WO2017081979A1 - Detection method and analysis chip - Google Patents

Detection method and analysis chip Download PDF

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Publication number
WO2017081979A1
WO2017081979A1 PCT/JP2016/080356 JP2016080356W WO2017081979A1 WO 2017081979 A1 WO2017081979 A1 WO 2017081979A1 JP 2016080356 W JP2016080356 W JP 2016080356W WO 2017081979 A1 WO2017081979 A1 WO 2017081979A1
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analyte
ligand
flow path
analysis chip
detection method
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PCT/JP2016/080356
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French (fr)
Japanese (ja)
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茂樹 松本
恵子 小畑
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ウシオ電機株式会社
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    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a detection method and an analysis chip used therefor.
  • Japanese Patent Application Nos. 2015-204728, 2015-204729, and 2015-204730 include capillary tubes. Extracting plasma using force is disclosed.
  • a mixture of a component to be examined (antigen) and an antibody labeled with a fluorescent substance is introduced onto a test surface on which an antibody not labeled with the fluorescent substance is fixed.
  • a conjugate is formed in such a manner that the antigen is sandwiched between the antibody immobilized on the test surface and the antibody labeled with the fluorescent substance, and as a result, only the antibody labeled with the fluorescent substance forming the conjugate is evanescent. Excited by light component. Therefore, in this method, B / F separation is not necessary, but it is difficult to carry out in a microchannel because it requires uniform mixing of an antigen and an antibody labeled with a fluorescent substance. Accurate optical measurement results cannot be obtained with this sample.
  • the present invention has been made based on the above circumstances, and provides a detection method capable of accurately performing optical measurement of a test target component from a very small amount of sample and an analysis chip used therefor. Objective.
  • the detection method of the present invention introduces an analyte that reacts with the ligand to the test surface of the analysis chip having the test surface on which the labeled ligand is immobilized,
  • a detection method for detecting the concentration of the analyte by measuring the degree of optical change that occurs in the labeled ligand by binding the analyte to the labeled ligand,
  • the analyte is an antigen and the ligand is an antibody against the antigen;
  • the labeled ligand is in a quenching state labeled with a fluorescent dye, and emits fluorescence when bound to an analyte.
  • the analyte is preferably contained in plasma.
  • the analysis chip of the present invention is an analysis chip used in the above detection method, Having a test surface to which a labeled ligand is immobilized;
  • the labeled ligand is characterized by causing an optical change when an analyte that reacts with the ligand is bound.
  • the analysis chip of the present invention has a substrate made of a light transmissive material having a test surface on which the labeled ligand is fixed, It is preferable that the optical change is caused by an evanescent light component on the test surface.
  • the analysis chip of the present invention may be configured such that the test surface is a surface of a member that divides a measurement space communicating with a flow path to which a specimen is supplied on the substrate.
  • the analysis chip of the present invention comprises a plate-like body having a first flow path for circulating a liquid specimen and a second flow path formed by branching from the first flow path and communicating with the first flow path. Become The second flow path has a width capable of separating the test target component from the sample flowing through the first flow path, It is preferable that the inner wall surface of the second flow path is a test surface.
  • an analyte that reacts with a ligand is introduced into an analysis chip having a test surface on which the labeled ligand is immobilized, and the optical generated in the labeled ligand in a state where they are bound. Change is detected. Therefore, an optical change can be caused in the labeled ligand only by introducing the sample into the test surface without performing a separate operation. As a result, it is possible to accurately perform optical measurement of a test target component from a very small amount of sample.
  • FIG. 2 is an AA cross-sectional end view of the configuration of the analysis chip shown in FIG. 1.
  • FIG. 2 is an explanatory view showing a main part of the analysis chip shown in FIG. 1, wherein (a) is an enlarged plan view showing a first flow path, a micro flow path and a reservoir, and (b) is a B- It is B sectional end view.
  • FIG. 5 is a cross-sectional end view taken along the line CC of the structure of the analysis chip shown in FIG. 4. It is a top view which shows another example of a structure of the analysis chip of this invention in the state arrange
  • FIG. 7 is a DD cross-sectional end view of the configuration of the analysis chip shown in FIG. 6.
  • an analyte that reacts with the ligand is introduced to the test surface of the analysis chip having the test surface on which the labeled ligand is immobilized, and the analyte is bound to the labeled ligand.
