  The above-mentioned physiologically active substances include biopolymers (nucleic acids, proteins, polysaccharides, etc.) that are basic materials constituting the living body, and nucleotides, nucleosides, amino acids, sugars, and lipids, vitamins, hormones, etc., which are constituents thereof It is a substance that changes the regulation of the living body and the function of the living body, and these are important in the development of pharmaceutical ingredients and functional foods.

  For example, low molecular weight compounds having a thiol group (SH group) include cysteine, homocysteine, glutathione, coenzyme A, and dihydrolipoic acid. Coenzymes such as coenzyme A and dihydrolipoic acid form thioesters with acyl groups. Work as a carrier of acyl group. Glutathione and cysteine are involved in the regulation of intracellular redox potential. In addition, SH groups in proteins and peptides are in cysteine residues and play an important role in the activity of enzymes, hormones and the like.

It is known that such a compound having an SH group binds to maleimide. As a biosensor chip using this bond, a chip in which maleimide is introduced into a carboxyl group on the surface of the sensor chip is known (Non-Patent Document 1). According to this chip, it is possible to detect a biological component having the SH group.
Biacore SPR Biosensor User's Guide to Biacore Experiments, page 14-15 2006.09.05

  However, when the adsorption amount of the biological component having the SH group was examined with the chip of Non-Patent Document 1, it was found that only half or less of the biological component having the SH group actually contained could be detected. In biosensor chips, if non-specific adsorption is large, accurate detection cannot be performed, so it is important how to suppress non-specific adsorption, but more precisely how specific adsorption can be detected. Whether it can be done is a fundamental issue, and the amount of adsorption is a very demanding factor.

  The present invention has been made in view of such circumstances, and is a biosensor that can accurately detect specific adsorption of a biological component having an SH group and can suppress nonspecific adsorption. An object of the present invention is to provide a chip for use and a manufacturing method thereof.

  The biosensor chip of the present invention comprises a substrate, a hydrogel disposed on the substrate surface, a polyamino group bonded to the hydrogel, and an α, β-unsaturated carbonyl group bonded to the polyamino group. It is characterized by.

The polyamino group is preferably a diaminoalkylene group or a di (aminoalkyl) ether group.
The α, β-unsaturated carbonyl group is preferably a maleimide group.

  The hydrogel is preferably a polymer having a carboxyl group, more preferably carboxymethyl-modified dextran.

  The hydrogel is preferably disposed on the substrate via a metal film. In this case, the metal film is preferably made of at least one metal selected from gold, silver, copper, platinum, or aluminum.

  In the method for producing a biosensor chip of the present invention, a hydrogel disposed on a substrate surface is activated, a compound having a polyamino group is bonded to the activated hydrogel, and then an α, β-unsaturated carbonyl group is bonded. It is characterized in that a compound having the same is bonded.

  The biosensor chip is preferably used as a sensor chip for surface plasmon resonance analysis.

  The biosensor chip of the present invention comprises a substrate, a hydrogel disposed on the surface of the substrate, a polyamino group bonded to the hydrogel, and an α, β-unsaturated carbonyl group bonded to the polyamino group. Since the α, β-unsaturated carbonyl group is fixed by the polyamino group bonded to the hydrogel, the specific adsorption amount of the biological component having the SH group can be greatly increased while suppressing non-specific adsorption. Therefore, it is possible to accurately detect the biological component having the SH group of the specimen.

  Details of the mechanism that can increase the specific adsorption amount of biological components having SH groups by immobilizing the α, β-unsaturated carbonyl group with the polyamino group bonded to the hydrogel is unknown, but the hydrogel and α, When a β-unsaturated carbonyl group is directly bonded, charge repulsion occurs there, and the amount of α, β-unsaturated carbonyl group binding is less than expected, but in the present invention, By using a polyamino group as a linking group, it is presumed that charge repulsion is reduced and the amount of α, β-unsaturated carbonyl groups bound is increased.

  Hereinafter, the biosensor chip of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram schematically showing the configuration of the biosensor chip of the present invention.

  As shown in FIG. 1, the biosensor chip of the present invention comprises a substrate, a hydrogel disposed on the surface of the substrate, a polyamino group bonded to the hydrogel, and an α, β-unbound bonded to the polyamino group. And a saturated carbonyl group.

