JP2006078213A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor for suppressing non-specific adsorption of a specimen to a flow channel member. <P>SOLUTION: The biosensor includes a substrate and a flow channel formed on it. An adhesion amount of physiologically active substances to the flow channel member is 100 pg/mm<SP>2</SP>or less. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

  Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations, etc., but conventional methods require complicated operations and labeling substances, so measurement without the need for labeling substances Several techniques that can detect a change in the amount of a substance bound with high sensitivity are used. For example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, and measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. SPR measurement technology detects adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a fixed wavelength. It is a method to do. The QCM measurement technique is a technique that can detect the adsorption / desorption mass at the ng level from the change in the oscillation frequency of the oscillator due to the adsorption / desorption of a substance on the gold electrode (device) of the crystal oscillator. In addition, by functionalizing the surface of gold ultrafine particles (nm level), immobilizing a physiologically active substance on the surface, and performing a specific recognition reaction between the physiologically active substances, it is possible to obtain a living body from the sedimentation and arrangement of gold fine particles. Related substances can be detected.

  In any of the above techniques, the surface on which the physiologically active substance is immobilized is important. Hereinafter, the surface plasmon resonance (SPR) most used in this technical field will be described as an example.

  A commonly used measurement chip is composed of a transparent substrate (eg, glass), a deposited metal film, and a thin film having a functional group capable of immobilizing a physiologically active substance thereon, and the metal surface is interposed through the functional group. Immobilize physiologically active substances. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance.

  As a thin film having a functional group capable of immobilizing a physiologically active substance, a functional group capable of binding to a metal, a linker having a chain length of 10 or more atoms, and a compound having a functional group capable of binding to a physiologically active substance, A measurement chip in which an active substance is immobilized has been reported (see Patent Document 1). In addition, a measurement chip comprising a metal film and a plasma polymerization film formed on the metal film has been reported (see Patent Document 2).

  When an experiment is performed using a sensor capable of detecting a trace amount, if non-specific adsorption to the flow path occurs, (1) change in the amount of immobilized ligand (that is, physiologically active substance) (deterioration of reproducibility), ( 2) Problems such as fluctuations in the binding amount due to fluctuations in the concentration of the analyte (ie, the test substance) and the accompanying deterioration in the reliability of kinetic analysis, and (3) the inability to perform adsorbate analysis using mass spectra. Therefore, there has been a demand for the construction of a measurement system that suppresses nonspecific adsorption including the flow path system.

Japanese Patent No. 2815120 JP-A-9-264843

  The present invention has been made to solve the above-described problems of the prior art. That is, this invention made it the subject which should be solved to provide the biosensor which suppressed the nonspecific adsorption | suction of the to-be-tested substance to a flow-path member.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have selected a flow path member that has a bioactive substance adhesion amount of 100 pg / mm ² or less, thereby providing The present inventors have found that a biosensor in which non-specific adsorption of an active substance is suppressed can be provided, and the present invention has been completed.

That is, according to the present invention, in the biosensor including the substrate and the flow path formed thereon, the amount of the physiologically active substance attached to the flow path member is 100 pg / mm ² or less. The biosensor is provided.

Preferably, the contact angle of the surface of the flow channel is 50 ° or more and 120 ° or less with respect to water.
Preferably, the flow path member is polydimethylsiloxane, polypropylene, polyethylene, polymethyl methacrylate, or polystyrene.
Preferably, the flow path member is treated with a fluorine-based surface treatment agent.

  Preferably, the flow path area is 5 to 100 times the ligand immobilization area.

Preferably, the substrate is 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 biosensor of the present invention is used for non-electrochemical detection, more preferably for surface plasmon resonance analysis.

  According to another aspect of the present invention, there is provided the biosensor of the present invention described above, wherein a physiologically active substance is covalently bonded to the surface of the substrate.

According to still another aspect of the present invention, the step of bringing the biosensor of the present invention into contact with a physiologically active substance and covalently bonding the physiologically active substance to the surface of the biosensor substrate; Contacting the test substance with a biosensor in which the active substance is covalently bound to the surface of the substrate;
A method for detecting or measuring a substance that interacts with the physiologically active substance is provided.

  Preferably, the step of binding the physiologically active substance to the biosensor and the step of detecting or measuring the substance that interacts with the physiologically active substance by bringing the test substance into contact with the biosensor are performed by different apparatuses.

Preferably, the measurement is performed without sequentially bringing a plurality of test substances into contact with the biosensor.
Preferably, the substance that interacts with the 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, it is possible to provide a biosensor that suppresses non-specific adsorption to a flow path member.

