US20190128882A1 - Biosensor and biochip - Google Patents

Biosensor and biochip Download PDF

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US20190128882A1
US20190128882A1 US16/089,007 US201716089007A US2019128882A1 US 20190128882 A1 US20190128882 A1 US 20190128882A1 US 201716089007 A US201716089007 A US 201716089007A US 2019128882 A1 US2019128882 A1 US 2019128882A1
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oxide
protective film
group
film
region
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Susumu Haratani
Sachio TSUBOIKE
Sumiko Kitagawa
Takashi Kikukawa
Haruki YUGA
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TDK Corp
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TDK Corp
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Assigned to TDK CORPORATION reassignment TDK CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HARATANI, SUSUMU, KIKUKAWA, TAKASHI, KITAGAWA, SUMIKO, TSUBOIKE, SACHIO, YUGA, HARUKI
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/0656—Investigating concentration of particle suspensions using electric, e.g. electrostatic methods or magnetic methods
    • 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
    • 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
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • 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
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
    • G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • G01R33/098—Magnetoresistive devices comprising tunnel junctions, e.g. tunnel magnetoresistance sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/12—Measuring magnetic properties of articles or specimens of solids or fluids
    • G01R33/1269—Measuring magnetic properties of articles or specimens of solids or fluids of molecules labeled with magnetic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/629—Detection means characterised by use of a special device being a microfluidic device
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/02—Measuring direction or magnitude of magnetic fields or magnetic flux
    • G01R33/06—Measuring direction or magnitude of magnetic fields or magnetic flux using galvano-magnetic devices
    • G01R33/09—Magnetoresistive devices
    • G01R33/093—Magnetoresistive devices using multilayer structures, e.g. giant magnetoresistance sensors

Definitions

  • the present disclosure relates to a biosensor and a biochip.
  • a magnetoresistance effect element such as a giant magnetoresistance effect (GMR) element, a magnetic tunnel junction (TMR) element, and an anisotropic magnetoresistance effect (AMR) element is often used (for example, refer to Published Japanese Translation No. S/H 2005-513475 of the PCT International Publication and Japanese Unexamined Patent Application, First Publication No. 2008-039782).
  • GMR giant magnetoresistance effect
  • TMR magnetic tunnel junction
  • AMR anisotropic magnetoresistance effect
  • a magnetoresistance effect element is an element whose output resistance value changes according to an input magnetic field, and it is possible to measure a change in the detected magnetic field on the basis of the output resistance value.
  • FIG. 6 and FIG. 7 are diagrams for explaining a biosensor 500 of the related art.
  • the biosensor 500 includes a substrate 101 , a magnetoresistance effect element 102 , a protective film 107 , and a biomolecule capturing layer 109 that captures target biomolecules in that order.
  • a magnetic field is then horizontally applied (an applied magnetic field 105 )
  • a stray magnetic field 111 is generated from magnetic beads 104 and the stray magnetic field 111 is input to the magnetoresistance effect element 102 .
  • FIG. 7 is a diagram showing details of the magnetoresistance element 102 of the related art used in the biosensor 500 of the related art. As shown in FIG. 7 , the magnetoresistance effect element 102 has a meander structure with sets of three lines.
  • the present disclosure has been made in view of the above circumstances and provides a biosensor through which a measurement error due to magnetic beads present both on thin lines and between thin lines of a magnetoresistance effect element having a meander structure is avoided and it is possible to detect biomolecules in a sample with high accuracy.
  • the inventors conducted extensive studies in order to address the above problem, and as a result, found that, when an adsorption prevention film is disposed between thin lines of a magnetoresistance effect element, a measurement error due to magnetic beads present both on thin lines and between thin lines of a magnetoresistance effect element can be avoided, and thereby completed the present disclosure.
  • a biosensor according to a first aspect of the present disclosure is a biosensor for detecting biomolecules in a sample, the biosensor including:
  • a substrate having a surface in which a first region and a second region disposed adjacent to the first region are formed
  • a magnetoresistance effect element that is disposed at least on the first region and is configured for a detected resistance value to be changed based on an input magnetic field
  • a protective film that is disposed on both the first region and the second region, covers a surface of the magnetoresistance effect element, is disposed on the top part of the first region and contains an affinity substance capable of recognizing the biomolecule on the outer surface of the first region exclusively;
  • an adsorption prevention film that is disposed on at least the top part of the second region and is substantially free of the affinity substance
  • the protective film and the adsorption prevention film are made of different materials.
  • FIG. 1 is a perspective view schematically showing a biosensor according to a first embodiment of the present disclosure.
  • FIG. 2 is a cross-sectional view schematically showing the biosensor according to the first embodiment of the present disclosure.
  • FIG. 3 is a cross-sectional view schematically showing a biosensor according to a second embodiment of the present disclosure.
  • FIG. 4 is a cross-sectional view schematically showing a biosensor according to a third embodiment of the present disclosure.
  • FIG. 5 is a cross-sectional view schematically showing a biosensor according to a fourth embodiment of the present disclosure.
  • FIG. 6 is a cross-sectional view of a magnetic detection type biosensor of the related art.
  • FIG. 7 is a perspective view of the magnetic detection type biosensor of the related art.
  • a biosensor includes a substrate, a magnetoresistance effect element, a protective film, and an adsorption prevention film.
  • the substrate has a surface in which a first area and a second area disposed adjacent to the first area are formed.
  • the magnetoresistance effect element is disposed on at least the first area and is configured such that a resistance value detected according to an input magnetic field varies.
  • the protective film is disposed on both the first area and the second area and covers a surface of the magnetoresistance effect element, and is disposed on the top part of the first area.
  • the protective film contains an affinity substance that allows recognition of the biomolecules on the outer surface only on the first area.
  • the adsorption prevention film is disposed on at least the top part of the second area and is substantially free of the affinity substance.
  • the protective film and the adsorption prevention film are made of different materials.
  • a measurement error due to magnetic beads present both on thin lines and between thin lines of the magnetoresistance effect element having a meander structure is avoided, and it is possible to detect biomolecules in a sample with high accuracy.
