JP4484626B2 - Biosensor - Google Patents

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 measuring a specific binding reaction between a physiologically active substance and a sample substance, the sample substance is not necessarily a single component, and it is also required to measure the sample substance in a heterogeneous system such as in a cell extract. The In that case, when various kinds of contaminants such as proteins and lipids cause nonspecific adsorption on the detection surface, the measurement and detection sensitivity is remarkably lowered. The above detection surface has a problem that non-specific adsorption is very likely to occur. Several methods have been investigated to solve this problem. For example, a method of suppressing physical adsorption by immobilizing a hydrophilic hydrogel on a metal surface via a linker has been used (see Patent Document 1, Patent Document 3, and Patent Document 4). However, even with this method, the suppression of nonspecific adsorption was not at a sufficient level.

  On the other hand, in the biosensor as described above, the measurement unit for measuring the specific binding reaction between the physiologically active substance and the sample substance and the control unit that does not perform such a binding reaction exist on the same plane. In order to reduce the baseline fluctuation by removing the influence of measurement disturbance (temperature change, concentration change, pressure change), it is preferable that they are as close as possible. For this purpose, it has become necessary to make the reference part and the measurement part coexist on the surface of the SPR sensor using a polymer thin film.

  Patent Document 5 discloses a method of microstamping a biological ligand onto a polymer surface, which includes at least hydrolysis, reduction, photoinitiated graft polymerization, amination, surface cross-polymerization of polyethylene oxide, and chemical reaction of terminal hydroxyl groups. Forming a first functional group on the polymer surface by a method selected from one of corona discharge, plasma etching, laser treatment, and ion beam treatment, and at least one biological function having at least a second functional group on the surface. A ligand-adsorbed stamp is contacted to form a covalent bond with the first functional group on the polymer surface, and the biochemical ligand is covalently attached directly to the polymer surface by separating the stamp from the polymer surface. A method of fixing is described. In the above method, patterning is performed by bringing a solid (PDMS) into contact with the polymer film. However, since the sensor for SPR is a surface with a polymer thin film on a metal thin film, the physical strength is small, and the contact of solids will damage the sensor surface. Is not suitable.

Japanese Patent No. 2815120 JP-A-9-264843 US Pat. No. 5,436,161 JP-A-8-193948 U.S. Pat.No. 6,444,254

  This invention made it the subject which should solve the above-mentioned problem to be solved. In particular, an object of the present invention is to provide a biosensor in which at least two types of surfaces are patterned without damaging the sensor surface, thereby suppressing nonspecific adsorption.

  As a result of intensive studies to solve the above problems, the present inventors provide a desired biosensor by coating the surface of the substrate with a hydrophobic compound having a photoactive group and patterning by light irradiation. The present inventors have found that this can be done and have completed the present invention.

  That is, according to the present invention, there is provided a biosensor comprising a substrate coated with a hydrophobic compound having a photoactive group.

  Preferably, in the biosensor of the present invention, at least two types of surfaces are patterned by irradiating light onto a substrate coated with a hydrophobic compound having a photoactive group.

Preferably, the biosensor of the present invention comprises a metal surface or metal film coated with a hydrophobic compound having a photoactive group.
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 or aluminum.

Preferably, the thickness of the metal film is 0.1 nm to 500 nm .
Preferably, the coating thickness of the hydrophobic compound having a photoactive group is 0.1 nm or more and 500 nm or less.

Preferably, the biosensor of the present invention has a functional group capable of immobilizing a physiologically active substance on the outermost surface on the substrate.
Preferably, the functional group capable of immobilizing the physiologically active substance is —OH, —SH, —COOH, —NR ¹ R ² (wherein R ¹ and R ² are each independently a hydrogen atom or a lower alkyl group. -CHO, -NR ³ NR ¹ R ² (wherein R ¹ , R ² and R ³ independently represent a hydrogen atom or a lower alkyl group), -NCO, -NCS, an epoxy group, or Vinyl group.

Preferably, it has a functional group capable of immobilizing a physiologically active substance in a predetermined region on the outermost surface of the substrate and patterned by light irradiation.
Preferably, the biosensor of the present invention is used for non-electrochemical detection, more preferably for surface plasmon resonance analysis.