  The polyamino group has 2 to 4 amino groups in one group, preferably 2 to 3 amino groups, and is preferably a diaminoalkylene group or a di (aminoalkyl) ether. Groups are preferred. In this case, the alkylene group or alkyl group connecting two amino groups preferably has 2 to 10 carbon atoms, and more preferably 2 to 4 carbon atoms. Specifically, ethylenediamine group, tetraethylenediamine group, octamethylenediamine group, decamethylenediamine group, triethylenediamine group, diethylenetriamine group, triethylenetetraamine group, dihexamethylenetriamine group, 1,4-diaminocyclohexane group, 2 -Aliphatic diamine groups such as aminoethyl ether and 3-aminopropyl ether, paraphenylenediamine group, metaphenylenediamine group, paraxylylenediamine group, metaxylylenediamine group, 4,4'-diamonobiphenyl group, 4 Aromatic diamine groups such as 4,4′-diaminodiphenylmethane group, 4,4′-diaminodiphenyl ketone group, 4,4′-diaminodiphenylsulfonic acid group can be preferably used.

 Examples of the α, β-unsaturated carbonyl group include α, β-unsaturated ketone groups such as 3-buten-2-one group, 2-cyclohexen-1-one group, and 2-cyclopenten-1-one group, acrylic acid (Meth) acrylic acid ester groups such as methyl group and ethyl methacrylate group, (meth) acrylic acid amide groups such as acrylic acid amide group and methacrylic acid diisopropylamide group, maleimide, 3,4-dehydro-1-methylpyrimidine- And α, β-unsaturated imide groups such as 2,6-dione and 3,4-dehydro-1-phenylpyrimidine-2,6-dione. In particular, from the viewpoint of reactivity with the SH group, an α, β-unsaturated imide group such as maleimide is more preferable.

  The biosensor chip shown in FIG. 1 is formed by bonding a polyamino group and an α, β-unsaturated carbonyl group to a hydrogel disposed on a substrate, but the hydrogel is disposed on the substrate via a metal film. Preferably, the hydrogel may be bound to a self-assembled film formed on the metal film. Hereinafter, other constituent materials constituting the biosensor chip of the present invention will be described in detail.

(Substrate and metal film)
When considering the surface plasmon resonance biosensor substrate, the substrate in the biosensor chip of the present invention is generally optical glass such as BK7 or synthetic resin, specifically polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cyclohexane. A material made of a material transparent to laser light such as an olefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  A metal film is preferably disposed on the substrate. Here, the term “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and the metal film is arranged through another layer without directly contacting the substrate. It also means including the case. The metal constituting the metal film is not particularly limited as long as surface plasmon resonance can occur. Preferably, the metal film is at least one metal selected from the group consisting of gold, silver, copper, platinum, palladium, and aluminum. It is preferred that gold be 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.

  The thickness of the metal film is arbitrary, but is preferably 0.1 nm or more and 500 nm or less, and particularly preferably 1 nm or more and 200 nm or less. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. In the case where an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 0.1 nm or more and 10 nm or less.

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

(Self-assembled film-forming molecule)
A self-assembled film is an ultra-thin film such as a monomolecular film or LB film with a certain ordered structure formed by the mechanism of the film material itself without fine control from the outside. Say. This self-organization forms ordered structures and patterns over long distances in a non-equilibrium situation.

  The self-assembled film is preferably formed of a compound represented by the general formula X—R—Y. Here, X represents a group capable of binding to the metal film, specifically, a group capable of binding to the metal film such as —SH, —SS, —SeH, —SeSe, and —COSH groups. Here, R is a divalent organic linking group, which may optionally be interrupted by a heteroatom, and is preferably straight-chain (unbranched) for suitably dense packing, and optionally double And / or a hydrocarbon chain containing a triple bond. The carbon chain can optionally be perfluorinated and the alkyl chain length of R is 6-18 carbons, preferably 6-11. Y represents a group capable of binding to the hydrogel, specifically, a functional group selected from the group consisting of a hydroxyl group, a hydroxycarbonyl group, an alkoxy group, and an alkyl group.

