JP2007085970A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor capable of evaluating the charge of a molecule of a protein or the like included in a measuring sample, and a method for analyzing the charge of a material to be measured using it. <P>SOLUTION: This biosensor comprises a substrate having the surface wherein the charge is changed according to a portion. Preferably, a functional group imparting the charge is introduced into the substrate surface, and the introduction rate of the functional group is changed according to a portion, and thereby the charge on the substrate surface is changed according to the portion. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biosensor for analyzing the charge of a molecule such as a protein contained in a measurement sample, and a method for analyzing the charge of a molecule using the biosensor. More specifically, the present invention relates to a biosensor for analyzing the charge of a molecule using surface plasmon resonance analysis and the like, and a method for analyzing the charge of a molecule using the same.

  In the development of a surface plasmon resonance (SPR) sensor, it is necessary to modify the metal thin film of the sensing part. For example, in order to apply an SPR sensor to a biosensor, a metal thin film is modified with a polymer having a functional group. When a gold thin film is used as the metal thin film, a modification method using self-adsorptivity (self-assembly) of thiols or sulfides to gold is known. For example, in order to immobilize a ligand on the sensor surface in an SPR biosensor, a carboxymethyl dextran (CMD) layer is introduced using a self-adsorbed alkanethiol as a linker layer on a gold thin film, and the ligand is added to the CMD layer. A method of immobilization has been reported. The role of the CMD layer used here is to increase the amount of ligand to be immobilized and to prevent nonspecific binding of biomolecules to the gold thin film. The modification method with thiol or sulfides is a simple method. For example, in Claire E. Jordan et al. (Non-patent Document 1), an adsorption rate of 90% is obtained by immersing a gold thin film in an ethanol solution of 1 mM thiol for 1 hour. Is described. Further, the adsorption can be evaluated by X-ray electron spectroscopy or measurement of SPR angle change.

  Patent Document 1 describes a detection surface in which a biocompatible porous matrix layer such as a hydrogel is provided on a layer bonded to a metal surface. Patent Document 2 describes a sensor unit using a bifunctional or polyfunctional molecule. Patent Document 3 describes a method for determining the characteristics of a macromolecule using a sensor having a ligand bound to the sensor surface. US Pat. No. 6,057,049 describes a method and apparatus for detecting an analyte of interest in a sample using a solid support comprising an immobilized binding partner comprising a reversible binding receptor. Furthermore, Patent Document 5 describes a method for producing a substrate surface in which a film-forming protein or lipid is bound on a self-adsorptive monolayer.

  Patent Document 6 discloses a biochip for measuring surface plasmon resonance (SPR) having a portion for immobilizing a biomolecule or a biomolecule assembly (an immobilization site) and a background portion other than the immobilization site. A phosphate buffer solution having a pH of 7.4 in which poly-L-glutamic acid having a concentration of 2000 to 20000 is dissolved at a concentration of 1 mg / ml is brought into contact with the chip surface at 30 ° C. for 10 minutes, and then the phosphate buffer solution is used. An SPR in which the signal ratio of the signal due to SPR obtained by poly-L-glutamic acid binding to the immobilization site and the signal due to nonspecific adsorption to a background part other than the immobilization site is 2 or more when washed for 10 minutes A biochip for measurement is described. This biochip for SPR measurement is characterized in that there is almost no non-specific adsorption in the background part and can be immobilized while maintaining the activity of the biomolecule or biomolecule assembly at the binding site.

  However, none of the above-mentioned documents describes the analysis of molecular charge using a biosensor.

Jordan, C.E. et al., Langmuir, 1994, 10, 3642-3648 Japanese National Patent Publication No. 4-501605 Japanese National Patent Publication No. 4-501606 Japanese National Patent Publication No. 4-501607 Japanese National Patent Publication No. 7-507865 International Publication WO96 / 10178 JP 2004-125625 A

  An object of the present invention is to provide a biosensor capable of evaluating the charge of a molecule such as a protein contained in a measurement sample, and a method for analyzing the charge of a substance to be measured using the biosensor. did.

  As a result of intensive investigations to solve the above problems, the present inventors have produced a biosensor comprising a substrate having a surface whose charge varies depending on the site, and brought the charged protein into contact with the surface of the biosensor. By investigating the degree of protein concentration to each part of the sensor chip surface, it was found that the charge of the protein can be evaluated by SPR measurement using a biosensor comprising a substrate having a surface whose charge varies depending on the part. It was. The present invention has been completed based on these findings.