  • the “biosensor” refers to a sensor that detects biological materials (that may be naturally derived or chemically synthesized) such as enzymes, antigens, antibodies, and nucleic acids (including not only DNA, RNA, and the like but also artificial nucleic acids, for example, LNA).
  • biological materials that may be naturally derived or chemically synthesized
  • enzymes such as enzymes, antigens, antibodies, and nucleic acids (including not only DNA, RNA, and the like but also artificial nucleic acids, for example, LNA).
  • adsorption prevention film refers to a film for preventing biomolecules or magnetic beads from being adsorbed between thin lines of the magnetoresistance effect element having a meander structure.
  • the biosensor of the present embodiment includes an adsorption prevention film that is disposed on at least the top part of the second area and is substantially free of the affinity substance, a measurement error due to magnetic beads present both on thin lines and between thin lines of the magnetoresistance effect element having a meander structure is avoided, and it is possible to detect biomolecules in a sample with high accuracy.
  • substantially free of an affinity substance refers to a case in which no affinity substance is contained or a case in which, for example, when an affinity substance is nonspecifically adsorbed onto an adsorption prevention film, the affinity substance is contained only in an amount at which it is not possible to capture biomolecules.
  • FIG. 1 is a perspective view schematically showing a biosensor according to a first embodiment of the present disclosure.
  • a magnetoresistance effect element 12 has a meander structure with sets of three lines, and an adsorption prevention film 16 is disposed between thin lines of the magnetoresistance effect element 12 .
  • FIG. 2 is a cross-sectional view schematically showing the biosensor according to the first embodiment of the present disclosure, and is a cross-sectional view of the biosensor taken along the line X-X′ in FIG. 1 .
  • a first area, a second area, a first plane, and a second plane which will be described below, are introduced for convenience in order to define a positional relationship between members on an area or plane in a virtual area or plane.
  • the first area and the second area are alternately repeatedly provided.
  • the biosensor 100 of the present embodiment detects biomolecules in a sample.
  • the biosensor 100 includes a substrate 11 , the magnetoresistance effect element 12 , a protective film 17 , and the adsorption prevention film 16 .
  • the substrate 11 has a surface in which a first area A and a second area B disposed adjacent to the first area A are formed.
  • the magnetoresistance effect element 12 is disposed on at least the first area A and is configured such that a resistance value detected according to an input magnetic field varies.
  • the protective film 17 is disposed on both the first area A and the second area B and covers a surface of the magnetoresistance effect element 12 and is disposed on the top part of the first area A.
  • the protective film 17 contains an affinity substance 19 (hereinafter referred to as a “first affinity substance”) that allows recognition of biomolecules on the outer surface only on the first area A.
  • the adsorption prevention film 16 is disposed on at least the top part of the second area B and is substantially free of the affinity substance 19 .
  • the protective film 17 and the adsorption prevention film 16 are made of different materials.
  • the adsorption prevention film 16 is substantially free of the affinity substance 19 , and is made of a material different from that of a protective layer 17 .
  • the adsorption prevention film 16 is substantially free of the affinity substance 19 , and is made of a material different from that of a protective layer 17 .
  • FIG. 2 a configuration in which the adsorption prevention film 16 extends in a width direction (left-right direction of the plane of the paper) on the entire second area B is formed.
  • this configuration is preferable in consideration of ease of production.
  • the magnetic beads 14 contain a second affinity substance (not shown) that allows recognition of a part different from the biomolecule recognition part of the first affinity substance 19 .
  • the magnetic beads 14 accumulate on the protective film 17 via a first affinity substance-biomolecule-second affinity substance complex. Then, when a magnetic field is horizontally applied (an applied magnetic field 15 ), a stray magnetic field is generated from the magnetic beads 14 and a stray magnetic field is input to the magnetoresistance effect element 12 .
  • the surface of the magnetoresistance effect element 12 is covered with the protective film 17 , and the outer surface of the protective film 17 contains the first affinity substance 19 that captures biomolecules to be detected.
  • the magnetic beads 14 also contain a second affinity substance (not shown) that captures biomolecules.
  • the first affinity substance 19 and the second affinity substance allow recognition of different parts in biomolecules. That is, they can form a first affinity substance-biomolecule-second affinity substance complex.
  • the electrode terminal is disposed on a plane which is positioned away from the main surface of the substrate 11 and immediately above the magnetoresistance effect element 12 .
  • the electrode terminal is connected when it is disposed at a position in contact with the magnetoresistance effect element 12 .
  • FIG. 3 is a cross-sectional view schematically showing a biosensor according to a second embodiment of the present disclosure.
  • components that are the same as those shown in the drawings explained above are denoted with the same reference numerals as in the drawings explained above, and details thereof will not be described.
  • a biosensor 200 is the same as the biosensor 100 shown in FIG. 1 except that a protective film is composed of a plurality of films. That is, in the biosensor 200 , a second protective film 20 is laminated on one surface of the substrate 11 . In addition, the magnetoresistance effect element 12 is disposed on a first plane a in the second protective film 20 . In addition, the second protective film 20 is laminated on a second plane b which is a surface of the magnetoresistance effect element 12 . In addition, the protective film 17 is laminated on both the first area A and the second area B on the surface of the second protective film 20 . In addition, the adsorption prevention film 16 is laminated on the surface of the protective film 17 on the second area B.
  • the surface of the protective film 17 on the first area A contains the affinity substance 19 .
  • the second protective film 20 and the protective film 17 are laminated on both the first area A and the second area B on the magnetoresistance effect element 12 in that order.
  • the adsorption prevention film 16 is laminated on the surface of the protective film 17 on the second area B.
  • the adsorption prevention film 16 is substantially free of the affinity substance 19 , and the adsorption prevention film 16 , the protective film 17 and the second protective film 20 are made of different materials.
  • the biosensor 200 shown in FIG. 3 is used to detect biomolecules in a sample based on the same principle as in the biosensor 100 shown in FIG. 2 .
  • FIG. 4 is a cross-sectional view schematically showing a biosensor according to a third embodiment of the present disclosure.