  Preferably, the biosensor of the present invention includes a dielectric block, a metal film formed on one surface of the dielectric block, a light source that generates a light beam, and the light beam to the dielectric block. An optical system that allows the total reflection condition to be obtained at the interface between the dielectric block and the metal film and that includes various incident angle components, and the intensity of the light beam that is totally reflected at the interface to measure the surface A measurement chip for use in a surface plasmon resonance measurement apparatus comprising a light detection means for detecting a state of plasmon resonance, comprising the dielectric block and the metal film, wherein the dielectric block is Formed as one block including all of the light beam incident surface, light exit surface, and one surface on which the metal film is formed, and the metal film is integrated with the dielectric block. It is formed on the measurement chip being.

According to another aspect of the present invention, there is provided the above-described method for producing the biosensor of the present invention, comprising the steps of coating a substrate with a hydrophobic compound having a photoactive group, and irradiating the substrate with light. .
Preferably, the step of coating the substrate with a hydrophobic compound having a photoactive group, the step of bringing the monomer compound having a functional group capable of immobilizing a physiologically active substance into contact with the substrate, and a predetermined region of the substrate only There is provided a method for producing the biosensor of the present invention described above, which comprises a step of polymerizing the monomer compound by irradiating light while patterning.

According to still another aspect of the present invention, there is provided the biosensor of the present invention as described above, wherein a physiologically active substance is covalently bonded to the surface.
Preferably, at least a measurement part in which a physiologically active substance or a substance that interacts with it is bound and a reference part that does not have a physiologically active substance or a substance that interacts with it exist in the same plane.

  According to still another aspect of the present invention, the biosensor includes a 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. A method for immobilizing a physiologically active substance is provided.

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

  According to the present invention, it is possible to provide a biosensor in which at least two types of surfaces are patterned without damaging the sensor surface, thereby suppressing nonspecific adsorption.

Embodiments of the present invention will be described below.
The biosensor of the present invention is characterized by comprising a substrate coated with a hydrophobic compound having a photoactive group, and more preferably, at least by subjecting the substrate coated with a hydrophobic compound having a photoactive group to light irradiation. Two or more types of surfaces are patterned.

  First, the hydrophobic compound having a photoactive group used in the present invention will be described. The method for obtaining a hydrophobic compound having a photoactive group used in the present invention includes (1) a method of introducing a photoactive group or a compound having a photoactive group into a general hydrophobic compound, and (2) a photoactive group. Examples thereof include a method of introducing a hydrophobic group into a polymer compound having, and (3) a method of mixing a compound having a photoactive group with a hydrophobic compound.

  Examples of the general hydrophobic compound used in the above-described method (1) of introducing a photoactive group or a compound having a photoactive group into a general hydrophobic compound include compounds having an aromatic group and an alkyl group. Is mentioned.

  Moreover, it is also a preferable aspect to apply what is obtained by superposing | polymerizing the hydrophobic monomer mentioned below. Examples of such hydrophobic monomers include monomers having a hydrophobic group such as (meth) acrylate and styrene, and homopolymers obtained by using at least one selected from these hydrophobic monomers, or two or more types A copolymer or the like obtained by copolymerization of may be selected according to the purpose. When copolymerizing, random copolymerization or block copolymerization may be used.

  The photoactive group used in the present invention refers to a group that is decomposed by light irradiation in the region of 150 nm to 1200 nm to generate a radical. Specific examples include an azide group that generates photonitrene, a diazo group that generally generates radicals by light, a benzophenone group, a trichloromethyl group, an α-hydroxyacetophenone group, an α-alkoxyacetophenone group, and the like. Examples of the compound having a photoactive group include an azide compound, a diazo compound, and a trichloromethyl-triazine compound.

  Examples of the polymer compound having a photoactive group used in the above method (2) of introducing a hydrophobic group into the polymer compound having a photoactive group include a diazo compound, an azide compound, and a benzophenone type compound. Moreover, (meth) acrylate, styrene, etc. are mentioned as a hydrophobic group introduce | transduced into the high molecular compound which has the said photoactive group.

  The compound having a photoactive group and the hydrophobic compound used in the above method (3) of mixing a compound having a photoactive group with a hydrophobic compound are described in the methods (1) and (2), respectively. Things.

