WO2016043078A1 - Biocapteur pour la détection simultanée d'un substrat et d'un antigène, électrode, procédé de détection simultanée de substrat et d'antigène, et programme - Google Patents

Biocapteur pour la détection simultanée d'un substrat et d'un antigène, électrode, procédé de détection simultanée de substrat et d'antigène, et programme Download PDF

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WO2016043078A1
WO2016043078A1 PCT/JP2015/075310 JP2015075310W WO2016043078A1 WO 2016043078 A1 WO2016043078 A1 WO 2016043078A1 JP 2015075310 W JP2015075310 W JP 2015075310W WO 2016043078 A1 WO2016043078 A1 WO 2016043078A1
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conductive polymer
antigen
substrate
biosensor
simultaneous detection
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PCT/JP2015/075310
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English (en)
Japanese (ja)
Inventor
暁 馬場
ラピパン ジャンマニー
山本　格
一成 新保
景三 加藤
双男 金子
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国立大学法人 新潟大学
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Priority to JP2016548838A priority Critical patent/JP6714278B2/ja
Publication of WO2016043078A1 publication Critical patent/WO2016043078A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a substrate antigen simultaneous detection biosensor, an electrode, a substrate antigen simultaneous detection method, and a program.
  • Non-Patent Document 1 discloses that an enzyme and an antigen are simultaneously detected by fluorescence using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Non-Patent Document 2 discloses that an antigen and glucose are detected using a conductive polymer.
  • Patent Document 1 has a problem in that it requires time for measurement and labor since it requires processing such as sample preparation in advance.
  • the flow cell described in Patent Document 2 has problems that it is impossible to simultaneously detect an antigen and an enzyme substrate and to immobilize an antibody and an enzyme on a conductive polymer at the same time. It was.
  • Non-Patent Document 1 has a problem that it is difficult to quantify both by one type of measurement because it tries to obtain two pieces of information only from fluorescence information. It was.
  • the present invention has been made in view of the above problems, and is a substrate antigen simultaneous detection bio that can simultaneously and quantitatively and easily measure a substrate such as a sugar and an antigen such as a protein in a liquid such as a body fluid.
  • An object is to provide a sensor, an electrode, a method for simultaneously detecting a substrate antigen, and a program.
  • the substrate antigen simultaneous detection biosensor of the present invention includes an electrode on which a conductive polymer on which an enzyme and an antibody are fixed is deposited, and an enzyme reaction in the conductive polymer. Based on the current value detection means for detecting a change in the current value on the conductive polymer, and detects a change in the reflectance of the light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer. And a reflectance detection means.
  • the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, further comprising a solution bottle filled with a liquid in contact with the conductive polymer.
  • the substrate antigen simultaneous detection biosensor of the present invention is characterized in that in the substrate antigen simultaneous detection biosensor described above, the liquid contains a body fluid.
  • the substrate antigen simultaneous detection biosensor of the present invention is characterized in that the body fluid is urine, biological fluid, or blood in the substrate antigen simultaneous detection biosensor described above.
  • the substrate antigen simultaneous detection biosensor of the present invention is characterized in that in the substrate antigen simultaneous detection biosensor described above, the enzyme is glucose oxidase or creatinase.
  • the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the antigen is albumin, a protein other than albumin, or a peptide that is a degradation product of the protein. To do.
  • the substrate antigen simultaneous detection biosensor of the present invention is characterized in that the conductive polymer is polypyrrolecarboxylic acid in the substrate antigen simultaneous detection biosensor described above.
  • the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the reflectance detection means uses the surface plasmon resonance method for the antigen-antibody reaction in the conductive polymer. Based on, detecting a change in the reflectivity of the light reflected by the electrode.
  • the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the current value detection means is based on the enzyme reaction in the conductive polymer using an electrochemical measurement method. And detecting a change in the current value on the conductive polymer.