  • a biosensor 300 is the same as the biosensor 200 shown in FIG. 3 except that an adsorption prevention film is disposed on both the first area A and the second area B. That is, in the biosensor 300 , the second protective film 20 is laminated on one surface of the substrate 11 . In addition, the magnetoresistance effect element 12 is disposed on the first plane a in the second protective film 20 . In addition, the second protective film 20 is laminated on the second plane b which is a surface of the magnetoresistance effect element 12 . In addition, the adsorption prevention film 16 is laminated on both the first area A and the second area B on the surface of the second protective film 20 .
  • the protective film 17 is laminated on the surface of the adsorption prevention film 16 on the first area A and the surface of the protective film 17 on the first area A contains the affinity substance 19 .
  • the adsorption prevention film 16 that is interposed between the second protective film 20 and the protective film 17 is disposed.
  • the adsorption prevention film is substantially free of the affinity substance 19 , and the adsorption prevention film 16 , the protective film 17 and the second protective film 20 are made of different materials.
  • the biosensor 300 shown in FIG. 4 is used to detect biomolecules in a sample based on the same principle as in the biosensor 100 shown in FIG. 2 .
  • FIG. 5 is a cross-sectional view schematically showing a biosensor according to a fourth embodiment of the present disclosure.
  • a biosensor 400 is the same as the biosensor 200 shown in FIG. 3 except that the adsorption prevention film 16 is disposed on the top part of the second area B and the protective film 17 is disposed on the top part of the first area A. That is, in the biosensor 400 , the second protective film 20 is laminated on one surface of the substrate 11 . In addition, the magnetoresistance effect element 12 is disposed on the first plane a in the second protective film 20 . In addition, the second protective film 20 is laminated on the second plane b which is a surface of the magnetoresistance effect element 12 . In addition, the adsorption prevention film 16 is laminated on the surface of the second protective film 20 on the second area B.
  • the protective film 17 is laminated on the surface of the second protective film 20 on the first area A.
  • the surface of the protective film 17 on the first area A contains the affinity substance 19 .
  • the adsorption prevention film 16 is disposed on the top part of the second area B, and the protective film 17 is disposed on the top part of the first area A.
  • the adsorption prevention film 16 is substantially free of the affinity substance 19 , and the adsorption prevention film 16 , the protective film 17 and the second protective film 20 are made of different materials.
  • the biosensor 400 shown in FIG. 5 is used to detect biomolecules in a sample based on the same principle as in the biosensor 100 shown in FIG. 2 .
  • the biosensor according to the present embodiment is not limited to those shown in FIGS. 1 to 5 , and a biosensor in which some components of those shown in FIGS. 1 to 5 are modified or deleted and a biosensor in which other components are additionally added to those described above may be used as long as effects thereof are not impaired.
  • the adsorption prevention film may be disposed on the entire top part in which there is no magnetoresistance effect element.
  • a semiconductor such as silicon and AlTiC or a conductor, or a material made of an insulator such as alumina or glass may be exemplified, and a form thereof is not particularly limited.
  • the thickness of the substrate is not particularly limited, but it may be, for example, 400 ⁇ m or more and 2000 ⁇ m or less. When the thickness of the substrate is in such a range, it is possible to obtain a thin and lightweight biosensor having an appropriate strength.
  • the thickness of the substrate refers to the thickness of the entire substrate.
  • the thickness of the substrate composed of a plurality of layers refers to the total thickness of all layers constituting the substrate.
  • the magnetoresistance effect element is not particularly limited as long as it is an element that uses a phenomenon in which a magnetic field influence is received and an electrical resistance changes.
  • An element of a type including a magnetization fixed layer having a magnetization direction fixed in a certain direction in the plane of the laminate and a magnetization free layer whose magnetization direction changes according to an external magnetic field is preferable.
  • a magnetization fixed direction of the magnetization fixed layer is substantially parallel or substantially antiparallel to a direction of a magnetic field (the applied magnetic field 15 ) applied for magnetic bead excitation, and is a film surface direction of the magnetoresistance effect element.
  • description including the terms substantially parallel or substantially antiparallel means approximately parallel or anti-parallel, and includes deviation within a range of 0.1° or more and 10° or less.
  • the magnetoresistance effect element includes a magnetization fixed layer, an intermediate layer made of a nonmagnetic conductor or an insulator, and a magnetization free layer, and preferably includes a laminate including the intermediate layer interposed between the magnetization fixed layer and the magnetization free layer.
  • the magnetoresistance effect element when the intermediate layer is made of a nonmagnetic conductor, the magnetoresistance effect element is generally called a giant magnetoresistance (GMR) effect element and when the intermediate layer is made of an insulator, the magnetoresistance effect element is called a tunnel type magnetoresistance (TMR) effect element.
  • GMR giant magnetoresistance
  • TMR tunnel type magnetoresistance
  • a resistance of the magnetoresistance effect element changes according to an angle between a magnetization direction of the magnetization fixed layer and an average magnetization direction of the magnetization free layer.
  • the magnetization direction of the magnetization fixed layer is defined as a magnetic sensing direction.
  • the magnetization free layer is made of, for example, a soft magnetic film of NiFe or the like.
  • the intermediate layer is made of, for example, a conductor film of Cu or the like or made of an insulator film of an alumina-magnesium oxide or the like.
  • the magnetization fixed layer is made of an antiferromagnetic film and a magnetization fixed film, and the magnetization fixed film is in contact with the intermediate layer.
  • the antiferromagnetic film is made of, for example, an antiferromagnetic Mn alloy such as IrMn and PtMn.
  • the magnetization fixed film is made of, for example, a ferromagnetic material such as CoFe and NiFe, or may have a configuration in which a Ru thin film layer is interposed between CoFe layers or the like.
  • Magnetic beads are not particularly limited as long as they are magnetic particles.
  • iron oxide particles may be exemplified.
  • the diameter of the magnetic beads depends on the balance with the area of the protective film.
  • the diameter is preferably 0.01 ⁇ m or more and 100 ⁇ m or less, more preferably 0.05 ⁇ m or more and 50 ⁇ m or less, and particularly preferably 0.1 ⁇ m or more and 5 ⁇ m or less.