  More specifically, preferred examples of the molecules forming the self-assembled film include alkanethiols on the gold surface, alkylsilanes on the glass surface, and alcohols on the silicon surface. Specific examples of alkanethiols include 7-carboxy-1-heptanethiol, 10-carboxy-1-decanethiol, 4,4′-dithiodibutyric acid, 11-hydroxy-1-undecanthiol, 11-amino-1 -Undecanethiol etc. can be used. As specific examples of the alkylsilanes, aminopropyltrimethoxysilane, aminoethylaminotriethoxysilane, hydroxypropyltriethoxysilane, and the like can be used.

(Hydrogel)
Preferred hydrogels include natural polymers such as dextran derivatives, starch derivatives, cellulose derivatives and gelatin, and synthetic polymers such as polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, polyacrylamide derivatives and polymethyl vinyl ether. A polymer containing a group is preferred.

  Specifically, a carboxyl group-containing synthetic polymer and a carboxyl group-containing natural polymer can be used. Examples of the carboxyl group-containing synthetic polymer include polyacrylic acid, polymethacrylic acid, and copolymers thereof, such as JP-A-59-44615, JP-B-54-34327, JP-B-58-12577, JP-B-54. No.-25957, JP-A-59-53836, JP-A-59-71048, methacrylic acid copolymer, acrylic acid copolymer, itaconic acid copolymer, crotonic acid copolymer Examples thereof include a polymer, a maleic acid copolymer, a partially esterified maleic acid copolymer, and a polymer having a hydroxyl group to which an acid anhydride is added. The carboxyl group-containing natural polymer may be any of an extract from a natural plant, a product of microbial fermentation, a synthetic product by enzyme, or a chemical synthetic product. Specifically, hyaluronic acid, chondroitin sulfate, Examples thereof include polysaccharides such as heparin, sulfuric acid dermatanate, carboxymethylcellulose, carboxyethylcellulose, ceurouronic acid, carboxymethylchitin, carboxymethyldextran and carboxymethyl starch, and polyamino acids such as polyglutamic acid and polyaspartic acid. As the carboxyl group-containing natural polysaccharide, a commercially available compound can be used. Specifically, carboxymethyl-modified dextran, CMD, CMD-L, CMD-D40 (manufactured by Meisho Sangyo Co., Ltd.), carboxymethylcellulose sodium (Manufactured by Wako Pure Chemical Industries, Ltd.), sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd.), and the like. More preferably, the hydrogel is carboxymethyl modified dextran.

  The molecular weight of the hydrogel used in the present invention is not particularly limited, but the average molecular weight is preferably 1000 to 5000000, the average molecular weight is more preferably 10,000 to 2000000, and the average molecular weight is further preferably 100,000 to 100,000. . 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.

  The hydrogel as described above may be bonded to the substrate via the above-described self-assembled film or hydrophobic polymer, or may be formed directly on the substrate from a solution containing a monomer. Furthermore, the hydrogel described above can also be crosslinked.

  Next, a method for producing the biosensor chip of the present invention will be described. The biosensor chip of the present invention activates a hydrogel disposed on the substrate surface prepared by the above method, and binds a compound having a polyamino group to the activated hydrogel, and then α, β-unsaturated carbonyl. It can be produced by bonding a compound having a group. Hereinafter, it demonstrates in order.

(Activation of hydrogel)
When a polymer containing a carboxyl group is used as the hydrogel, it can be bonded to a substrate coated with a self-assembled film (or metal film) by activating the carboxyl group. 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) Can be preferably used.

Instead of the NHS, the following general formula I (wherein R ¹ and R ² independently represent a carbonyl group, a carbon atom, or a nitrogen atom which may have a substituent, and R ¹ and R ² are A nitrogen-containing compound represented by the above formula:

A nitrogen-containing compound represented by the following formula,

A nitrogen-containing compound represented by the following general formula II (wherein Y and Z independently represent CH or a nitrogen atom),

Specifically, the following compounds

を用いることができる。 In addition, the following nitrogen-containing compounds

Can be used.

Instead of NHS, a uronium salt represented by the following general formula III, a phosphonium salt represented by the following general formula IV, or a triazine derivative represented by the following general formula V can also be used.

(In the general formula III, R ¹ and R ² 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 ³ represents an aromatic 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. .)

Further, a water-soluble carbodiimide derivative can be used in combination with the EDC, and preferably the following compound (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride) can be used.