  • the substrate antigen simultaneous detection biosensor of the present invention is the above-described substrate antigen simultaneous detection biosensor, wherein the analysis means for acquiring the analysis result based on the change in the current value and the change in the reflectance, and the analysis An analysis result output means for outputting the results is further provided.
  • the electrode of the present invention is characterized by depositing a conductive polymer in which an enzyme and an antibody are immobilized.
  • the substrate antigen simultaneous detection method of the present invention is based on the enzyme reaction in the conductive polymer on the electrode on which the conductive polymer on which the enzyme and the antibody are immobilized is deposited.
  • the program of the present invention provides a current value on the conductive polymer based on an enzyme reaction in the conductive polymer on the electrode on which the conductive polymer on which the enzyme and the antibody are fixed is deposited.
  • the change in the current value on the conductive polymer based on the enzyme reaction in the conductive polymer is detected, and the reflection of light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer is detected. Because it detects the change in rate, it can simultaneously measure quantitatively the enzyme substrate and antigen, which are different information that had to be measured separately, and shorten the measurement time. There is an effect.
  • the solution tank filled with the liquid in contact with the conductive polymer since the solution tank filled with the liquid in contact with the conductive polymer is provided, there is an effect that simple measurement can be performed.
  • the liquid contains body fluid, it is possible to simultaneously measure the main components in the body fluid that have been separately measured so far, and it can be applied to future simple body fluid sensors. Play.
  • the body fluid is urine, biological fluid, or blood
  • a biosensor that matches the purpose of use such as a simple urine sensor, biological fluid sensor, or blood sensor in the future. There is an effect that can be done.
  • the enzyme is glucose oxidase or creatinase
  • the amount of sugar such as urine sugar which is a main component in urine, can be quantitatively measured.
  • the antigen is albumin, a protein other than albumin, or a peptide that is a degradation product of protein
  • the amount of protein such as urine protein that is a major component of urine can be quantitatively measured. There is an effect.
  • the conductive polymer is polypyrrole carboxylic acid, it not only has stability, but also has an effect of facilitating electrochemical measurement and facilitating quantitative measurement.
  • the surface plasmon resonance method is used to detect a change in the reflectance of light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer, so that the optical measurement can be easily performed. Play.
  • the electrochemical measurement method is used to detect a change in the current value on the conductive polymer based on the enzyme reaction in the conductive polymer, so that the electrochemical measurement can be easily performed. Play.
  • the analysis result based on the change in the current value and the change in the reflectance is acquired and the analysis result is output, it is possible to promote the quick utilization of the disease diagnosis and sensing. There is an effect.
  • FIG. 1 is a flowchart showing the basic principle of this embodiment.
  • FIG. 2 is a block diagram showing an example of the configuration of the substrate antigen simultaneous detection biosensor in the present embodiment.
  • FIG. 3 is a block diagram illustrating an example of the configuration of the control unit in the present embodiment.
  • FIG. 4 is a flowchart showing an example of processing of the substrate antigen simultaneous detection biosensor of the present embodiment.
  • FIG. 5 is a diagram showing an example of antibody / glucose oxidase immobilization in the present embodiment.
  • FIG. 6 is a diagram showing an example of the surface plasmon dispersion relationship at the prism / gold thin film boundary / dielectric 10 nm interface in this embodiment, and an example of a reflectance theoretical calculation curve using the Fresnel equation.
  • FIG. 1 is a flowchart showing the basic principle of this embodiment.
  • FIG. 2 is a block diagram showing an example of the configuration of the substrate antigen simultaneous detection biosensor in the present embodiment.
  • FIG. 3 is
  • FIG. 7 is a diagram illustrating an example of simultaneous antigen / glucose detection in the present embodiment.
  • FIG. 8 is a diagram illustrating an example of current value changes detected simultaneously in the present embodiment.
  • FIG. 9 is a diagram showing an example of the reflectance change detected simultaneously in the present embodiment.