  • the magnetic beads contain a second affinity substance that specifically binds to biomolecules, and capture biomolecules via the second affinity substance.
  • the magnetic beads may be magnetic beads to which a second affinity substance is added by a coating treatment or the like or magnetic beads composed of the second affinity substance itself.
  • the surface of the magnetic beads is coated with a polymer or silica matrix according to biomolecules to be captured.
  • the surface of the magnetic beads is preferably hydrophilic, and when antibodies are desired to be captured as biomolecules, the surface of the magnetic beads is preferably hydrophobic.
  • the protective film is not particularly limited as long as it can protect the magnetoresistance effect element.
  • the material of the protective film include oxides such as alumina, silica, titanium oxide, zirconium oxide, indium oxide, tartaric oxide, zinc oxide, gallium oxide, and tin oxide; noble metals such as gold, silver, platinum, rhodium, ruthenium, and palladium; and inorganic substances such as aluminum nitride, and silicon nitride, and organic substances such as a polyimide.
  • the protective film may be composed of one layer (single layer) or a plurality of layers such as two or more layers.
  • the plurality of layers may be the same as or different from each other, and combinations of the plurality of layers are not particularly limited.
  • the thickness of the protective film is preferably 1 nm or more and 1000 nm or less, more preferably 1 nm or more and 100 nm or less, and particularly preferably 1 nm or more and 15 nm or less.
  • the thickness of the protective film refers to the thickness of the entire protective film.
  • the thickness of the protective film composed of a plurality of layers refers to the total thickness of all layers constituting the protective film.
  • the outer surface of the protective film is a surface that comes in contact with biomolecules in a sample.
  • the outer surface contains a first affinity substance that specifically binds to biomolecules to be detected.
  • the magnetic beads also contain a second affinity substance that specifically binds to biomolecules.
  • biomolecules are fixed to the outer surface of the protective film via the first affinity substance only if there are biomolecules to be detected in a sample (in a specimen).
  • magnetic beads bind to biomolecules via the second affinity substance and thus the magnetic beads are fixed to the surface of the protective film.
  • biomolecules to be detected examples include nucleic acids (that may be naturally derived or chemically synthesized) such as DNA, mRNA, miRNA, siRNA, and artificial nucleic acids (for example, locked nucleic acid (LNA), bridged nucleic acid (BNA)); peptides such as ligands, cytokines, and hormones; proteins such as receptors, enzymes, antigens, and antibodies; cells, viruses, bacteria, and fungi.
  • nucleic acids that may be naturally derived or chemically synthesized
  • DNA mRNA
  • miRNA miRNA
  • siRNA siRNA
  • artificial nucleic acids for example, locked nucleic acid (LNA), bridged nucleic acid (BNA)
  • peptides such as ligands, cytokines, and hormones
  • proteins such as receptors, enzymes, antigens, and antibodies
  • cells viruses, bacteria, and fungi.
  • sample containing biomolecules to be detected examples include blood, serum, plasma, urine, buffy coat, saliva, semen, thoracic exudates, cerebrospinal fluids, tears, sputum, mucosa, lymph fluids, abdominal fluids, pleural effusion, amniotic fluids, bladder irrigation fluids, bronchoalveolar lavage fluids, cell extraction liquids, and cell culture supernatants.
  • the biomolecules to be detected may be biomolecules to be detected with which other biomolecules are complexed or biomolecules to be detected which are converted into other biomolecules.
  • a complex obtained by hybridizing DNA having biotin at the end with RNA (hereinafter referred to as an “RNA-DNA-biotin complex”) may be exemplified.
  • biotin is added to RNA by complexation, it can be specifically bound to streptavidin. Therefore, for example, when RNA or DNA that is hybridizable with a nucleic acid part in which RNA or DNA contained in the RNA-DNA-biotin complex is not hybridized is used as the first affinity substance, they are captured on the biosensor of the present embodiment.
  • streptavidin is used as the second affinity substance, it is possible to specifically detect the RNA-DNA-biotin complex.
  • nucleic acids complementary to the nucleic acids may be exemplified.
  • biomolecules to be detected are antigens
  • antibodies having affinity for antigens may be exemplified.
  • biomolecules to be detected are primary antibodies, antigens having affinity for primary antibodies and secondary antibodies may be exemplified.
  • biomolecules to be detected are cells, viruses, bacteria, fungi, or the like, antibodies that recognize antigens presented on surfaces thereof may be exemplified.
  • a first nucleic acid complementary to 10 bases at the 5′ end of miRNA may be exemplified as the first affinity substance and a second nucleic acid complementary to 10 bases at the 3′ end of miRNA may be exemplified as the second affinity substance.
  • first antibodies that recognize the antigen proteins may be exemplified as the first affinity substance
  • second antibodies that recognize the antigen proteins and have a different epitope from the first antibodies may be exemplified as the second affinity substance.
  • the antibody can be prepared, for example, by immunizing a rodent animal such as a mouse with a labeled peptide as an antigen. In addition, for example, this can be prepared by phage library screening.
  • the antibody may be an antibody fragment and Fv, Fab, scFv, and the like may be exemplified as the antibody fragment.
  • Biomolecules to be detected may be bound to magnetic beads in advance, and they may be brought into contact with the surface of the protective film as a sample.
  • any technology that has been developed in the past or will be developed in the future can be applied and any method may be used as long as it is possible to indirectly detect the presence of biomolecules to be detected by measuring magnetic beads.
  • an affinity substance for example, when a constituent material of the protective film disposed on the top part is a noble metal, a thiol group, an isothiocyanate group or a disulfide group derived from the affinity substance or introduced into the affinity substance forms a thiolate bond with the surface of the noble metal, and the affinity substance can be fixed.
  • a constituent material of the protective film disposed on the top part is an oxide
  • the functional group is not particularly limited as long as it is a group that can be covalently bonded or non-covalently bonded to the affinity substance.
  • a chemically active (that is, activated so that the reactivity with the first affinity substance becomes higher) group, a receptor group, and a ligand group may be exemplified.