Hereinafter, embodiments of the present invention will be described.
The biosensor of the present invention includes a substrate and a flow path formed thereon.
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 for each other is immobilized on a substrate and used as a molecular recognition substance to selectively measure the other substance to be matched.

  The flow path referred to in the present invention does not include a syringe or pipette for injecting a drug or protein. However, from the viewpoint of preventing contamination, it is preferable to inject chemicals and proteins using a disposable pipette. An example of the flow path of the present invention is shown in FIG.

In the present invention, it is preferable that a physiologically active substance (eg, protein) does not substantially adhere to the flow path. Specifically, the amount of the physiologically active substance attached to the flow path member is 100 pg / mm ² or less. , more preferably not more than 50 pg / mm ^2, further preferably not more than 20 pg / mm ^2, particularly preferably not more than 10 pg / mm ^2, or less and most preferably the detection limit.

  Here, the adhesion amount of a physiologically active substance (for example, protein etc.) can be detected, for example, after the channel member is immersed in the labeled protein and washed. As an example, after immersing a member in avidin-FITC as a labeled protein and storing it for 10 minutes, it is washed with a buffer to be measured (for example, PBS), and in the fluorescence measurement mode of LAS-1000 (manufactured by Fuji Photo Film). The amount of adhesion can be measured.

  In the present invention, the contact angle of the surface of the flow path is preferably 50 ° or more and 120 ° or less with respect to water, more preferably 60 ° or more and 120 ° or less, and further preferably 70 ° or more and 120 ° or less. is there. By setting the contact angle of the surface of the flow channel to water within the above range, it is possible to form a flow channel in which the adhesion of the physiologically active substance is suppressed.

  The contact angle with water is an angle formed between a droplet and a solid plane when the droplet is placed on a horizontal solid and is stationary. The contact angle with respect to water can be measured with a commercially available contact angle meter.

The material for forming the flow path used in the present invention is not particularly limited as long as the physiologically active substance (for example, protein) does not substantially adhere to the flow path as described above. For example, polydimethylsiloxane Polypropylene, polyethylene, polymethyl methacrylate, polystyrene and the like can be used.

  In addition, a physiologically active substance can be prevented from adhering to the material forming the flow path by surface treatment. For example, the treatment of the flow path member with a fluorine-based surface treatment agent can suppress the adhesion of a physiologically active substance. As the fluorine-based surface treatment agent, for example, Novec EGC-1700 (manufactured by Sumitomo 3M) and the like can be used.

  The substrate of the biosensor of the present invention is preferably a metal surface or a metal film. 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 the use for a surface plasmon resonance biosensor, it is preferably 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 2000 angstrom or less. If it exceeds 5000 angstroms, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 1 angstrom or more and 100 angstrom 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.

  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, when considering use for a surface plasmon resonance biosensor, generally, optical glass such as BK7, or synthetic resin, specifically, polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  The substrate of the present invention preferably has a functional group capable of immobilizing a physiologically active substance on the outermost surface of the substrate. The “outermost surface of the substrate” here means “the side farthest from the substrate”.

Preferred functional groups include —OH, —SH, —COOH, —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group), —CHO, —NR ³ NR ^1. R ² (wherein R ¹ , R ² and R ³ each independently represents a hydrogen atom or a lower alkyl group), —NCO, —NCS, an epoxy group, or a vinyl group can be mentioned. Here, the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.

As a method for introducing these functional groups on the outermost surface, a polymer containing a precursor of those functional groups is coated on a metal surface or a metal film, and then the precursor is located on the outermost surface by chemical treatment. The method of producing | generating the functional group of is mentioned. For example, after polymethylmethacrylate containing —COOCH ₃ groups is coated on a metal film, the surface is brought into contact with an aqueous NaOH solution (1N) at 40 ° C. for 16 hours to form —COOH groups on the outermost surface.

  On the biosensor surface obtained as described above, the physiologically active substance can be immobilized on the metal surface or metal film by covalently bonding the physiologically active substance via the functional group.

  The physiologically active substance immobilized on the biosensor surface 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-molecular compounds, and the like. Examples include immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand binding ability.

  Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.

  The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme, etc. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. 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, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
Examples of the low-molecular 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.

  When the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme, the immobilization can be performed by covalently bonding to a functional group on the metal surface using the amino group, thiol group or the like of the physiologically active substance. .

  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.

  That is, according to the present invention, the biosensor of the present invention on which a physiologically active substance is immobilized is brought into contact with a test substance to thereby interact with the physiologically active substance immobilized on the biosensor. A method of detecting and / or measuring a substance to be provided is provided.