  • FIG. 1 is a flowchart showing the basic principle of this embodiment.
  • the present embodiment schematically has the following basic features.
  • the user injects (sets) a liquid to be a specimen (sample) into the substrate antigen simultaneous detection biosensor (step SA-1).
  • the substrate antigen simultaneous detection biosensor is a current value on the conductive polymer based on an enzyme reaction in the conductive polymer on the electrode on which the conductive polymer on which the enzyme and the antibody are fixed is deposited. Is detected (electrochemical detection) (step SA-2).
  • the substrate antigen simultaneous detection biosensor detects (optical detection) a change in the reflectance of the light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer (step SA-3), and ends the processing. To do. Note that step SA-2 and step SA-3 may be executed at the same time, and either may be executed first.
  • FIG. 2 is a block diagram showing an example of the configuration of the substrate antigen simultaneous detection biosensor 100 in the present embodiment, and conceptually shows only the portion related to the present invention in the configuration.
  • the substrate antigen simultaneous detection biosensor 100 schematically includes a substrate 10, a working electrode 20, a potentiostat 30, a counter electrode 31, a reference electrode 32, a solution tank 40, a glass prism 50, and a laser oscillator 51. , A polarizer 52, and a detector 53.
  • the substrate 10 forms the working electrode 20 on the surface, and is made of metal, glass, plastic, metal oxide, or the like.
  • the working electrode 20 is an electrode on which a conductive polymer 21 to which an enzyme 22 and an antibody 23 are fixed is deposited (see FIG. 5).
  • the enzyme 22 may be glucose oxidase, creatinase, or the like.
  • the antigen acting on the antibody 23 may be albumin, a protein other than albumin, a peptide that is a degradation product of protein, creatinine, or the like.
  • the conductive polymer 21 may be polypyrrole carboxylic acid (PP3C) or the like.
  • the working electrode 20 may be composed of a gold thin film or the like. The conductive polymer 21 on the working electrode 20 may be in contact with the liquid in the solution tank 40.
  • the potentiostat 30 is a current detector that is connected to the working electrode 20, the counter electrode 31, and the reference electrode 32 and measures a current (current value) between the working electrode 20 and the counter electrode 31.
  • the potentiostat 30 may make the potential of the working electrode 20 constant with respect to the reference electrode 32. Further, the potentiostat 30 may be configured so that no current flows through the reference electrode 32.
  • the counter electrode 31 may be platinum, gold, carbon, mercury, or the like.
  • the reference electrode 32 may be a hydrogen electrode or a silver-silver chloride electrode.
  • the solution tank 40 is a container filled with a liquid in contact with the working electrode 20.
  • the liquid may include a body fluid.
  • the body fluid may be urine, biological fluid, or blood.
  • the liquid in the solution tank 40 may be in contact with the counter electrode 31 and the reference electrode 32.
  • the glass prism 50 is in contact with the surface of the substrate 10 where the working electrode 20 is not provided.
  • the dielectric constant ( ⁇ p) of the glass prism 50 may be larger than the dielectric constant ( ⁇ d) of the liquid in the solution tank 40.
  • the laser oscillator 51 is a device that generates laser light.
  • the laser oscillator 51 may irradiate the working electrode 20 with a laser.
  • the polarizer 52 creates linearly polarized light from natural light or circularly polarized light.
  • the polarizer 52 may be installed between the laser oscillator 51 and the glass prism 50.
  • the detector 53 is a reflected light detector that detects the light reflected by the working electrode 20 and / or the reflectance of the light.
  • the substrate antigen simultaneous detection biosensor 100 may further include a control unit 102, a storage unit 106, an input / output unit 112, an input / output interface unit, and a communication interface unit. Good. Each part of these substrate antigen simultaneous detection biosensors 100 is communicably connected via an arbitrary communication path.