  • the affinity substance may be modified so that a covalent bond or non-covalent bond with a functional group can be made.
  • an activated carboxyl-derived group a carboxyl group, an aldehyde group, an epoxy group, a vinyl sulfone group, a biotinyl group, a thiol group, an amino group, an isocyanate group, an isothiocyanate group, a hydroxyl group, an acrylate group, a maleimide group, a hydrazide group, an aminooxy group, an azide group, an amide group, a sulfonate group, avidin, streptavidin, and a metal chelate may be exemplified, but the present disclosure is not limited thereto.
  • an aldehyde group, an activated carboxyl-derived group, an epoxy group, and a vinylsulfone group are preferable and a biotinyl group having a high coupling constant is preferable.
  • an activated carboxyl-derived group is preferable in consideration of the balance between the reactivity with the amino group and the storage stability.
  • an aminooxy group or a hydrazide group is preferable.
  • phosphonic acid derivatives for example, alkylphosphonic acid, alkenylphosphonic acid, and phenylphosphonic acid may be exemplified.
  • phosphonic acid derivatives more specifically, vinylphosphonic acid (CH 2 ⁇ CH—PO 3 H 2 ), propene-1-phosphonic acid (CH 3 —CH ⁇ CH—PO 3 H 2 ), and propene-2-phosphonic acid (CH 2 ⁇ CH(CH 3 )—PO 3 H 2 ) may be exemplified and those into which a functional group is introduced may be used.
  • phosphonic acid derivatives having a functional group for example, 2,5-dicarboxyphenylphosphonic acid, 3,5-dicarboxyphenylphosphonic acid, and 2,5-bisphosphonoterephthalic acid may be exemplified.
  • silane coupling agent having a functional group for example, trimethoxysilylbenzoic acid, ⁇ -methacryloxypropyltrimethoxysilane, vinyltriacetoxysilane, vinyltrimethoxysilane, ⁇ -isocyanatopropyltriethoxysilane, ⁇ -glycidoxypropyltrimethoxysilane, ⁇ -(3,4-epoxycyclohexyl)ethyltrimethoxysilane, tris-(3-trimethoxysilylpropyl)isocyanurate, methacryloxypropyldimethylmethoxysilane, methacryloxypropyldimethylethoxysilane, methacryloxypropylmethyldimethoxysilane, methacryloxypropylmethyldiethoxysilane, methacryloxypropyltrimethoxysilane, methacryloxypropyltriethoxysilane, 3-mercaptopropy
  • a more specific method of fixing the affinity substance to the surface of the protective film can be determined by those skilled in the art using known methods according to a type of the affinity substance. For example, a method in which a solution containing an affinity substance is brought into contact with a protective film together with a silane coupling agent or phosphonic acid derivatives having a functional group that covalently bonds with the affinity substance may be exemplified.
  • a material constituting the protective film is an oxide and an affinity substance having an amino group is fixed via a silane coupling agent having a carboxyl group
  • a solution in which a first affinity substance and a silane coupling agent are mixed into a general buffer solution with a pH of 7.0 or more and 10.0 or less is incubated for a predetermined time
  • the amino group of the affinity substance and the carboxyl group of the silane coupling agent react to form an amide bond
  • the silane coupling agent and the surface of the protective film react to form an ether bond, and thus the affinity substance can be fixed to the outer surface of the protective film.
  • the buffer solution include a phosphate buffer solution, and a tris buffer solution.
  • the adsorption prevention film is not particularly limited as long as it can prevent biomolecules or magnetic beads from being adsorbed between thin lines of the magnetoresistance effect element having a meander structure.
  • Examples of the material of the adsorption prevention film include oxides such as alumina, silica, titanium oxide, zirconium oxide, indium oxide, tartaric oxide, zinc oxide, gallium oxide, and tin oxide; and inorganic substances of noble metals such as gold, silver, platinum, rhodium, ruthenium, and palladium.
  • a material constituting the adsorption prevention film is preferably an oxide.
  • a material constituting the adsorption prevention film is preferably a noble metal.
  • the affinity substance is selectively fixed only to the protective film.
  • the adsorption prevention film is substantially free of the affinity substance, and nonspecific adsorption of biomolecules or magnetic beads on the adsorption prevention film is reduced.
  • a measurement error due to magnetic beads present both on thin lines and between thin lines of the magnetoresistance effect element having a meander structure is avoided, and it is possible to detect biomolecules in a sample with high accuracy.
  • the adsorption prevention film may be composed of one layer (single layer) or a plurality of layers such as two or more layers.
  • the adsorption prevention film is composed of a plurality of layers, the plurality of layers may be the same as or different from each other, and combinations of the plurality of layers are not particularly limited.
  • the thickness of the adsorption prevention film is preferably 1 nm or more and 1000 nm or less, more preferably 1 nm or more and 100 nm or less, and particularly preferably 1 nm or more and 15 nm or less.
  • the thickness of the adsorption prevention film refers to the thickness of the entire adsorption prevention film.
  • the thickness of the adsorption prevention film composed of a plurality of layers refers to the total thickness of all layers constituting the adsorption prevention film.
  • the adsorption prevention film preferably contains a substance (nonspecific adsorption inhibiting substance) that reduces nonspecific adsorption of biomolecules on the outer surface.
  • a substance nonspecific adsorption inhibiting substance
  • the nonspecific adsorption inhibiting substance may be any monomer or polymer as long as it has a fixing group on the adsorption prevention film at an end and has a biocompatible group at the other end or in a compound.
  • examples of the fixing group include a thiol group, an isothiocyanate group, and a disulfide group.
  • examples of the fixing group include an alkoxysilane group and a phosphonic acid group.
  • the biocompatible group has an excellent nonspecific adsorption inhibiting effect.
  • a biocompatible group specifically, a phosphorylcholine group, (poly)alkylene glycol residues, a sulfoalkyl amino group, and the like may be exemplified.
  • a nonspecific adsorption inhibiting substance in the present embodiment for example, a polymer compound having a biocompatible group that is produced by polymerizing monomers having such a biocompatible group may be produced and used.