  As the test substance, for example, a sample containing a substance that interacts with the above physiologically active substance can be used. The test substance may be a single substance or a mixture of a plurality of substances.

  In the present invention, detection and / or measurement are preferably performed without sequentially bringing a plurality of test substances into contact with the biosensor. The fact that a plurality of test substances are not sequentially brought into contact with the biosensor means that the test substances are not sequentially passed through the flow path, and after the physiologically active substance (ligand) is immobilized, the binding measurement is performed only once. means.

  Preferably, the step of binding the bioactive substance to the biosensor and the step of contacting the test substance with the biosensor to detect or measure a substance that interacts with the bioactive substance are performed with different devices.

  In the present invention, it is preferable to detect and / or measure the interaction between the physiologically active substance immobilized on the biosensor surface 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, an apparatus using a system called Kretschmann arrangement can be cited (for example, Japanese Patent Laid-Open No. 6-167443). See the official gazette). A surface plasmon measuring apparatus using the above system basically includes a dielectric block formed in a prism shape, for example, and a metal film formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a sample liquid. A light source that generates a light beam; an optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the metal film; and It comprises light detecting means for detecting the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface.

  In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or 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 substance to be measured 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 light energy is transferred to the surface plasmon. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means. The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.

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

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

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

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

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

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

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

  Note that, in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of the total reflection attenuation angle (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 amount of change in angle. If the above-described arrayed light detecting means and differentiating means are applied to a measuring apparatus 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: Measurement of non-specific adsorption A chip of a 1 cm x 1 cm x 0.5 cm channel member (shown in Table 1) was prepared using a mold. Avidin-FITC as a labeled protein is immersed in 1 mg / ml PBS buffer (pH 6.8), stored for 10 minutes, washed 10 times with PBS buffer (pH 6.8), dried, and then subjected to LAS-1000. The fluorescence intensity was measured in the fluorescence measurement mode (manufactured by Fuji Photo Film). A calibration curve was created separately to estimate the amount of adhesion. The measurement results are shown in Table 1. It can be seen that non-specific adsorption increases when the contact angle to water is low.

  Further, polystyrene and fluorinated rubber chips were immersed in Novec EGC-1700 (manufactured by Sumitomo 3M) for 10 minutes, then dried at 60 ° C for 4 hours, then dried at 25 ° C for 18 hours, and the same test as above was performed. . The measurement results are shown in Table 2. It can be seen that a surface with reduced nonspecific adsorption can be obtained by increasing the contact angle by surface treatment.

Example 2: Immobilization of ligand (bioactive substance) and measurement of analyte (test substance) Using the materials shown in Table 3 and the surface-treated material, a flow path as shown in FIG. ) And the analyte (test substance) were measured.

<Creation of measurement surface>
Dielectric block with 50nm gold deposited as metal film is treated with Model-208UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, then 11-hydroxy-1-undecanethiol in ethanol / water (80/20) A 5.0 mM solution was added so as to contact the metal film, and surface treatment was performed at 25 ° C. for 18 hours. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water.

  Next, the surface coated with 11-hydroxy-1-undecanthiol in medium was brought into contact with a 10% by weight epichlorohydrin solution (solvent: 1: 1 mixed solution of 0.4M sodium hydroxide and diethylene glycol dimethyl ether) at 25 ° C. The reaction was allowed to proceed for 4 hours in a shaking incubator. The surface was washed twice with ethanol and five times with water.

  Next, 4.5 ml of 1 M sodium hydroxide was added to 40.5 ml of a 25 wt% aqueous dextran (T500, Pharmacia) solution and the solution was contacted on the epichlorohydrin treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. The surface was washed 10 times with 50 ° C. water. Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated for 16 hours in a shaking incubator at 28 ° C. The surface was washed with water and then the above procedure was repeated once. A measurement surface was thus created.