  • FIG. 3 is a block diagram illustrating an example of the configuration of the control unit 102 in the present embodiment.
  • the control unit 102 has an internal memory for storing a control program such as an OS (Operating System), a program defining various processing procedures, and necessary data. And the control part 102 performs the information processing for performing various processes by these programs.
  • the control unit 102 may control the input / output unit 112, the input / output interface unit, and the communication interface unit.
  • control unit 102 includes a current value detection unit 102a, a reflectance detection unit 102b, an analysis unit 102c, and an analysis result output unit 102d in terms of functional concept.
  • the current value detection unit 102a is current value detection means for detecting a change in current value on the conductive polymer 21 based on an enzyme reaction in the conductive polymer 21.
  • the current value detection unit 102a may detect a change in the current value on the conductive polymer 21 based on an enzyme reaction in the conductive polymer 21 using an electrochemical measurement method.
  • the current value detection unit 102a may be provided in the potentiostat 30.
  • the reflectance detector 102b is a reflectance detector that detects a change in reflectance of light reflected by the working electrode 20 based on an antigen-antibody reaction in the conductive polymer 21.
  • the reflectance detection unit 102b may detect a change in reflectance of light reflected by the working electrode 20 based on an antigen-antibody reaction in the conductive polymer 21 by using a surface plasmon resonance method.
  • the reflectance detection unit 102b may be provided in the detector 53.
  • the analysis unit 102c is an analysis unit that acquires an analysis result based on a change in current value and a change in reflectance.
  • the analysis unit 102c may acquire an analysis result regarding the substrate of the enzyme 22 based on the change in the current value and the amount of the antigen based on the change in the reflectance.
  • the analysis result output unit 102d is an analysis result output unit that outputs an analysis result.
  • the analysis result output unit 102 d may output the analysis result via the input / output unit 112.
  • the storage unit 106 is a device that stores various databases and tables.
  • the various databases and tables stored in the storage unit 106 are storage means such as a fixed disk device.
  • the storage unit 106 stores various programs, tables, files, databases, web pages, and the like used for various processes.
  • the storage unit 106 may store a current value, a change in the current value, a light reflectance, a light reflectance change, and / or an analysis result.
  • the input / output unit 112 performs data input / output (I / O).
  • the input / output unit 112 may be, for example, a key input unit, a touch panel, a control pad (for example, a touch pad and a game pad), a mouse, a keyboard, and a microphone.
  • the input / output unit 112 may be a display unit that displays a display screen of an application or the like (for example, a display, a monitor, a touch panel, or the like including a liquid crystal or an organic EL).
  • the input / output unit 112 may be an audio output unit (for example, a speaker) that outputs audio information as audio.
  • the input / output interface unit is an interface connected to the input / output unit 112.
  • the input / output interface unit connects the control unit 102 and the input / output unit 112 and controls the input / output unit 112.
  • the communication interface unit is an interface connected to a communication device such as a router connected to a communication line or the like.
  • the communication interface unit performs communication control between the substrate antigen simultaneous detection biosensor 100 and a network (or a communication device such as a router). That is, the communication interface unit has a function of communicating data with an external system and other terminals via a communication line.
  • the substrate antigen simultaneous detection biosensor 100 is communicably connected to a network (such as the Internet) via a communication device such as a router and a wired or wireless communication line such as a dedicated line.
  • FIG. 4 is a flowchart showing an example of processing of the substrate antigen simultaneous detection biosensor 100 in the present embodiment.
  • the user puts a specimen (sample) in the solution tank 40 of the substrate antigen simultaneous detection biosensor 100 filled with a liquid (buffer solution) in contact with the conductive polymer 21 on the working electrode 20.
  • a body fluid is injected (step SB-1).
  • the body fluid may be urine, biological fluid, or blood.
  • FIG. 5 is a diagram showing an example of antibody / glucose oxidase immobilization in the present embodiment.