  That is, according to the present invention, there is provided a biosensor composed of a substrate having a surface whose charge varies depending on the site.

Preferably, a functional group that imparts charge is introduced on the surface of the substrate, and the introduction rate of the functional group varies depending on the site, whereby the charge on the surface of the substrate varies depending on the site.
Preferably, the substrate is a substrate made of a metal surface or a metal film.
Preferably, the metal surface or the metal film is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum, and aluminum.

  Preferably, the substrate is a metal surface or metal film coated with a hydrophobic polymer compound or a water-soluble polymer compound, or a metal surface or metal film having a self-assembled film.

Preferably, the biosensor of the present invention is produced by coating the surface of a substrate with a polymer compound having a functional group that imparts a charge.
Preferably, the functional group that imparts a charge is a carboxyl group, a sulfate group, a phosphate group, or a sulfonate group.

Preferably, the biosensor of the present invention is used for analyzing the charge of a substance to be measured.
Preferably, the charge of the substance to be measured is analyzed by surface plasmon resonance analysis.

According to another aspect of the present invention, the charge of the substance to be measured includes contacting the substance to be measured with the surface of the biosensor of the present invention and detecting the presence or absence of the interaction between the substance to be measured and the surface. A method of analyzing the is provided.
Preferably, presence / absence of interaction between the substance to be measured and the surface is detected by surface plasmon resonance analysis.

  According to the biosensor of the present invention, it is possible to evaluate the charge of molecules such as proteins contained in a measurement sample.

Hereinafter, embodiments of the present invention will be described in detail.
The biosensor of the present invention is characterized by comprising a substrate having a surface whose charge is changed depending on a site.

  A physiologically active substance is either positively or negatively charged when dissolved in a buffer having a pH other than its isoelectric point. On the other hand, when a charged polymer is prepared in the same buffer at the same time, the physiologically active substance is concentrated on the polymer by electrostatic attraction. At this time, when the charge amount or the plus or minus value of the polymer is changed, the degree of concentration to the polymer changes according to the charge amount of the physiologically active substance. If such a polymer is prepared on the surface of the SPR sensor, the degree of concentration in the polymer can be detected. That is, it is possible to evaluate the charge of the physiologically active substance contained in the measurement sample by using a biosensor having a membrane whose charge is changed. When a protein is considered as a physiologically active substance, there is isoelectric focusing as a method for evaluating the charge of the protein. In the present invention, the charge of the protein contained in the measurement sample can be evaluated based on a completely new principle. it can.

  As a substrate having a surface whose charge varies depending on the part, a substrate having a surface whose charge varies depending on the part may be constructed by arranging a plurality of substrates having the same charge on the same plane. Alternatively, in a single substrate, a substrate having a surface whose charge is changed depending on the portion may be manufactured.

  The functional group that gives charge to the membrane is preferably any of a carboxyl group, a sulfate ester group, a phosphate ester group, and a sulfonic acid group.

There is no particular limitation on the method of introducing a functional group that gives charge to the membrane for the polymer used in the present invention, and a polymer (polymer) is obtained by carrying out a copolymerization reaction between a normal monomer molecule and a monomer having a functional group that gives charge. The functional group which gives the above-mentioned charge may be introduced by a so-called polymer reaction after the polymer has been produced in advance. The polymer used in the present invention may be copolymerized with other monomer components in addition to the functional group monomer component that imparts a charge.

  In order to produce a membrane with a changed charge, a functional group having a charge is attached to a surface to which a water-soluble polymer is bound, a surface to which a hydrophobic polymer is bound, or a self-assembled monolayer. A functional group can be introduced with a concentration gradient.

  In order to produce a membrane with varying charge, a method of creating a concentration gradient based on the difference in diffusion rate by introducing a functional group having a charge from one point on the membrane surface when a charged functional group is introduced. Can be used.

  In order to produce a membrane with a changed charge, a method of introducing a functional group by changing the concentration of the functional group when introducing a functional group having a charge can be used. Further, in order to produce a membrane with changed charge, a method of changing the type of functional group at the time of introduction of a functional group having charge can be used.