  • the labeled ligand is in a quenching state labeled with a fluorescent dye, and emits fluorescence when bound to an analyte, and a change in fluorescence intensity generated in the labeled ligand This is a method for calculating the concentration of the analyte by measuring.
  • FIG. 1 is a plan view showing an example of the configuration of the analysis chip of the present invention
  • FIG. 2 is an end view taken along the line AA of the configuration of the analysis chip shown in FIG. 1
  • FIG. 3 is a schematic diagram of the analysis chip shown in FIG. 4A is an explanatory plan view showing an enlarged view of the first flow path, the micro flow path, and the reservoir
  • FIG. 4B is an end view taken along the line BB in FIG.
  • This analysis chip 10 is a bio used in a detection method in which a specific component such as plasma containing a test target component (analyte) is separated from a liquid sample such as blood and the concentration of the analyte is measured by optical measurement. It is a sensor chip.
  • the analysis chip 10 includes a chip base 11 including a first substrate 12 and a second substrate 15, and is configured by a plate-like body in which the first substrate 12 and the second substrate 15 are joined.
  • substrate 15 is not specifically limited, For example, they are 0.1 mm or more and 5.0 mm or less.
  • a material constituting the first substrate 12 of the chip base 11 a material having light absorption and self-adhesiveness, for example, a black resin material can be used.
  • light transmissive materials such as glass and a cycloolefin copolymer, which permeate
  • the analysis chip 10 includes a first flow path 20 through which a liquid sample is circulated, and a plurality of second flow paths (hereinafter referred to as “the first flow path 20”) into which a specific component separated from the sample flows. 25).
  • Each of the micro flow paths 25 is formed in a state where the micro flow paths 25 are branched from the first flow path 20 and extend in the vertical direction with respect to the first flow paths 20 and are spaced apart at equal intervals.
  • the upstream end of the first flow path 20 is connected to a sample storage unit 22 that stores the sample introduced from the sample introduction unit 21.
  • the downstream end of the first flow path 20 is connected to the first discharge part 23.
  • the downstream end of each of the micro flow paths 25 is connected to the accumulation portion 26.
  • One end of the storage part 26 is connected to the second discharge part 28.
  • the first flow path 20 is formed by being partitioned by the inner wall surface of the first flow path groove 13 a formed in the first substrate 12 and the second substrate 15.
  • the reservoir 26 is formed by being partitioned by the inner wall surface of the reservoir recess 13 b formed in the first substrate 12 and the second substrate 15.
  • the microchannel 25 is formed in a state where the first channel 12 and the reservoir recess 13b are communicated with the first substrate 12 and shallower than these depths. It is formed by being partitioned by the inner wall surface of the first substrate 12 and the first substrate 12.
  • the first flow path 20 has a width that allows a liquid specimen (for example, blood) to circulate.
  • the “width” of the flow channel means the smallest width of the flow channel in a cross section perpendicular to the direction in which the flow channel extends.
  • the width in the thickness direction of the analysis chip 10 is the smallest width.
  • the width of the first flow path 20 is preferably 10 ⁇ m or more and 1000 ⁇ m or less, more preferably 50 ⁇ m or more and 300 ⁇ m or less, and particularly preferably 100 ⁇ m.
  • the width of the first flow path 20 is too small, the amount of a specific component (for example, plasma) that can be supplied to the micro flow path 25 is reduced, and a considerably long time is required for extracting the specific component. It may take.
  • the width of the first flow path 20 is excessive, the capillary force becomes small, so that the specimen flows slowly and may take a considerably long time to reach the micro flow path 25.
  • the length from the upstream end of the first flow path 20 to the branch point with the micro flow path 25 is not particularly limited, but is, for example, 10 ⁇ m or more and 100 mm or less.
  • the microchannel 25 has a width capable of separating a specific component (plasma) including a test target component (analyte) from a specimen (blood) flowing through the first channel 20.
  • the width of such a microchannel 25 is preferably 1 ⁇ m or more and 5 ⁇ m or less, more preferably 1 ⁇ m or more and 2 ⁇ m or less.
  • the width of the microchannel 25 is too small, the amount of a specific component that can be extracted may be reduced.
  • the width of the microchannel 25 is excessive, components (for example, blood cell components such as red blood cells) other than a specific component (for example, plasma) may be mixed.
  • the length of the microchannel 25 is not particularly limited, but is, for example, 1 mm or more and 10 mm or less.