  • a conductive polymer (polypyrrolecarboxylic acid (PP3C) thin film is formed on the working electrode (gold thin film) 20 by electrolytic polymerization.
  • P3C polypyrrolecarboxylic acid
  • 21 was deposited (step SC-1). That is, in this sample preparation, the conductive polymer 21 having a carboxyl group is deposited on the working electrode 20 by an electrolytic polymerization method.
  • the electrolytic polymerization may be performed by a cyclic voltammetry method (for example, 0.1 M pyrrolecarboxylic acid (P3C) in a 0.5 M sulfuric acid aqueous solution is subjected to a potential range of 0 V to 0.8 V and a sweep rate of 20 mV / 5 cycles may be performed with s).
  • a cyclic voltammetry method for example, 0.1 M pyrrolecarboxylic acid (P3C) in a 0.5 M sulfuric acid aqueous solution is subjected to a potential range of 0 V to 0.8 V and a sweep rate of 20 mV / 5 cycles may be performed with s).
  • the PP3C thin film 21 was once again deposited by electrolytic polymerization under the same conditions (Step SC-3). That is, in this sample preparation, the conductive polymer 21 having a carboxyl group is further deposited by electrolytic polymerization.
  • the carboxyl group of the newly deposited PP3C thin film 21 is activated using 0.4 MEDC / 0.1 MNHS, and the antibody (Anti-IgG) 23 is immobilized on the activated site. (Step SC-4). That is, in this sample preparation, the antibody 23 is immobilized for antigen detection.
  • the antibody 23 that causes an adsorption reaction specifically with the antigen and the glucose oxidase 22 necessary for the detection of glucose are simultaneously immobilized in the conductive polymer 21.
  • pyrrole having a carboxyl group is electropolymerized in an electrolyte solution, A polypyrrole dielectric may be deposited on the gold electrode.
  • the antibody 23 may be immobilized after the carboxyl group is activated, glucose oxidase is immobilized, and the polypyrrole dielectric is deposited and activated.
  • the current value detection unit 102 a uses the current (current value) between the working electrode 20 and the counter electrode 31 measured by the potentiostat 30 by using an electrochemical measurement method to detect the antibody 23.
  • a change in the current value on the conductive polymer 21 based on the enzyme reaction in the conductive polymer 21 to which is fixed is detected (electrochemical detection) (step SB-2).
  • the reflectance detection unit 102b irradiates the working electrode 20 from the laser oscillator 51 through the polarizer 52 through the glass prism 50 using the surface plasmon resonance method, and is reflected by the working electrode 20 to be reflected by the glass prism 50.
  • the reflectance of the light detected by the detector 53 based on the light reaching through A change in reflectance is detected (optical detection) (step SB-3).
  • the enzyme 22 may be glucose oxidase, creatinase, or the like.
  • step SB-2 and step SB-3 may be executed at the same time, either of which may be executed first.
  • FIG. 6 is a diagram showing an example of the surface plasmon dispersion relationship at the prism / gold thin film boundary / dielectric 10 nm interface in this embodiment, and an example of a reflectance theoretical calculation curve using the Fresnel equation.
  • the surface plasmon resonance excitation measurement method (surface plasmon resonance method) is a measurement method capable of obtaining optical information of the metal thin film / dielectric interface with high sensitivity.
  • the surface plasmon resonance method combined with the electrochemical measurement in this embodiment is that the metal thin film used for surface plasmon excitation can be used as a working electrode in the electrochemical measurement at the same time, so that the surface of the metal thin film Optical information and electrochemical information can be obtained simultaneously.
  • the surface plasmon resonance method combined with the electrochemical measurement method in this embodiment is a very effective measurement that can detect both light and electrochemical signals with high sensitivity in sensing mediated by a conductive polymer. It is also a law.
  • an electromagnetic wave and an evanescent wave (surface plasmon wave) 1 that attenuates exponentially in the z direction and propagates in the x direction exist near the surface of the prism 50.