  The activated hydrogel may be reacted with the substrate as a solution, or may be reacted with a thin film formed on the substrate using a technique such as spin coating. The reaction in a state where a thin film is formed is preferable.

(Binding of compounds having a polyamino group)
In quantifying a biological component having an SH group, it is important to bind a large amount of a compound having a polyamino group. Therefore, this step of bonding a compound having a polyamino group to the hydrogel activated by the above-described method is performed using 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide (EDC) and N-Hydroxysuccinimide (NHS) used in the activation of the hydrogel. ), And the following compounds that can be used instead of NHS and EDC are preferably used as an activator and bonded (in the following chemical formula, R and X in the general formula are the same as above).

を用いることが好ましい。 Particularly preferably,

Is preferably used.

 The compound having a polyamino group is a diaminoalkylene or di (aminoalkyl) ether having the polyamino group described above, specifically, ethylenediamine, tetraethylenediamine, octamethylenediamine, decamethylenediamine, triethylenediamine. , Aliphatic diamines such as diethylenetriamine, triethylenetetraamine, dihexamethylenetriamine, 1,4-diaminocyclohexane, 2-aminoethyl ether, and 3-aminopropyl ether, paraphenylenediamine, metaphenylenediamine, paraxylylenediamine, Aromatic diamine groups such as metaxylylenediamine, 4,4'-diamonobiphenyl, 4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenyl ketone, 4,4'-diaminodiphenylsulfonic acid It can be used.

From the viewpoint of improving the hydrophilicity of the biosensor surface, it is also possible to use a compound in which two amino groups are linked by an ethylene glycol unit (wherein n and m are integers of 1 to 3). .

  In particular, ethylenediamine and 2-aminoethyl ether are more preferable from the viewpoint of preferable reactivity with hydrogels and α, β-unsaturated carbonyl compounds.

(Coupling with a compound having an α, β-unsaturated carbonyl group)
This step of bonding a compound having an α, β-unsaturated carbonyl group to a polyamino group can be achieved by contacting (adding) a compound having an α, β-unsaturated carbonyl group. In addition, you may couple | bond using the activator used when the said compound which has a polyamino group was couple | bonded.

 Examples of the α, β-unsaturated carbonyl compound include 3-buten-2-one, 2-cyclohexen-1-one and 2-cyclopenten-1-one having the α, β-unsaturated carbonyl group described above. Α, β-unsaturated ketone compounds, (meth) acrylic acid ester compounds such as methyl acrylate and ethyl methacrylate, (meth) acrylic acid amide compounds such as acrylic amide and methacrylic acid diisopropylamide, α such as maleimide Examples thereof include β-unsaturated imide compounds. In particular, from the viewpoint of reactivity with the SH group, α, β-unsaturated imide compounds such as maleimide are more preferable.

(Bioactive substance)
Examples of physiologically active substances include SH proteins, immune proteins, enzymes, microorganisms, nucleic acids, low molecular weight organic compounds, non-immune proteins, immunoglobulin binding proteins, sugar binding proteins, sugar chains that recognize sugars, lipids, fatty acids Alternatively, fatty acid esters, polypeptides or oligopeptides having ligand binding ability, and the like can be mentioned. Immobilization of the physiologically active substance can be performed by applying a solution containing the physiologically active substance or by dipping in a solution containing the physiologically active substance.

(Biosensor)
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. In the biosensor, one of these substances having affinity for each other is immobilized on a substrate and used as a molecular recognition substance, thereby utilizing the principle of selectively measuring the other substance to be matched.

  The biosensor chip of the present invention can be used to detect and / or measure an interaction between a physiologically active substance immobilized on a biosensor chip substrate and a test substance. As described above, biosensors include, for example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. Etc.

  According to a preferred embodiment, the biosensor chip of the present invention can be used, for example, as a chip for a surface plasmon resonance biosensor provided with a metal film disposed on a transparent substrate. The surface plasmon resonance biosensor is composed of a member that includes a portion that transmits and reflects light emitted from the sensor and a portion that immobilizes the bioactive substance. The biosensor chip of the present invention immobilizes the bioactive substance. It can be used as a member including a portion.

  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 detection means for detecting the state of surface plasmon resonance, that is, the state of total reflection attenuation (ATR) by measuring the intensity of the light beam totally reflected at the interface.