  • the nonspecific adsorption inhibiting substance is a monomer
  • the nonspecific adsorption inhibiting substance is subjected to molecular self assembly (MSA) and forms a monomolecular film.
  • MSA molecular self assembly
  • molecular self assembly means that tissues or structures are naturally formed by molecules themselves without being controlled by external factors.
  • nonspecific adsorption inhibiting substance in the present embodiment a weak intermolecular bond such as Van der Waals coupling is used, nonspecific adsorption inhibiting substances are aligned and bonded to form one monomolecular film (self-assembled monolayers (SAMs)).
  • SAMs self-assembled monolayers
  • Examples of the monomer having a phosphorylcholine group include (meth)acryloyloxyalkylphosphorylcholines such as 2-methacryloyloxyethyl phosphorylcholine and 6-methacryloyloxyhexylphosphorylcholine; (meth)acryloyloxyalkoxyalkylphosphorylcholines such as 2-methacryloyloxyethoxyethyl phosphorylcholine and 10-methacryloyloxyethoxy nonyl phosphorylcholine; and alkenyl phosphorylcholines such as allyl phosphorylcholine, butenylphosphorylcholine, hexenyl phosphorylcholine, octenyl phosphorylcholine, and decenyl phosphorylcholine.
  • the nonspecific adsorption inhibiting substance of the present embodiment those in which the fixing group is introduced into an end opposite to the phosphocholine group may be used.
  • alkylene glycol residue refers to an alkyleneoxy group (—R—O—, here, R denotes an alkylene group) which remains after hydroxyl groups at one end or both ends of an alkylene glycol (HO—R—OH, here, R denotes an alkylene group) undergo a condensation reaction with another compound.
  • HO—CH 2 —OH alkylene glycol
  • the alkylene glycol residue is a methyleneoxy group (—CH 2 —O—)
  • ethylene glycol HO—CH 2 CH 2 —OH
  • polyalkylene glycol residue refers to a structure in which a plurality of alkyleneoxy groups are repeated.
  • Examples of the monomer having an alkylene glycol residue include methoxy polyethylene glycol (meth)acrylate, ethoxypolyethylene glycol (meth)acrylate, 2-hydroxyethyl (meth)acrylate and monosubstituted esters of its hydroxyl group, 2-hydroxypropyl (meth)acrylate and monosubstituted esters of its hydroxyl group, 2-hydroxybutyl (meth)acrylate and monosubstituted esters of its hydroxyl group, glycerol mono (meth)acrylate, (meth)acrylate having polypropylene glycol as side chain, 2-methoxyethyl (meth)acrylate, 2-ethoxyethyl (meth)acrylate, methoxydiethylene glycol (meth)acrylate, ethoxydiethyleneglycol (meth)acrylate, and ethoxypolyethylene glycol (meth)acrylate.
  • the average number of repetitions of the alkylene glycol residue is preferably
  • nonspecific adsorption inhibiting substance of the present embodiment those in which the fixing group is introduced into any end of the monomer having the alkylene glycol residue may be used.
  • Examples of the monomer having a sulfoalkyl amino group include N-methyl-N-(3-sulfopropyl)acrylamide, 3-(N,N-dimethylmyristylammonio)propanesulfonic acid (3-(N,N-dimethylmyristylammonio)propanesulfonate, and SB3-14, myristyl sulfobetaine).
  • N-methyl-N-(3-sulfopropyl)acrylamide 3-(N,N-dimethylmyristylammonio)propanesulfonic acid (3-(N,N-dimethylmyristylammonio)propanesulfonate
  • SB3-14 myristyl sulfobetaine
  • a known method may be used according to a fixing type to be introduced.
  • the fixing group is a thiol group
  • at least one hydrogen bonded to carbon into which a fixing group is introduced is substituted with a halogen atom (for example, chlorine, bromine, iodine, etc.), and hydrogen sulfide is then reacted in the presence of alkali, and thus a thiol group can be introduced.
  • a halogen atom for example, chlorine, bromine, iodine, etc.
  • the biocompatible group may be introduced into the silane coupling agent or phosphocholine derivatives exemplified in the above ⁇ Method of fixing affinity substance to protective film>> using a known method.
  • nonspecific adsorption inhibiting substance of the present embodiment more specifically, for example, 3-[(11-mercaptoundecyl)-N,N-dimethylammonio] propanesulfonate which is a sulfobetaine type alkane thiol may be exemplified.
  • a method of fixing the nonspecific adsorption inhibiting substance to the surface of the adsorption prevention film for example, when a constituent material of the adsorption prevention film is a noble metal, a nonspecific adsorption inhibiting substance having a thiol group, an isothiocyanate group or a disulfide group as a fixing group may be used, the fixing group and the surface of the noble metal form a thiolate bond, and the nonspecific adsorption inhibiting substance can be fixed.
  • a constituent material of the adsorption prevention film is an oxide
  • a nonspecific adsorption inhibiting substance having an alkoxysilane group or a phosphonic acid group as a fixing group may be used and the fixing group and the surface of the oxide form an ether bond, and the nonspecific adsorption inhibiting substance can be fixed.
  • a more specific method of fixing the nonspecific adsorption inhibiting substance to the surface of the adsorption prevention film can be determined by those skilled in the art by a known method according to a constituent material of the adsorption prevention film. For example, a method in which a solution containing a nonspecific adsorption inhibiting substance is brought into contact with an adsorption prevention film may be exemplified.
  • a material constituting the protective film is an oxide and a nonspecific adsorption inhibiting substance having an alkoxysilane group is fixed
  • the surface of the adsorption prevention film that is in contact with a solution in which a nonspecific adsorption inhibiting substance is mixed into a general buffer solution with a pH of 7.0 or more and 10.0 or less is incubated for a predetermined time
  • the nonspecific adsorption inhibiting substance can be fixed to the surface of the adsorption prevention film via an ether bond.
  • the buffer solution include a phosphate buffer solution, and a tris buffer solution.
  • the electrode terminal may be disposed on the same plane as the magnetoresistance effect element or disposed on a plane different from the magnetoresistance effect element.