  Furthermore, in order to produce a membrane with a changed charge, the reaction time between the water-soluble polymer, the hydrophobic polymer, or the self-assembled monolayer and the functional group should be reduced when the charged functional group is introduced. A changing method can also be used.

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

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

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

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

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

  In the present invention, the substrate is preferably a metal surface or metal film coated with a hydrophobic polymer compound or a water-soluble polymer compound, or a metal surface or metal film having a self-assembled film. Hereinafter, the hydrophobic polymer compound, the water-soluble polymer compound, and the self-assembled film will be described.

  The hydrophobic polymer compound that can be used in the present invention is generally a polymer compound that has no water absorption or low water absorption, and its solubility in water (25 ° C.) is preferably 10% or less. More preferably, it is 1% or less, and most preferably 0.1% or less.

  Specific examples of the hydrophobic polymer include polyacrylic acid derivatives, polymethacrylic acid derivatives, polyethylene (PE), polypropylene (PP), polybutadiene, polymethylpentene, cycloolefin polymer, polystyrene (PS), acrylonitrile / butadiene. / Styrene copolymer (ABS), styrene / maleic anhydride copolymer / polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), nylon 6, nylon 66, cellulose acetate (TAC), Polycarbonate (PC), modified polyphenylene ether (m-PPE), polyphenylene sulfide (PPS), polyether ketone (PEK), polyether ether ketone (PEEK), polysulfone (PSF), polyether Sulfone (PES), polyphenylene sulfide (PPS), and the like a liquid crystal polymer (LCP). Even when a functional group imparting charge is introduced into the surface of these hydrophobic polymers, it becomes possible to classify the physiologically active substance by the charge of the substance on the two-dimensional surface.

  Coating of the hydrophobic polymer compound on the substrate can be performed by a conventional method, for example, spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, spraying, vapor deposition, casting, It can be performed by an immersion method or the like.

  The coating thickness of the hydrophobic polymer compound is not particularly limited, but is preferably 0.1 nm to 500 nm, particularly preferably 1 nm to 300 nm.

  Examples of the water-soluble polymer compound used in the present invention include polyhydroxy polymer compounds such as polysaccharides (for example, agarose, dextran, carrageenan, alginic acid, starch, cellulose), or synthetic polymer compounds (for example, Polyvinyl alcohol). In the present invention, polysaccharides are preferably used, and dextran is most preferred.

  In the present invention, a polyhydroxy polymer compound having an average molecular weight of 10,000 to 2,000,000 is preferably used. Preferably, a polyhydroxy polymer compound having a molecular weight of 20,000 to 2,000,000, more preferably 30,000 to 1,000,000, and most preferably 200,000 to 800,000 can be used.

  The polyhydroxy polymer compound can be carboxylated, for example, by reacting with bromoacetic acid under basic conditions. By controlling the reaction conditions, it is possible to carboxylate a certain proportion of the hydroxy groups that the polyhydroxy compound contains in the initial state. In the present invention, for example, 1 to 90% of hydroxy groups can be carboxylated. In addition, about the surface coat | covered with arbitrary polyhydroxy polymer compounds, a carboxylation rate is computable with the following method, for example. The membrane surface was gas-phase modified with trifluoroethanol at 50 ° C. for 16 hours using a di-tert-butylcarbodiimide / pyridine catalyst, and the amount of fluorine derived from trifluoroethanol was measured by ESCA (electron spectroscopy for chemical analysis). A ratio with the amount of oxygen on the film surface (hereinafter referred to as F / O value) is calculated. Carboxylation rate at that time by measuring the F / O value when carboxylation is carried out under arbitrary conditions with the theoretical F / O value when all hydroxy groups are carboxylated as 100% carboxylation rate Can be calculated.

The polyhydroxy polymer compound can be attached to the metal film via the organic molecule X ¹ —R ¹ —Y ¹ . The organic molecule X ¹ —R ¹ —Y ¹ will be described in detail.

X ¹ is a group having a binding property to the metal film. Specifically, asymmetric or symmetric sulfide (—SSR ¹¹ Y ¹¹ , —SSR ¹ Y ¹ ), sulfide (—SR ¹¹ Y ¹¹ , —SR ¹ Y ¹ ), diselenide (—SeSeR ¹¹ Y ¹¹ , —SeSeR ¹ Y) ¹ ), selenide (SeR ¹¹ Y ¹¹ , —SeR ¹ Y ¹ ), thiol (—SH), nitrile (—CN), isonitrile, nitro (—NO ₂ ), selenol (—SeH), trivalent phosphorus compound, iso Thiocyanate, xanthate, thiocarbamate, phosphine, thioacid or dithioacid (-COSH, -CSSH) is preferably used.