  The composition ratio of the hydrophobic group in the hydrophobic compound having a photoactive group and the photoactive group synthesized by the methods (1) and (2) is preferably 1:99 to 90:10. 5: 95-60: 40 is more preferable. These composition ratios can be controlled during synthesis by adjusting the amount of photoactive groups added to the above general hydrophobic compounds or the amount of hydrophobic groups added to polymer compounds having photoactive groups. can do.

  In addition, when the photoactive group or the compound having a photoactive group used in the present invention is introduced into the hydrophobic compound having a polymer structure obtained by polymerization of the hydrophobic monomer, the introduction position is a side chain. However, it is preferable to have at least the terminal from the viewpoint of hydrophobic expression. Here, although the specific example (hydrophobic compound 1-hydrophobic compound 15) of the hydrophobic compound which has a photoactive group is shown below with a composition ratio, this invention is not limited to these.

  Such a hydrophobic compound having a photoactive group can be synthesized by a known method as described in JP-A-2003-345038.

Coating of a hydrophobic compound having a photoactive group on a 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, It can be performed by a casting method, a dipping method, or the like.
The coating thickness of the hydrophobic compound having a photoactive group is not particularly limited, but is preferably 0.1 nm to 500 nm , and particularly preferably 1 nm to 300 nm .

There is no particular limitation on the energy application method for pattern formation in the present invention, and any method of exposure and heating can be used as long as it can generate radicals by decomposing photoactive groups. From the viewpoint of simplicity of the apparatus, a method of irradiating actinic rays is preferable. When actinic light irradiation is applied to image-like exposure, both scanning exposure based on digital data and pattern exposure using a lith film can be used. Examples of the method used for pattern formation include a method of writing by radiation irradiation such as heating and exposure. For example, light irradiation with an infrared laser, an ultraviolet lamp, visible light, etc., electron beam irradiation such as γ rays, thermal recording with a thermal head, and the like are possible. Examples of these light sources include mercury lamps, metal halide lamps, xenon lamps, chemical lamps, and carbon arc lamps. Examples of radiation include electron beams, X-rays, ion beams, and far infrared rays. Also, g-line, i-line, deep-UV light, and high-density energy beam (laser beam) are used. Specific examples generally used include direct image-like recording with a thermal recording head, scanning exposure with an infrared laser, high-illuminance flash exposure such as a xenon discharge lamp, and infrared lamp exposure. In order to perform direct pattern formation using digital data of a computer, it is preferable to apply energy by laser exposure. Lasers include gas lasers such as carbon dioxide lasers, nitrogen lasers, Ar lasers, He / Ne lasers, He / Cd lasers, Kr lasers, solid state lasers such as liquid (pigment) lasers, ruby lasers, Nd / YAG lasers, GaAs / Semiconductor lasers such as GaAlAs and InGaAs lasers, KrF lasers, XeCl lasers, XeF lasers, and excimer lasers such as Ar ₂ can be used. In particular, exposure with a solid-state high-power infrared laser such as a semiconductor laser or a YAG laser that emits infrared light with a wavelength of 700 to 1200 nm is preferable. Thereafter, the polymer in the unexposed area is dissolved and removed by washing with water.

  In the present invention, at least two types of surfaces are patterned on the biosensor surface coated with a hydrophobic compound having a photoactive group by light irradiation. The patterning can be performed, for example, to pattern a portion where the physiologically active substance is immobilized and a portion where the physiologically active substance is not immobilized.

  For example, at least two types of surfaces formed in the same plane include, for example, a measurement unit combined with a physiologically active substance or a substance that interacts with it and a reference part that does not have the physiologically active substance or a substance that interacts with it. Combinations are listed.

  In the present invention, by providing the measurement unit and the reference unit in the same plane as described above, it is possible to cancel the baseline fluctuation due to the disturbance and substantially stabilize it.