  • the electrode terminal is connected to the magnetoresistance effect element through contact therewith and can output a change in resistance of the magnetoresistance effect element as an output to the outside.
  • the electrode terminal when the electrode terminal is disposed on a plane different from the magnetoresistance effect element, it may be disposed directly above the magnetoresistance effect element, and connected to the magnetoresistance element through contact therewith (refer to FIG. 1 ), or it may be disposed directly below the magnetoresistance effect element and connected to the magnetoresistance element through contact therewith.
  • a conductive metal such as Au, Al, Ag, or Cu or an alloy thereof is preferably used as a material of the electrode terminal.
  • the insulating layer is formed on the main surface of the substrate and an electrical short circuit via the substrate can be prevented.
  • an inorganic substance such as alumina, aluminum nitride, silicon oxide, or silicon nitride or an organic substance such as a polyimide is preferably used as a material of the insulating layer.
  • a detection magnetic field (stray magnetic field) is input to the magnetoresistance effect element.
  • a direction of the applied magnetic field is preferably a direction crossing the main surface of the magnetoresistance effect element.
  • the applied magnetic field is not particularly limited, and is preferably 0.1 m tesla or more and 100 m tesla or less and more preferably 1 m tesla or more and 10 m tesla or less.
  • the detection magnetic field (stray magnetic field) is influenced by a proportion of magnetic beads occupying the main surface of the magnetoresistance effect element via the protective film. As the number of magnetic beads accumulated on the protective film increases, a detected resistance value changes. The number of magnetic beads accumulated on the protective film and the detected resistance value via the stray magnetic field are linearly correlated.
  • the titer for example, the number of biomolecules that the second affinity substance captures
  • the number of biomolecules accumulated on the protective film it is possible to calculate the number of biomolecules accumulated on the protective film.
  • the number of biomolecules contained in a sample can be calculated. In this manner, in the biosensor of the present embodiment, it is possible to secure quantitation of biomolecules in a sample with high accuracy.
  • the biosensor of the present embodiment has high sensitivity, and can perform detection at a level of tens of nanotesla. Specifically, it is possible to detect an increase or decrease of 10 with respect to 1500 magnetic beads. That is, a change of about 0.5% can be detected.
  • the biosensor of the present embodiment uses magnetic beads, it has higher sensitivity and a longer lifespan compared to fluorescence. Therefore, it is much better than a detection method such as ELISA.
  • the biosensor of the present embodiment can be produced using a known method according to sequential lamination so that it has a corresponding positional relationship among the above components.
  • a method of fixing an affinity substance to a protective film is the same as the ⁇ Method of fixing affinity substance to protective film>> described above.
  • a method of fixing a nonspecific adsorption inhibiting substance to an adsorption prevention film is the same as the ⁇ Method of fixing nonspecific adsorption inhibiting substance to adsorption prevention film>> described above.
  • the biosensor of the present embodiment can be used for, for example, a method of detecting biomolecules to be described below.
  • a sample containing biomolecules is brought into contact with a protective film, and the biomolecules accumulate on the protective film via the first affinity substance (Process 1).
  • magnetic beads are brought into contact with the protective film and accumulate on the protective film via the biomolecules (Process 2).
  • a magnetic field is applied in a direction crossing the main surface of the magnetoresistance effect element, a detection magnetic field is input to the magnetoresistance effect element, and a resistance value is detected (Process 3).
  • Process 1 is a process in which a sample containing biomolecules is brought into contact with a protective film and the biomolecule accumulate on the protective film via a first affinity substance.
  • the biosensor is preferably used in a microfluidic device.
  • Process 1 first, a sample containing biomolecules flow through a micro flow path.
  • the sample not particularly limited as long as it contains biomolecules to be detected.
  • a sample derived from a subject such as a person in whom onset of a disease was confirmed or a person in whom onset of a disease was suspected, or a sample derived from a subject such as a patient being treated for a disease may be exemplified.
  • a sample derived from a subject such as a person in whom onset of a disease was confirmed or a person in whom onset of a disease was suspected, or a sample derived from a subject such as a patient being treated for a disease
  • ⁇ Protective film may be used.
  • a sample may be caused to directly flow through the micro flow path.
  • miRNA is involved in onset and progress of cancer, cardiovascular diseases, neurodegenerative diseases, mental illness, chronic inflammatory diseases and the like.
  • nucleic acids such as genomic DNA, cDNA, Total RNA, mRNA, and rRNA including miRNA are to be detected, a nucleic acid is preferably extracted from the biological sample.
  • the extraction method is appropriately selected from conventional methods according to a type of nucleic acid.
  • Biomolecules in a sample which flows through a micro flow path are captured by the first affinity substance on the protective film and accumulate on the protective film.
  • the first affinity substance nucleic acids, antibodies, and the like may be exemplified as described above.
  • the biomolecules form a complex with the first affinity substance on the protective film according to hybridization, an antigen and antibody reaction, and the like.
  • the protective film is preferably washed using a buffer solution or the like. According to washing, impurities that are nonspecifically bound to the protective film can be removed and detection accuracy of the biomolecules can be improved.
  • the buffer solution include a phosphate buffer solution, and a tris buffer solution.
  • Process 2 is a process in which magnetic beads are brought into contact with a protective film and accumulate on the protective film via the biomolecules.
  • the magnetic beads contain a second affinity substance that captures biomolecules.
  • a first affinity substance-biomolecule-second affinity substance complex is formed on the protective film. That is, the magnetic beads containing the second affinity substance accumulate on the protective film.
  • the protective film is preferably washed with a buffer solution or the like as in Process 1. According to washing, the magnetic beads that are nonspecifically bound to the protective film can be removed, and detection accuracy of the biomolecule can be improved.
  • a buffer solution the same ones exemplified in [Process 1] may be exemplified.
  • Process 3 is a process in which a magnetic field is applied in a direction crossing the main surface of the magnetoresistance effect element, a detection magnetic field is input to the magnetoresistance effect element, and a resistance value is detected.