R ¹ (and R ¹¹ ) is optionally interrupted by a heteroatom, preferably straight-chain (unbranched) for reasonably close packing, and optionally carbon containing double and / or triple bonds It is a hydrogen chain. The chain length is preferably greater than 10 atoms. The carbon chain can optionally be perfluorinated.

Y ¹ and Y ¹¹ are groups for binding the polyhydroxy polymer compound. Y ¹ and Y ¹¹ are preferably the same and have the property that they can be bonded directly or after activation to the polyhydroxy polymer compound. Specifically, hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, epoxy, vinyl group, or the like can be used.

Specific examples of the organic molecule X ¹ -R ¹ -Y ¹ include 10-carboxy-1-decanethiol, 4,4′-dithiodibutylic acid, 11-hydroxy-1-undecanethiol, 11-amino-1- And undecanethiol.

  The self-assembled film (self-assembled monomolecular film) will be described. Sulfur compounds such as thiols and disulfides spontaneously adsorb on a noble metal substrate such as gold to give an ultra-thin film of single molecular size. The aggregate is called a self-assembled film because it shows an arrangement depending on the crystal lattice of the substrate and the molecular structure of adsorbed molecules. Examples of the self-assembled monolayer include alkanethiols on the gold surface, alkylsilanes on the glass surface, alcohols on the silicon surface, and the like. Specific examples of alkanethiols include 7-carboxy-1-heptanethiol, 10-carboxy-1-decanethiol, 4,4′-dithiodibutyric acid, 11-hydroxy-1-undecanethiol, 11-amino- 1-undecanethiol and the like can be used.

  Examples of the substance to be measured in the present invention include immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-immune proteins, immunoglobulin binding proteins, sugar binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters. Or a polypeptide or oligopeptide having a ligand binding ability.

  In the present invention, the interaction between the substance to be measured and the surface is preferably detected and / or measured 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.

  A surface plasmon resonance measuring apparatus is an apparatus for analyzing the characteristics of a substance to be measured using a phenomenon in which surface plasmons are excited by light waves. The surface plasmon resonance measuring apparatus used in the present invention is provided with a metal film, a light source for generating a light beam, a total reflection condition at the interface between the light beam and the metal film, and various incident angle components. And an optical system for detecting the surface plasmon resonance state by measuring the intensity of the light beam totally reflected at the interface.

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

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

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

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

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

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

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

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

  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 present invention is not limited to the examples.

Example 1: Preparation of sensor chip for evaluating protein charge (1) Preparation of sample 1 (comparative example)
Biacore sensor chip CM-5 (research grade) was used as it was as the surface to which carboxymethyldextran was bound.

(2) Preparation of samples 2, 3 and 4 (Example)
In order to produce a sensor having a membrane with a changed charge on the surface, a surface in which the carboxyl group of Biacore sensor chip CM-5 (research grade) was replaced with taurine was used.

  CM-5 was set in Biacore3000, a surface plasmon resonance device manufactured by Biacore, and 0.4M EDC (1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide: Dojindo) and 2.8mM HODhbt (3,4- Contact with an aqueous solution containing Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Tokyo Kasei) for 5 minutes, and 1.0M of taurine (2-aminoethane-1-sulfonic acid: Wako Pure Chemical Industries, Ltd.) By contacting with a solution (adjusted to pH 10.0 with NaOH), a surface in which the carboxyl groups of carboxymethyldextran are partially substituted with sulfonic acid groups is produced. At this time, CM-5 was prepared in which the contact time was changed to 5 minutes (sample 2), 10 minutes (sample 3), and 20 minutes (sample 4) to change the substitution rate to sulfonic acid groups. The substitution rate to the sulfonic acid group can be controlled by the activation time by EDC / HODhbt, the reaction time of taurine, and the number of repetitions of this operation.