  • the number of the micro flow paths 25 is 10 or more and 1000 or less, for example.
  • variety of the thickness direction in the storage part 26 is 10 micrometers or more and 1000 micrometers or less, More preferably, they are 100 micrometers or more and 500 micrometers or less. Moreover, it is preferable that the width
  • a cavity 29 for introducing light to the surface to be examined is formed at a position between the light source 30 and the microchannel 25 in the first substrate 12.
  • the cavity 29 is for totally reflecting the light from the light source 30 on the lower surface when the light is guided to the test surface in the second substrate 15.
  • test surface by which the labeled ligand which reacts specifically with a test object component (analyte) was fixed on the surface is formed.
  • the test surface is specifically the surface of a member that defines the micro flow path 25 that is a measurement space communicating with the first flow path 20 to which the specimen is supplied, specifically, the micro flow in the second substrate 15. It consists of a microchannel region surface 25A facing the channel groove 16.
  • the analyte is an antigen
  • the ligand is an antibody against the antigen
  • the analyte include those contained in plasma, such as proteins such as C-reactive protein (CRP) and prostate specific antigen (PSA), and low molecular weight compounds such as methamphetamine at the time of drug testing. Its concentration can be measured.
  • a labeled ligand is one that produces an optical change when an analyte that specifically reacts with the ligand is bound.
  • the labeled ligand is labeled with a fluorescent dye and is in a quenching state when no analyte is bound, and emits fluorescence when bound to the analyte (Q-body (registered trademark)). It is preferable that Here, “in a quenching state” does not mean a state in which light is not completely emitted, but means a state in which fluorescence of a desired wavelength is not emitted.
  • the labeled ligand may be a system using fluorescence resonance energy transfer (FRET), which includes a first ligand labeled with an acceptor dye and a second ligand labeled with a donor dye.
  • FRET fluorescence resonance energy transfer
  • the first ligand labeled with the acceptor dye and the second ligand labeled with the donor dye are fixed at a predetermined interval, and only when the analyte binds to the FRET. And fluorescence from the donor dye is observed.
  • the labeled ligand may have a lower fluorescence intensity by binding the analyte than in a state where the analyte is not bound.
  • the optical change of the labeled ligand is preferably caused by an evanescent light component on the test surface.
  • an evanescent light component on the test surface.
  • the optical change of the labeled ligand is preferably such that the optical intensity changes depending on the concentration of the analyte.
  • fluorescent dyes such as TAMRA (excitation wavelength 555 nm, fluorescence wavelength 580 nm), Cy5 (excitation wavelength 643 nm, fluorescence wavelength 667 nm), ATTO 655 (excitation wavelength 663 nm, fluorescence wavelength 684 nm) are used. Can do.
  • the labeled ligand can be fixed to the microchannel region surface 25A facing the microchannel groove 16 in the second substrate 15 constituting the microchannel 25 as follows. That is, first, the microchannel region surface 25A is surface-treated with a silane coupling agent to give a carboxyl group.
  • a silane coupling agent for example, (CH 3 ) 2 SiCl— (CH 2 ) m —CONHS (manufactured by Altec) can be used.
  • the labeled ligand can be fixed to the microchannel region surface 25A by amine coupling the amino group in the labeled ligand to the carboxyl group fixed to the microchannel region surface 25A.
  • the detection apparatus for carrying out the detection method of the present invention includes a light source 30 that irradiates a test surface of the microchannel 25 of the analysis chip 10 with a sheet-like light extending in the plane direction of the analysis chip 10, and a microchannel. And a detector 40 disposed at a position facing the end face 15A of the second substrate 15 for receiving light emitted from the labeled ligands on the 25 test surfaces.
  • a dichroic mirror 34 that transmits light from the analysis chip 10 and reflects light from the light source 30 is located at the intersection of the optical path of light from the light source 30 and the optical path of light from the analysis chip 10 to the detector 40.
  • the optical path of the light from the light source 30 and the optical path of the light from the analysis chip 10 to the detector 40 are arranged in a state inclined at 45 °.
  • a lens 38 for collimating incident light is disposed between the light source 30 and the dichroic mirror 34.
  • An optical filter 35 that restricts the wavelength range of the transmitted light is disposed on the optical path between the dichroic mirror 34 and the detector 40 in the optical path from the analysis chip 10 to the detector 40.
  • the light source 30 is not particularly limited, and for example, a semiconductor laser, a He—Ne laser, an LED, a halogen lamp, or the like can be used.