<Immobilization of ligand (bioactive substance)>
The flow path was fixed on the measurement surface created by the above method, and the following operation was performed.
Ligand solution: Protein A (manufactured by Nacalai Tesque) 10 μg dissolved in 1 ml of pH 5.5 acetate buffer Activation solution: 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) Mixed solution with

  The channel was filled with HBS-EP buffer, and the signal was measured. The composition of the HBS-EP buffer is as follows: HEPES (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4), NaCl 0.15 mol / l, EDTA 0.003 mol / l, Surfactant P20 0.005 % By weight. Thereafter, the flow path was filled with the activation solution, allowed to stand for 15 minutes, and then washed 3 times with HBS-EP buffer. Thereafter, the flow path was filled with the ligand solution, allowed to stand for 15 minutes, and then washed three times with HBS-EP buffer. Thereafter, the flow path was filled with an ethanolamine / HCl solution (1 M, pH 8.5), allowed to stand for 15 minutes, and then washed three times with HBS-EP buffer. Further, a 10 mM sodium hydroxide solution was filled in the flow path, left for 1 minute, and then washed 5 times with HBS-EP buffer. The channel was filled with HBS-EP buffer, and the signal was measured. (Signal after ligand fixation-signal before ligand fixation) was defined as a fixed amount. The above-described operation was performed 10 times using 10 similar flow paths. Average values and standard deviations are shown in Table 3. It was found that there was little variation in the fixed amount when a flow path with little ligand adhesion was used.

<Measurement of analyte (test substance)>
Analyte solution: Mouse IgG (purchased from Cosmo Bio) 10 μg dissolved in 1 ml of HBS-EP buffer

  The channel was filled with HBS-EP buffer, signal measurement was started, and the binding constant (KD) was calculated by the following method.

  That is, from the measurement result of the signal change of surface plasmon resonance, the adsorption rate coefficient (ka) and the desorption rate coefficient (kd) are obtained by using the following formulas (1), (2) and (3), and the obtained adsorption rate is obtained. From the coefficient (ka) and the separation rate coefficient (kd), the dissociation constant (KD) was calculated by the equation represented by KD = kd / ka.

_{dθ / dt = ka × c s} × (1-θ) -kd × θ (1)
(In the formula, θ represents the adsorption rate (= adsorption amount / saturated adsorption amount), ka represents the adsorption rate coefficient, kd represents the desorption rate coefficient, and c _s represents the concentration of the molecule to be analyzed in the vicinity of the metal surface.)
^{∂c / ∂t = D × ∂ 2} c / ∂x 2 (2)
(Distance from wherein, x is a metal surface, D is the diffusion coefficient of the analyte molecule, c is represents the concentration of the analyte molecule, and c = c _s when x = 0.)
θ = R / Rmax (3)
(In the formula, θ represents an adsorption rate (= adsorption amount / saturated adsorption amount), R represents a surface plasmon signal, and Rmax represents a signal when the molecule to be analyzed is saturated adsorbed.)

  The binding constant (KD) was calculated for the n = 10 measurement surface with the ligand immobilized thereon, and the average value and standard deviation were obtained. The results are shown in Table 3. It was found that there was little variation in the value of the coupling constant (KD) in the channel with little adhesion.

FIG. 1 shows an example of the flow path of the present invention.

Claims (15)

A biosensor including a substrate and a flow path formed thereon, wherein the amount of physiologically active substance attached to the flow path member is 100 pg / mm ² or less. The biosensor according to claim 1, wherein the contact angle of the surface of the channel is 50 ° or more and 120 ° or less with respect to water. The biosensor according to claim 1 or 2, wherein the flow path member is polydimethylsiloxane, polypropylene, polyethylene, polymethyl methacrylate, or polystyrene. The biosensor according to any one of claims 1 to 3, wherein the flow path member is treated with a fluorine-based surface treatment agent. The biosensor according to any one of claims 1 to 4, wherein the flow path area is 5 to 100 times the ligand-immobilized area. The biosensor according to any one of claims 1 to 5, wherein the substrate is a metal surface or a metal film. The biosensor according to claim 6, wherein the metal surface or metal film is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum, and aluminum. The biosensor according to any one of claims 1 to 7, which is used for non-electrochemical detection. The biosensor according to any one of claims 1 to 8, which is used for surface plasmon resonance analysis. The biosensor according to any one of claims 1 to 9, wherein the physiologically active substance is bound to the surface of the substrate by a covalent bond. A step of bringing the biosensor according to any one of claims 1 to 9 and a physiologically active substance into contact with each other and covalently binding the physiologically active substance to the surface of the biosensor substrate; Contacting the test substance with the biosensor bound to the surface of the substrate by
A method for detecting or measuring a substance that interacts with the physiologically active substance. The method according to claim 11, wherein the step of binding the physiologically active substance to the biosensor and the step of detecting or measuring a substance that interacts with the physiologically active substance by bringing the test substance into contact with the biosensor are performed in different apparatuses. . The method according to claim 11 or 12, wherein a plurality of test substances are not sequentially brought into contact with the biosensor. The method according to claim 11, wherein a substance that interacts with a physiologically active substance is detected or measured by a non-electrochemical method. The method according to claim 11, wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.

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