  This will be described with reference to the drawings. FIG. 2 is a schematic view showing one embodiment of a surface plasmon resonance analysis sensor equipped with the biosensor chip of the present invention in a side view. The surface plasmon resonance analysis sensor is composed of a plurality of surface plasmon measurement units configured as shown in FIG. 2, and the measurement unit includes a biosensor chip 10, a laser light source 14 that is a light source that generates a light beam 13, and the above-mentioned An incident optical system 15 that makes the light beam 13 incident on the chip 10, a collimator lens 16 that collimates the light beam 13 reflected by the chip 10 and emits it toward the photodetector 17, and emits from the collimator lens 16 A light detector 17 for detecting the light intensity by receiving the light beam 13, a differential amplifier array 18 connected to the light detector 17, a driver 19 connected to the differential amplifier array 18, and a driver 19 And a signal processing unit 20 including a computer system or the like connected to the computer. The signal processing unit 20 also functions as a sensitivity correction unit that performs sensitivity correction performed for each chip 10. Note that the photodetector 17, the differential amplifier array 18, the driver 19, and the signal processing unit 20 constitute measuring means for measuring the dark line position in the light beam.

  The chip 10 includes a dielectric block 11 having a shape in which a part including an apex angle where four ridge lines of a quadrangular pyramid gather is cut out and a concave portion functioning as a sample holding mechanism for storing a sample liquid is formed on the bottom surface of the quadrangular pyramid. The metal film 12 is formed on the bottom surface of the concave portion of the dielectric block 11 and is not shown in the drawing, but the polymer is bonded onto the metal film 12 as described above, and the polymer surface Has a hydrogel, and a polyamino group and an α, β-unsaturated carbonyl group are sequentially bonded to the hydrogel.

  Moreover, 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 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. An optical system to be incident and an optical 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. The biosensor chip can also be used in such a leakage mode measuring apparatus.

In addition, the biosensor chip of the present invention is a biosensor that detects a change in refractive index using a waveguide, for example, having a waveguide structure having a diffraction grating and possibly an additional layer on the surface of the substrate. It can also be used as a chip. Regarding the structure of this type of biosensor, for example, from JP 4-77703, page 4, line 48 to page 14, line 15 and FIGS. 1 to 8, US Pat. No. 6,829,073, column 6, line 6 It is described in the 47th line of column7 and FIGS. 9A and B. 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.
Examples of the biosensor chip of the present invention are shown below.

2.8mM HODhbt（3,4-ジヒドロ-３-ヒドロキシ-４-オキソ-1,2,3-ベンゾトリアジン）水溶液50μlと0.4M EDC水溶液50μlを混合し、この混合液をCM5(Biacore社製)のセンサー表面に添加し室温で10分間反応させた。反応後センサー表面をミリQ水で洗滌し、真空乾燥機で10分間乾燥させた。続いてエチレンジアミンをセンサー表面に100μl添加し、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩（（1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride））とともに室温で10分間反応させた。反応後センサー表面を0.1N-NaOH、続いてミリQ水で洗滌した。 Example 1

50 μl of 2.8 mM HODhbt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine) aqueous solution and 50 μl of 0.4 M EDC aqueous solution were mixed, and this mixture was mixed with CM5 (Biacore). It was added to the sensor surface and allowed to react for 10 minutes at room temperature. After the reaction, the sensor surface was washed with milli-Q water and dried for 10 minutes with a vacuum dryer. Subsequently, 100 μl of ethylenediamine was added to the sensor surface, and 10 minutes at room temperature with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride ((1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride)) Reacted. After the reaction, the sensor surface was washed with 0.1 N NaOH, followed by milliQ water.

  Next, 300 μl of 0.8 mM SSMCC (sulfidosuccinimidyl-4- (N-maleimidomethyl) cyclohexanecarboxylate sodium salt) was added to the sensor surface and reacted at room temperature for 30 minutes. After the reaction, it was washed with PBS (0.01 M, pH 7.2 + 150 mM NaCl), milli-Q water, and dried by nitrogen flow.