  • a semiconductor laser a He—Ne laser, an LED, a halogen lamp, or the like
  • a light source 30 having a peak wavelength of 520 nm can be used
  • Cy5 or ATTO655 is used, a light source 30 having a peak wavelength of 635 nm can be used.
  • a band-pass filter or a long-pass filter that limits the wavelength range of transmitted light to a wavelength range necessary for measuring the concentration of the component to be inspected is used.
  • TAMRA fluorescent dye for labeling a ligand
  • a long-pass filter that transmits light in a wavelength range of 570 nm or more is used.
  • Cy5 or ATTO655 is used, a long-pass filter has a wavelength range of 670 nm or more. Those that transmit light can be used.
  • the detector 40 a detector capable of detecting light including a wavelength region necessary for measuring the concentration of the analyte is used.
  • a photomultiplier tube photomultiplier
  • Si A highly sensitive photodetector such as a photodiode can be used.
  • a liquid sample (blood) is introduced from the sample introduction unit 21 of the analysis chip 10 and stored in the sample storage unit 22.
  • the sample stored in the sample storage unit 22 flows through the first flow path 20 by capillary action and reaches the branch point with the micro flow path 25.
  • a component blood cell having a size larger than the width of the microchannel 25 in the sample cannot enter the microchannel 25, and therefore flows through the first channel 20 toward the downstream side.
  • a specific component having a size smaller than the width of the microchannel 25 in the sample (plasma containing the analyte, which is the analyte), can enter the microchannel 25, and thus flows through the microchannel 25. Then, it flows into the storage part 26. At this time, on the test surface of the microchannel 25, the labeled ligand immobilized on the test surface binds to the analyte contained in the specific component, and components other than the analyte in the specific component are present. It flows into the storage part 26.
  • light emitted from the light source 30 is incident on the end surface 15A of the second substrate 15 of the chip base 11 of the analysis chip 10 via the lens 38 and the dichroic mirror 34, and is totally reflected in the second substrate 15.
  • Light is guided.
  • the fluorescent dye of the labeled ligand to which the analyte is bound is excited by the evanescent light component generated on the test surface of the microchannel 25, and fluorescence is emitted.
  • the emitted fluorescence is guided through the second substrate 15 while being totally reflected, and is emitted from the end face 15A, and the intensity of the fluorescence emitted by the detector 40 is detected via the dichroic mirror 34 and the optical filter 35.
  • the concentration of the analyte, which is the component to be examined is calculated.
  • Components other than the evanescent light component in the light guided while totally reflecting in the second substrate 15 are irradiated onto the first substrate 12 and absorbed by the first substrate 12.
  • an analyte that reacts with the ligand is introduced into the analysis chip 10 having the test surface on which the labeled ligand is immobilized, and the optical generated in the labeled ligand in a state where they are bound. Change is detected. Therefore, an optical change can be caused in the labeled ligand only by introducing the sample into the test surface without performing a separate operation. As a result, it is possible to accurately perform optical measurement of a test target component from a very small amount of sample.
  • the detection device used in the detection method of the present invention is not limited to the detector 40 provided at a position facing the end surface 15 ⁇ / b> A of the second substrate 15. It may be provided at a position facing the micro flow path 25 on the lower surface of the second substrate 15 (lower surface in FIG. 5).
  • the light source 30 is provided at a position facing the end surface 15 ⁇ / b> A of the second substrate 15.
  • a lens 38 that collimates incident light is disposed on the optical path between the light source 30 and the second substrate 15, and is incident on the optical path between the second substrate 15 and the detector 40.
  • a lens 39 for collimating the light and an optical filter 35 for limiting the wavelength range of the transmitted light are arranged.
  • Other configurations of the detection device shown in FIGS. 4 and 5 are the same as those of the detection device shown in FIGS. 1 and 2.
  • the first flow path 20 extending from the sample introduction part 21 branches into branch paths 20 ⁇ / b> A and 20 ⁇ / b> B having the same width, and the respective branch paths 20 ⁇ / b> A and 20 ⁇ / b> B.
  • branch paths 20 ⁇ / b> A and 20 ⁇ / b> B branch paths 20 ⁇ / b> A and 20 ⁇ / b> B.
  • each microchannel 25 is formed in a state extending in the same direction as the width direction of the sheet-like light and at a reflection position on the upper surface in the second substrate 15 of the sheet-like light. Yes.