  • This evanescent wave 1 can be detected experimentally using a near-field microscope and can be derived theoretically from Maxwell's equations.
  • a sufficiently thin noble metal thin film 45 to 50 nm
  • the evanescent wave 1 oozes out to the metal / liquid interface.
  • the wave vector obtained from the dispersion relation of surface plasmons in the metal is always larger than the wave vector of incident light from the air, the wave vector of incident light can be increased by passing through the prism 50 having a large refractive index. It becomes possible.
  • the surface plasmon when the incident angle is changed and the x component of the wave number vector of the incident light coincides with the wave number vector of the surface plasmon localized in the metal, the surface plasmon is resonantly excited.
  • the metal thin film serves as the working electrode 20.
  • the thin film of the conductive polymer 21 is also actively applied to a biosensor or a chemical sensor.
  • the reversible doping and dedoping of the conductive polymer 21 is performed electrochemically, causing a metal-insulator transition by transferring electrons to and from the electrode, and its dielectric constant is large. In many cases, the optical characteristics change greatly.
  • the working electrode 20 in this embodiment can also be used as a sensing sensitizing material for surface plasmon resonance characteristics by utilizing characteristic changes, and can be used for detection of both electrical (chemical) signals and optical signals. It can be used.
  • FIG. 7 is a diagram illustrating an example of simultaneous antigen / glucose detection in the present embodiment.
  • an antibody 23 and an enzyme (glucose oxidase) 22 are immobilized on a gold electrode on which a conductive polymer 21 having a carboxyl group is deposited. It is used as a working electrode 20 for electrochemical measurement and at the same time as a substrate for surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • two phenomena that occur on the working electrode 20 and the gold electrode that is the surface plasmon resonance substrate are detected as a current value and a change in light reflectance.
  • FIG. 8 is a diagram illustrating an example of current value changes detected simultaneously in the present embodiment.
  • FIG. 9 is a diagram showing an example of the reflectance change detected simultaneously in the present embodiment.
  • an antigen (5 ⁇ g / mL) and glucose (20 mM) are simultaneously injected into a solution tank (cell) 40 filled with PBS (Phosphate Buffered Saline).
  • PBS Phosphate Buffered Saline
  • the analysis unit 102c acquires the analysis result regarding the substrate of the enzyme 22 based on the change in the current value and the amount of the antigen based on the change in the reflectance (step SB-4).
  • the substrate may be glucose or creatinine.
  • the antigen may be albumin, a protein other than albumin, a peptide that is a degradation product of protein, creatinine, or the like.
  • the analysis result output unit 102d outputs the analysis result via the input / output unit 112 (step SB-5), and ends the process.
  • a doctor (or veterinarian) or the like refers to the analysis result that is output, so that various diseases or the like (for example, diabetes, gout, pregnancy) with respect to the human (or animal) that provided the specimen Diagnosis of heart disease, various lifestyle-related diseases, etc.) or health condition.
  • an antibody and glucose for detecting an antigen on a conductive polymer are detected.
  • Glucose oxidase is simultaneously immobilized.
  • the reaction between glucose and glucose oxidase does not change the refractive index on the surface of the conductive polymer, but involves movement of electrons.
  • the antigen-antibody reaction is not accompanied by electron transfer, but is accompanied by a change in refractive index because it is chemically bonded to the surface of the conductive polymer.
  • electrochemical information and optical information are obtained by electrochemical-surface plasmon resonance spectroscopy combining electrochemical measurement and surface plasmon resonance spectroscopy. Each is detected separately at the same time.
  • the glucose-glucose oxidase reaction promoted by the conductive polymer is performed by current detection, and the antigen-antibody reaction is performed by light detection by the surface plasmon resonance method. , It is possible to obtain information separately.