  In the present invention, for example, after coating a substrate with a hydrophobic compound having a photoactive group, a monomer compound having a functional group capable of immobilizing a physiologically active substance is brought into contact with the substrate, and thereafter The monomer compound can be bonded by irradiating light while patterning only the region. By this operation, a linker having a functional group capable of immobilizing a physiologically active substance only in the region irradiated with light is generated, but no linker is formed in the region not irradiated with light. After the light irradiation, the surface of the substrate is washed with an appropriate liquid (for example, water) to remove the monomer compound, whereby the surface of the substrate has a functional group capable of immobilizing a physiologically active substance, and such a region. Patterning can be performed on a region having no functional group.

In the present specification, the type of functional group capable of immobilizing a physiologically active substance is not particularly limited, but 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 independently represent a hydrogen atom or a lower alkyl group) ), -NCO, -NCS, an epoxy group, or a vinyl group. 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.

  Specific examples of the monomer compound having a functional group capable of immobilizing a physiologically active substance that can be used in the present invention include acrylic acid, methacrylic acid, polyethylene glycol, polyethylene glycol acrylic acid monoester, polyethylene glycol methacrylate monoester. Examples include, but are not limited to, esters.

  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 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.

  The biosensor of the present invention is preferably a metal surface or metal film coated with a hydrophobic compound having a photoactive group. 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 0.1 nm or more and 500 nm or less, more preferably 0.5 nm or more and 500 nm or less, particularly It is 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. Moreover, when an interstitial layer consisting of chrome or the like, the thickness of the intervening layer, 0.1 nm or more, preferably 10nm 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, in the case of a surface plasmon resonance biosensor, generally, optical glass such as BK7, or synthetic resin, specifically polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  The 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, and IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O 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 and the like can be used. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. Also, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit a specific reaction 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 weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

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

  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.

  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, 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.

  The biosensor of the present invention includes a dielectric block, a metal film formed on one surface of the dielectric block, a light source that generates a light beam, and the light beam with respect to the dielectric block. An optical system that allows the total reflection condition to be obtained at the interface between the surface and the metal film and includes various incident angle components, and the intensity of the light beam totally reflected at the interface to measure surface plasmon resonance. A measurement chip for use in a surface plasmon resonance measurement apparatus comprising a light detection means for detecting a state is preferable, and is composed of the dielectric block and the metal film, and the dielectric block is formed of the light beam. It is formed as one block including all of the entrance surface, the exit surface, and one surface on which the metal film is formed, and the metal film is integrated with the dielectric block. Formed in the serial measurement chip, it can be used.

  In the present invention, specifically, the surface plasmon resonance measuring apparatus described in FIGS. 1 to 32 described in JP 2001-330560 A and described in FIGS. 1 to 15 described in JP 2002-296177 A are described. A surface plasmon resonance measuring apparatus which is used can be preferably used. The contents described in Japanese Patent Laid-Open No. 2001-330560 and Japanese Patent Laid-Open No. 2002-296177 are all cited in this specification as part of the disclosure of this specification.

  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.

  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 angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.

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

When the biosensor of the present invention is used for surface plasmon resonance analysis, it can be applied as a part of various surface plasmon measurement devices as described above.
The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

  The apparatus shown in FIG. 1 (surface plasmon resonance measuring apparatus of the present invention) was used in the experiment. Details of the apparatus shown in FIG. 1 are described in Japanese Patent Application Laid-Open No. 2003-254906.

Example 1: Production of measurement chip (invention)
(1) Preparation of hydrophobic film After processing the dielectric block of the present invention on which 50 nm gold is deposited as a metal film for 30 minutes with Model-208 UV-ozone cleaning system (TECHNOVISION INC.), 1 mg / ml of the following P 5 μl of a methyl ethyl ketone solution of a compound of -1 or P-2 was added so as to contact the metal film, and allowed to stand at 25 ° C. for 15 minutes. Thereafter, it was dried at 40 ° C. for 2 hours under reduced pressure.