  • the detection method of the present invention can be effectively used as a method for analyzing the concentration of a specific component contained in plasma in blood.

Abstract

The present invention addresses the problem of providing a detection method and an analysis chip used therefor such that optical measurement can be accurately performed for a component to be inspected from a trace amount of a specimen. The detection method according to the present invention is a method for detecting the concentration of the analyte by introducing to an inspection surface of an analysis chip, in which a labelled ligand is affixed to the inspection surface, an analyte that reacts with the ligand and measuring the extent of optical changes produced in the labelled ligand by causing the analyte to bind to the labelled ligand, and is characterized in that: the analyte is an antigen and the ligand is an antibody to the antigen; and the labelled ligand is in a non-light-emitting state when labelled with a fluorescent dye and emits fluorescence due to the analyte binding.

Description

検出方法および分析チップDetection method and analysis chip
　本発明は、検出方法およびこれに用いる分析チップに関する。 The present invention relates to a detection method and an analysis chip used therefor.
　血漿中に存在する特定の成分を分析する際、検体である血液に含まれる赤血球、白血球、血小板などの細胞成分を除去する必要がある。細胞成分を除去する方法としては、遠心分離方式の方法が広く一般的に用いられているが、数分から十数分の時間が必要であること、装置が大型であること、検体（血液）が多量に必要であることなどのデメリットがある。遠心分離方式の方法の他には、フィルタ方式の方法などがあるが、先述のデメリットを解決することはできない。 When analyzing specific components present in plasma, it is necessary to remove cell components such as red blood cells, white blood cells, and platelets contained in the blood sample. As a method for removing cellular components, a centrifugal separation method is widely used. However, it requires a time of several minutes to several tens of minutes, a large apparatus, and a specimen (blood). There are disadvantages such as being necessary in large quantities. In addition to the centrifugal method, there is a filter method, but the above-mentioned disadvantages cannot be solved.
　一方、近年、微細加工技術を利用したマイクロ流路チップの開発が盛んに行われており、例えば、特願２０１５－２０４７２８号、特願２０１５－２０４７２９号、特願２０１５－２０４７３０号には、毛細管力を用いて血漿を抽出することが開示されている。 On the other hand, in recent years, microchannel chips utilizing microfabrication technology have been actively developed. For example, Japanese Patent Application Nos. 2015-204728, 2015-204729, and 2015-204730 include capillary tubes. Extracting plasma using force is disclosed.
　しかしながら、毛細管力による血漿の抽出方法においては、抽出される血漿が微量であり、さらに、検体（血液）の送液のための駆動力が存在しないため、混合や洗浄といった操作が分析チップ内では行い難い、という問題がある。そのため、Ｂ／Ｆ分離（抗体と結合している抗原と、抗体と結合していない抗原との分離）が必要となるイムノアッセイ法はマイクロ流路中で行うことが困難である、という課題があった。 However, in the method of extracting plasma by capillary force, the amount of plasma to be extracted is very small, and there is no driving force for feeding the specimen (blood), so operations such as mixing and washing are not performed in the analysis chip. There is a problem that it is difficult to do. Therefore, an immunoassay method that requires B / F separation (separation of antigen bound to antibody and antigen not bound to antibody) is difficult to perform in a microchannel. It was.
　Ｂ／Ｆ分離が不要であるイムノアッセイ法としては、被検表面において蛍光物質によって標識された抗体をエバネッセント光成分によって励起させる方法が提案されている（例えば、特許文献１参照。）。 As an immunoassay method that does not require B / F separation, a method of exciting an antibody labeled with a fluorescent substance on a test surface with an evanescent light component has been proposed (for example, see Patent Document 1).