  • the detection magnetic field (stray magnetic field) is influenced by a proportion of magnetic beads occupying the main surface of the magnetoresistance effect element via the protective film. As the number of magnetic beads accumulated on the protective film increases, a detected resistance value increases.
  • Process 3 it is possible to quantify accurately the number of magnetic beads accumulated on the protective film. Then, based on the titer (for example, the number of biomolecules that the second affinity substance captures) of the second affinity substance included in the magnetic beads, it is possible to calculate the number of all biomolecules accumulated on the protective film. That is, according to the detection method of the present embodiment, it is possible to calculate the number of biomolecules contained in a sample. Therefore, when there is a positive correlation between the number of biomolecules in a sample and a disease state, if the number of biomolecules in the sample is successively calculated, it is possible to perform follow-up observation of the disease state.
  • the titer for example, the number of biomolecules that the second affinity substance captures
  • biosensor of the present embodiment a method of detecting biomolecules to be described below may be used.
  • a sample containing biomolecules and magnetic beads are mixed tighter and the biomolecules are captured by the magnetic beads via the second affinity substance (Process 4).
  • magnetic beads that have captured biomolecules are brought into contact with a protective film and the magnetic beads accumulate on the protective film via the biomolecules (Process 5).
  • a magnetic field is applied in a direction crossing the magnetoresistance effect element, a detection magnetic field is input to the magnetoresistance effect element, and a resistance value is detected (Process 3).
  • this method is the same as the method of detecting biomolecules including the above [Process 1] to [Process 3] except that, when a first affinity substance-biomolecule-second affinity substance complex is formed, a biomolecule-second affinity substance complex is formed in advance, description thereof will be omitted.
  • the biosensor of the present embodiment can be applied to a biochip.
  • the biochip of the present embodiment can comprehensively analyze properties of a sample.
  • biochip for example, a biochip for cancer diagnosis, a biochip for carcinoma diagnosis, and a biochip for detecting influenza virus may be exemplified.
  • a nucleic acid complementary to a nucleic acid derived from a cancer gene or a cancer inhibiting gene may be exemplified.
  • a nucleic acid complementary to the nucleic acid containing the mutation is preferable.
  • a gene group that encodes a growth factor such as sis a gene group that encodes a receptor type tyrosine kinase such as erbB, fms, and ret; a gene group that encodes a non-receptor type tyrosine kinase such as fes; a gene group that encodes a GTP/GDP binding protein such as ras; a gene group that encodes a serine/threonine kinase such as src, mos, and raf; a gene group that encodes a nuclear protein such as myc, myb, fos, jun, and erbA; a gene group that encodes a signal transducing adapter molecule such as crk; and a fusion gene such as Bcr-Abl may be exemplified.
  • a Ras-MAP kinase pathway-linked gene such as Shc, Grb2, Sos, MEK, Rho, and Rac genes
  • a phospholipase C gamma-protein kinase C pathway-linked gene such as PLCy and PKC
  • a PI3K-Akt pathway-linked gene such as PI3K, Akt, and Bad
  • a JAK-STAT pathway-linked gene such as JAK and STAT
  • a GAP-related pathway-linked gene such as GAP, p180, and p62
  • cancer inhibiting gene examples include RB, p53, WT1, NF1, APC, VHL, NF2, p16, p19, BRCA1, BRCA2, PTEN, and E cadherin gene.
  • the first affinity substance a substance that captures a protein which is a genetic product of the above genes, for example, an antibody (including an antibody fragment), an aptamer, a ligand, and a receptor may be used.
  • a first affinity substance provided on a protective film may be a nucleic acid complementary to a plurality of nucleic acids that are extracted from one type of cancer. That is, the biochip of the present embodiment may be a biochip for diagnosis of a specific type of cancer.
  • the target cancer is not particularly limited, and, for example, breast cancer (for example, invasive ductal carcinoma, noninvasive ductal carcinoma, Inflammatory breast cancer, etc.), prostate cancer (for example, hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (for example, pancreatic duct cancer, etc.), stomach cancer (for example, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), lung cancer (for example, non small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (for example, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor, etc.), rectal cancer (for example, gastrointestinal stromal tumor, etc.), small intestine cancer (for example, non-Hodgkin's lymphoma, gastrointestinal stromal tumor, etc.), small intestine cancer (for example, non
  • the biochip of the present embodiment when used, it is possible to predict the susceptibility/resistance of an anti-cancer agent. For example, it has been reported that, in the case of gefinitib which is an EGFR inhibitor, when EGFR in a test sample has an L858R mutation or G719X mutation, the mutation exhibits gefinitib susceptibility.
  • the biochip of the present embodiment as the first affinity substance provided on the protective film, a nucleic acid complementary to a nucleic acid derived from influenza virus or a carbohydrate chain to which influenza virus specifically binds may be exemplified. That is, the biochip of the present embodiment may be a biochip for detecting influenza virus.
  • a nucleic acid that recognizes a certain mutation site including a reported mutation and is fixed to a protective film may be exemplified.
  • an antibody that can specifically recognize A type, B type and C type viruses may be used as the first affinity substance.
  • influenza virus binds to a sialic acid residue when a cell is infected therewith, and since binding modes of a sialic acid to which virus can bind and a sugar differ according to a type of virus, as the first affinity substance, a sialic acid-containing carbohydrate chain to which A type, B type and C type viruses bind may be used.
  • sialic acid generally refers to a substance in which an amino group or a hydroxy group of a nine-carbon sugar neuraminic acid is substituted.
  • N-acetylneuraminic acid (Neu5Ac) acetylated in position 5
  • N-glycolylneuraminic acid (Neu5Gc) modified with a glycolic acid may be exemplified.
  • biochip of the present embodiment it is possible to detect infection of influenza virus at an early stage.
  • biochip of the present embodiment when used over time, it is possible to perform follow-up observation of a disease state after viral infection.
  • the biosensor of the present embodiment is used as a biochip, it is possible to perform diagnosis easily and rapidly, and it can be applied for, for example, cancer diagnosis, diagnosis for a specific type of cancer, diagnosis of a degree to which cancer has progressed, detection of influenza virus, identification of a type of influenza virus, and observation of a state of an influenza disease.

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