(2) Creation of 2-partitioned surface 10 μl of 10% by weight aqueous solution of acrylic acid is added to each of the samples obtained in (1) above (ie, the sample using Compound P-1 and the sample using Compound P-2). added. Further, half of the measurement area was shielded with a metal mask, treated with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, and then washed with water. Next, 100 μl of a 1: 1 mixture of an ethanol solution of 400 mM 1-ethyl-2,3-dimethylaminopropylcarbodiimide and an ethanol solution of 100 mM pentafluorophenol was added and allowed to stand at 25 ° C. for 30 minutes. After washing with ethanol 5 times, 20 μl of 10 mM biotin-LC-amine (PIERCE) ethanol solution was added and left at 25 ° C. for 20 minutes. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water. Further, the exposed portion was shielded with a metal mask having a pattern opposite to the above, and the same exposure as described above was performed. Next, 100 μl of a 1: 1 mixture of an ethanol solution of 400 mM 1-ethyl-2,3-dimethylaminopropylcarbodiimide and an ethanol solution of 100 mM pentafluorophenol was added and allowed to stand at 25 ° C. for 30 minutes. After washing 5 times with ethanol, 20 μl of 1M ethanolamine ethanol solution was added and left at 25 ° C. for 20 minutes. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water. This chip is called an optical two-divided surface chip.

Comparative Example 1: Creation of a two-divided surface by contact A stamp that contacts half of the measurement part of the dielectric block used for measurement was prepared by PDMS. The surface was plasma ozone treated to ensure the wettability of the solution.

The dielectric block of the present invention on which 50 nm of gold was deposited was cleaned with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, and 5 μl of 1 mg / ml PMMA in methyl ethyl ketone was brought into contact with the metal film. And allowed to stand at 25 ° C. for 15 minutes. The thickness of the obtained PMMA film was 20 nm.
1N NaOH aqueous solution was added so as to be in contact with the PMMA film, allowed to stand at 60 ° C. for 5 hours, and then washed with water three times. Through this treatment, carboxyl groups were introduced on the surface of the PMMA film.

  400 mM 1-ethyl-2,3-dimethylaminopropylcarbodiimide ethanol solution and 100 mM pentafluorophenol ethanol solution were mixed in a 1: 1 ratio, and 100 μl of the mixture solution was introduced onto the surface of the PMMA film. And allowed to stand at 25 ° C. for 30 minutes. Thereafter, it was washed 5 times with ethanol.

  To the sample obtained above, 100 μl of a 1: 1 mixture of an ethanol solution of 400 mM 1-ethyl-2,3-dimethylaminopropylcarbodiimide and an ethanol solution of 100 mM pentafluorophenol was added and allowed to stand at 25 ° C. for 30 minutes. I put it. After washing with ethanol 5 times, a stamp soaked with an ethanol solution of 10 mM biotin-LC-amine (PIERCE) was contacted for 20 minutes. Thereafter, the stamp was removed, 40 μl of 1M ethanolamine in ethanol was added, and the mixture was allowed to stand at 25 ° C. for 20 minutes. Then, after washing once with ethanol, the partition was removed, and washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water. This sample is called a PMMA contact treatment chip.

Test Example 1:
(1) Evaluation of bipartite surface (non-specific adsorption measurement)
100 μl of HBS-EP solution containing 1 wt% DMSO (dimethyl sulfoxide) was added to each of the optically split surface chip prepared in Example 1 and the PMMA contact-treated chip prepared in Comparative Example 1 to obtain a baseline. Was measured for 1 minute, and this point was taken as the starting point. Thereafter, the solution was exchanged, and an HBS-EP solution containing 100 μg / ml bovine serum albumin (HBS-EP solution) and 1 wt% DMSO (dimethyl sulfoxide) was measured. The composition of the HBS-EP solution 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. The measurement was terminated with the end point being left for 15 minutes. After subtracting the values of the measurement surface and the reference surface, the end point-start point value was determined, and this was designated as NSB. Since the measurement surface surface-modified with a biotin derivative does not interact with bovine serum albumin, NSB is preferably close to zero.

(2) Evaluation of split surface (bond measurement)
100 μl of HBS-EP solution containing 1 wt% DMSO (dimethyl sulfoxide) was added to each of the optically split surface chip prepared in Example 1 and the PMMA contact-treated chip prepared in Comparative Example 1 to obtain a baseline. Was measured for 1 minute, and this point was taken as the starting point. Thereafter, the solution was exchanged, and an HBS-EP solution containing 0.1 μg / ml avidin, 100 μg / ml bovine serum albumin, and 1 wt% DMSO (dimethyl sulfoxide) was measured. The measurement was terminated with the end point being left for 15 minutes. After subtracting the values of the measurement surface and the reference surface, the end point-start point value was determined, and this was defined as BIND.