  In a glycine buffer having a pH of 1.5 lower than the pKa of carboxylic acid, the carboxyl group is positively charged and the sulfonic acid group is negatively charged. Therefore, as the samples 1, 2, 3, and 4, the negative charge increases. Sample 1 is positively charged. As described above, four films with different charges could be produced.

Example 2: Preparation of protein solution As proteins to be evaluated for charge, p53 (Calbiochem) and p38 (Upstate) were used. The isoelectric point (pI) of the two proteins was compared with a marker (Broad pI Kit, pH 3.5-9.3: Amersham Biosciences) simultaneously measured in an electrophoresis experiment using AE-8150 of ATTO. It was confirmed that p53 was about pI = 4.0, and p38 was about pI = 5.6.

  10 μl of a solution of 1 mg of p53 dissolved in 1 ml of HBS-EP buffer (Biacore, pH 7.4) is weighed and 90 μl of glycine buffer (Biacore, pH 1.5) is added to give 0.1 mg / ml P53 solution (pH 1.5, 0.1 mg / ml) was prepared.

  10 μl of a solution of 1 mg of p38 dissolved in 1 ml of HBS-EP buffer (Biacore, pH 7.4) was weighed, and 90 mg of glycine buffer (Biacore, pH 1.5) was added to give 0.1 mg / ml. P38 solution (pH 1.5, 0.1 mg / ml) was prepared.

Example 3: Concentration of protein on the surface of the sensor chip Each of the sensor chips (samples 1, 2, 3, and 4) prepared in Example 1 was sequentially set in a Biacore 3000 surface plasmon resonance apparatus manufactured by Biacore, and p53 And p38 were allowed to flow for 90 seconds each, and protein concentration on the surface of the sensor chip was examined. The results for p53 are shown in FIG. 1, and the results for p38 are shown in FIG.

  Concentration of p53 was not observed in the sensor of sample 1, and the concentration increased as samples 2, 3, and 4 increased. This is because in pH 1.5, p53 is positively charged, and the concentration effect increases as the negative charge on the sensor surface increases.

  Concentration of p38 was not observed in the sensor of sample 1, and the concentration increased as samples 2, 3, and 4 were obtained. This is also because p38 is positively charged in pH 1.5.

  When the concentration effects of p53 and p38 in Samples 2 and 3 were compared, p38 had a higher concentration effect. It was concluded that p38 has a higher isoelectric point and is therefore more strongly positively charged than p53 even at the same pH of 1.5.

  From the above results, it was proved that the charge of the protein contained in the measurement sample can be evaluated by the SPR measurement using the membrane whose charge is changed.

FIG. 1 shows the results of examining protein concentration on the sensor chip surface when p53 was passed through the sensor chips of samples 1 to 4. FIG. 2 shows the results of examining protein concentration on the sensor chip surface when p38 is passed through the sensor chips of Samples 1 to 4.

Claims (11)

A biosensor comprising a substrate having a surface whose charge varies depending on the site. The functional group which gives electric charge is introduce | transduced into the surface of the board | substrate, The charge of the surface of a board | substrate is changing with the site | parts by the introduction rate of this functional group changing with the site | parts. Biosensor. The biosensor according to claim 1 or 2, wherein the substrate is a substrate made of a metal surface or a metal film. The biosensor according to claim 3, wherein the metal surface or the metal film is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum, and aluminum. 5. The substrate according to claim 1, wherein the substrate is a metal surface or metal film coated with a hydrophobic polymer compound or a water-soluble polymer compound, or a metal surface or metal film having a self-assembled film. The biosensor described. The biosensor according to any one of claims 1 to 5, which is prepared by coating a surface of a substrate with a polymer compound having a functional group that imparts a charge. The biosensor according to any one of claims 1 to 6, wherein the functional group that imparts charge is a carboxyl group, a sulfate ester group, a phosphate ester group, or a sulfonic acid group. The biosensor according to any one of claims 1 to 7, which is used for analyzing the charge of a substance to be measured. The biosensor according to claim 8, wherein the charge of the substance to be measured is analyzed by surface plasmon resonance analysis. 10. Analyzing the charge of a substance to be measured, comprising contacting the substance to be measured with the surface of the biosensor according to any one of claims 1 to 9 and detecting the presence or absence of an interaction between the substance to be measured and the surface. how to. The method according to claim 10, wherein presence or absence of interaction between the substance to be measured and the surface is detected by surface plasmon resonance analysis.

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