  • the substrate antigen simultaneous detection biosensor 100 performs processing in a stand-alone form
  • the substrate antigen simultaneous detection biosensor 100 is a client terminal (a separate housing from the substrate antigen simultaneous detection biosensor 100).
  • the processing result may be returned to the client terminal.
  • all or part of the processes described as being automatically performed can be performed manually, or the processes described as being performed manually can be performed. All or a part can be automatically performed by a known method.
  • each illustrated component is functionally conceptual and does not necessarily need to be physically configured as illustrated.
  • each device of the substrate antigen simultaneous detection biosensor 100 is assigned to a CPU (Central Processing Unit) and the CPU. It may be realized by a program that is interpreted and executed, or may be realized as hardware by wired logic.
  • the program is recorded on a non-transitory computer-readable recording medium including programmed instructions for causing a computer to execute the method according to the present invention, which will be described later. It is mechanically read by the detection biosensor 100. That is, in the storage unit 106 such as a ROM or an HDD (Hard Disk Drive), a computer program for giving instructions to the CPU in cooperation with an OS (Operating System) and performing various processes is recorded. This computer program is executed by being loaded into the RAM, and constitutes a control unit in cooperation with the CPU.
  • OS Operating System
  • the computer program may be stored in an application program server connected to the substrate antigen simultaneous detection biosensor 100 via an arbitrary network, and may be downloaded in whole or in part as necessary. Is also possible.
  • the program according to the present invention may be stored in a computer-readable recording medium, or may be configured as a program product.
  • the “recording medium” includes a memory card, USB memory, SD card, flexible disk, magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, DVD, and Blu-ray (registered trademark). It includes any “portable physical medium” such as Disc.
  • program is a data processing method described in an arbitrary language or description method, and may be in any form such as source code or binary code.
  • program is not necessarily limited to a single configuration, but is distributed in the form of a plurality of modules and libraries, or in cooperation with a separate program typified by an OS (Operating System). Including those that achieve the function.
  • OS Operating System
  • a well-known configuration and procedure can be used for a specific configuration for reading a recording medium, a reading procedure, an installation procedure after reading, and the like in each device described in the embodiment.
  • Various databases and the like stored in the storage unit 106 are storage devices such as a memory device such as a RAM and a ROM, a fixed disk device such as a hard disk, a flexible disk, and an optical disk. Programs, tables, databases, web page files, and the like.
  • the substrate antigen simultaneous detection biosensor 100 may be configured by connecting an arbitrary peripheral device to a known device.
  • the substrate antigen simultaneous detection biosensor 100 may be realized by installing software (including a program, data, and the like) that realizes the method of the present invention in a known apparatus.
  • the specific form of distribution / integration of the devices is not limited to that shown in the figure, and all or a part of them may be functional or physical in arbitrary units according to various additions or according to functional loads. Can be distributed and integrated. In other words, the above-described embodiments may be arbitrarily combined and may be selectively implemented.
  • a substrate antigen simultaneous detection bio that can simultaneously and quantitatively and easily measure a substrate such as a sugar and an antigen such as a protein in a fluid such as a body fluid. Since a sensor, an electrode, a substrate antigen simultaneous detection method, and a program can be provided, it is extremely useful particularly in various fields such as medicine, pharmaceuticals, drug discovery, and biological research.

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Abstract

Le procédé selon la présente invention consiste à détecter un changement dans une valeur de courant sur un polymère électroconducteur, ladite modification étant basée sur une réaction enzymatique dans le polymère électroconducteur auquel une enzyme et un antigène sont immobilisés, et à détecter un changement dans la réflectance de la lumière réfléchie par une électrode de travail, ledit changement étant basé sur une réaction antigène-anticorps dans le polymère électroconducteur.
PCT/JP2015/075310 2014-09-19 2015-09-07 Biocapteur pour la détection simultanée d'un substrat et d'un antigène, électrode, procédé de détection simultanée de substrat et d'antigène, et programme WO2016043078A1 (fr)

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