(3) Evaluation results Table 1 shows the results of the above measurements.

  When the two-divided surface was created by contact, the thin film on the gold was destroyed and the gold surface appeared, so the non-specific adsorption deteriorated. This adds a nonspecific adsorption component to the actual binding value, resulting in a decrease in measurement accuracy. This shows that the object is achieved by the configuration of the present invention.

FIG. 1 shows a surface plasmon resonance measuring apparatus used in the present invention.

Claims (15)

A biosensor comprising a substrate coated with a hydrophobic compound having a photoactive group , wherein: (a) -OH, -SH, -COOH, -NR ¹ R ² (wherein R ¹ and R ² are independently of each other) A hydrogen atom or a lower alkyl group), —CHO, —NR ³ NR ¹ R ² (wherein R ¹ , R ² and R ³ independently represent a hydrogen atom or a lower alkyl group), —NCO, — A functional group capable of immobilizing a physiologically active substance selected from NCS, epoxy group, or vinyl group in a predetermined region on the outermost surface of the substrate and patterned by light irradiation; (B) After coating the substrate with a hydrophobic compound having a photoactive group, the monomer compound having a functional group capable of immobilizing a physiologically active substance is brought into contact with the substrate, and then only on a predetermined region of the substrate. Patter Training can be obtained by coupling the monomer compound is irradiated with light while the above biosensor. The biosensor according to claim 1, wherein at least two kinds of surfaces are patterned by irradiating light onto a substrate coated with a hydrophobic compound having a photoactive group. The biosensor according to claim 1 or 2, comprising a metal surface or metal film coated with a hydrophobic compound having a photoactive group. 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 or aluminum. The biosensor according to claim 3 or 4, wherein the metal film has a thickness of 0.1 nm to 500 nm . The biosensor according to any one of claims 1 to 5, wherein the coating thickness of the hydrophobic compound having a photoactive group is 0.1 nm or more and 500 nm or less. The biosensor according to any one of claims 1 to 6, which is used for surface plasmon resonance analysis. A dielectric block, a metal film formed on one surface of the dielectric block, a light source that generates a light beam, and the light beam with respect to the dielectric block at an interface between the dielectric block and the metal film An optical system that allows the total reflection condition to be obtained and includes various incident angle components, and a light detection means that detects the surface plasmon resonance state by measuring the intensity of the light beam totally reflected at the interface. A measuring chip for use in a surface plasmon resonance measuring apparatus comprising the dielectric block and the metal film, wherein the dielectric block comprises an incident surface, an exit surface, and an optical beam. It is formed as one block including all of the one surface on which the metal film is formed, and is formed on the measurement chip in which the metal film is integrated with the dielectric block. That, biosensor according to any one of claims 1 to 7. Coating a substrate with a hydrophobic compound having a photoactive group, comprising the step of irradiating light to the substrate, a manufacturing method of a biosensor according to any one of claims 1-8. Coating the substrate with a hydrophobic compound having a photoactive group, contacting the substrate with a monomer compound having a functional group capable of immobilizing a physiologically active substance, and patterning only a predetermined region of the substrate by irradiating light including a step of binding the monomer compounds, a manufacturing method of a biosensor according to any one of claims 1 to 8. The biosensor according to any one of claims 1 to 8 , wherein the physiologically active substance is bound to the surface by a covalent bond. The bio of claim 11 , wherein there are at least a measurement part in which a physiologically active substance or a substance that interacts with it and a reference part that does not have a physiologically active substance or a substance that interacts with it in the same plane. sensor. Contacting the biosensor with a physiologically active substance according to any one of claims 1 to 8, comprising the step of covalently coupling the physiologically active substance on the surface of the biosensor, a physiologically active substance on a biosensor How to immobilize. Detecting or measuring a substance that interacts with the bioactive substance, comprising the step of bringing the biosensor according to any one of claims 1 to 8 into contact with the test substance, wherein the bioactive substance is covalently bonded to the surface. how to. The method according to claim 14 , wherein a substance that interacts with a physiologically active substance is detected or measured by surface plasmon resonance analysis.

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