０．１ｍＭ 11−カルボニルウンデカンチオール塩酸塩（同仁化学研究所製）水溶液にSIA Kit Au (Biacore社製)の金チップを浸漬し40 ℃で20時間反応させ、反応後、40℃のミリQで洗滌し、チッソフローにより乾燥したものをCM5(Biacore社製)のかわりに用いた以外は実施例１と同様な操作を行った。 (Comparative Example 1)

A SIA Kit Au (Biacore) gold chip is immersed in an aqueous solution of 0.1 mM 11-carbonylundecanethiol hydrochloride (manufactured by Dojindo Laboratories) and reacted at 40 ° C. for 20 hours. The same operation as in Example 1 was carried out except that the product washed and dried by nitrogen flow was used instead of CM5 (Biacore).

2.8mM HODhbt水溶液50μlと0.4M EDC水溶液50μlを混合し、この混合液をCM5(Biacore社製)のセンサー表面に添加し室温で10分間反応させた。反応後センサー表面をミリQ水で洗滌し、真空乾燥機で10分間乾燥させた。さらに300μlの100ｍＭCMDBMPH（N-[β-maleimidopropionic acid] hydrazide, TFA salt）とともに室温で10分間反応させた。反応後センサー表面を0.1N-NaOH、続いてミリQ水で洗滌し、チッソフローにより乾燥した。 (Comparative Example 2)

50 μl of a 2.8 mM HODhbt aqueous solution and 50 μl of a 0.4 M EDC aqueous solution were mixed, and this mixed solution was added to the sensor surface of CM5 (Biacore) and allowed to react at room temperature for 10 minutes. After the reaction, the sensor surface was washed with milli-Q water and dried for 10 minutes with a vacuum dryer. Furthermore, it was made to react at room temperature for 10 minutes with 300 microliters of 100 mMCMDBMPH (N-[(beta) -maleimidopropionic acid] hydrazide, TFA salt). After the reaction, the sensor surface was washed with 0.1N-NaOH, followed by milli-Q water, and dried by nitrogen flow.

(Evaluation of sensor chip)
The sensor chip produced in Example 1 and Comparative Examples 1 and 2 was set in an SPR measuring device (Biacore 3000, manufactured by Biacore), and 100 mM glutathione (Glutathione) at a flow rate of 10 μl / min in a flow path stabilized with HBS-EP buffer. ) Was allowed to react for 5 minutes, and the fixed amount of the target product was evaluated from the adsorbed amount. Similarly, 0.1 mg / ml fibrinogen was reacted for 5 minutes, and nonspecific adsorption on the surface was evaluated from the amount of adsorption. The results are shown in Table 1.

 As is clear from Table 1, the amount of glutathione immobilized in the chip of Example 1 is more than double that of the chip of Comparative Example 2, and the amount of fibrinogen adsorbed indicating non-specific adsorption is the chip of Comparative Example 1. 2 orders of magnitude were significantly suppressed compared to. From this result, while the chip of the present invention can dramatically increase the amount of the biological component having an SH group, nonspecific adsorption is suppressed, and an extremely high level of measurement is possible.

Schematic schematic diagram showing one embodiment of a chip for biosensor of the present invention Schematic of a sensor for surface plasmon resonance analysis provided with the biosensor chip of the present invention

Claims (9)

  A biosensor comprising: a substrate; a hydrogel disposed on the surface of the substrate; a polyamino group bonded to the hydrogel; and an α, β-unsaturated carbonyl group bonded to the polyamino group. Chip.   The biosensor chip according to claim 1, wherein the polyamino group is a diaminoalkylene group or a di (aminoalkyl) ether group.   The biosensor chip according to claim 1, wherein the α, β-unsaturated carbonyl group is a maleimide group.   The biosensor chip according to claim 1, wherein the hydrogel is a polymer having a carboxyl group.   The biosensor chip according to claim 4, wherein the polymer having a carboxyl group is carboxymethyl-modified dextran.   The biosensor chip according to claim 1, wherein the hydrogel is disposed on the substrate via a metal film.   The biosensor chip according to claim 6, wherein the metal film is made of at least one metal selected from gold, silver, copper, platinum, or aluminum.   A biosensor characterized by activating a hydrogel disposed on a substrate surface, binding a compound having a polyamino group to the activated hydrogel, and then binding a compound having an α, β-unsaturated carbonyl group. Method for manufacturing chips.   A surface plasmon resonance analysis sensor comprising the biosensor chip according to claim 1.

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