　しかしながら、このような方法においては、蛍光物質によって標識されていない抗体が固定された被検表面に、検査対象成分（抗原）と蛍光物質によって標識された抗体とを混合したものが導入される。そして、抗原が被検表面に固定された抗体と蛍光物質によって標識された抗体とに挟まれる形で結合体が形成され、その結果、結合体を形成した蛍光物質によって標識された抗体のみがエバネッセント光成分によって励起される。従って、この方法においてはＢ／Ｆ分離は不要であるが、抗原と蛍光物質によって標識された抗体との均一な混合が必要となるなど、マイクロ流路中で行うことは困難であり、やはり微量の検体によって精確な光学測定結果を得ることができない。 However, in such a method, a mixture of a component to be examined (antigen) and an antibody labeled with a fluorescent substance is introduced onto a test surface on which an antibody not labeled with the fluorescent substance is fixed. Then, a conjugate is formed in such a manner that the antigen is sandwiched between the antibody immobilized on the test surface and the antibody labeled with the fluorescent substance, and as a result, only the antibody labeled with the fluorescent substance forming the conjugate is evanescent. Excited by light component. Therefore, in this method, B / F separation is not necessary, but it is difficult to carry out in a microchannel because it requires uniform mixing of an antigen and an antibody labeled with a fluorescent substance. Accurate optical measurement results cannot be obtained with this sample.
特許第３３６２２０６号明細書Japanese Patent No. 3362206
　本発明は、以上のような事情に基づいてなされたものであって、微量の検体から検査対象成分についての光学測定を精確に行うことができる検出方法およびこれに用いる分析チップを提供することを目的とする。 The present invention has been made based on the above circumstances, and provides a detection method capable of accurately performing optical measurement of a test target component from a very small amount of sample and an analysis chip used therefor. Objective.
　本発明の検出方法は、標識されたリガンドが固定された被検表面を有する分析チップの当該被検表面に、前記リガンドと反応するアナライトを導入し、
　前記標識されたリガンドにアナライトを結合させることによって当該標識されたリガンドに生じる光学的変化の程度を測定することにより、前記アナライトの濃度を検出する検出方法であって、
　前記アナライトが抗原であり、前記リガンドが前記抗原に対する抗体であり、
　前記標識されたリガンドは、蛍光色素によって標識された消光状態のものであり、アナライトを結合させることにより蛍光を放射するものであることを特徴とする。 The detection method of the present invention introduces an analyte that reacts with the ligand to the test surface of the analysis chip having the test surface on which the labeled ligand is immobilized,
A detection method for detecting the concentration of the analyte by measuring the degree of optical change that occurs in the labeled ligand by binding the analyte to the labeled ligand,
The analyte is an antigen and the ligand is an antibody against the antigen;
The labeled ligand is in a quenching state labeled with a fluorescent dye, and emits fluorescence when bound to an analyte.
　本発明の検出方法においては、前記アナライトは、血漿中に含まれているものであることが好ましい。 In the detection method of the present invention, the analyte is preferably contained in plasma.
　本発明の分析チップは、上記の検出方法に使用される分析チップであって、
　標識されたリガンドが固定された被検表面を有し、
　前記標識されたリガンドは、当該リガンドと反応するアナライトを結合させたときに光学的変化を生じるものであることを特徴とする。 The analysis chip of the present invention is an analysis chip used in the above detection method,
Having a test surface to which a labeled ligand is immobilized;
The labeled ligand is characterized by causing an optical change when an analyte that reacts with the ligand is bound.
　本発明の分析チップは、前記標識されたリガンドが固定された被検表面を有する、光透過性材料からなる基板を有し、
　前記光学的変化が、前記被検表面におけるエバネッセント光成分により生じるものであることが好ましい。 The analysis chip of the present invention has a substrate made of a light transmissive material having a test surface on which the labeled ligand is fixed,
It is preferable that the optical change is caused by an evanescent light component on the test surface.
　本発明の分析チップは、前記被検表面が、前記基板における、検体が供給される流路に連通する測定用空間を区画する部材の表面である構成とすることができる。
　本発明の分析チップは、液状の検体を流通させる第一流路と、この第一流路から分岐して形成された、当該第一流路に連通する第二流路とを内部に有する板状体よりなり、
　前記第二流路は、前記第一流路を流通する検体から検査対象成分を分離することが可能な幅を有し、
　当該第二流路の内壁面が被検表面とされることが好ましい。 The analysis chip of the present invention may be configured such that the test surface is a surface of a member that divides a measurement space communicating with a flow path to which a specimen is supplied on the substrate.
The analysis chip of the present invention comprises a plate-like body having a first flow path for circulating a liquid specimen and a second flow path formed by branching from the first flow path and communicating with the first flow path. Become
The second flow path has a width capable of separating the test target component from the sample flowing through the first flow path,
It is preferable that the inner wall surface of the second flow path is a test surface.
　本発明の検出方法によれば、標識されたリガンドが固定された被検表面を有する分析チップにこのリガンドと反応するアナライトを導入し、これらを結合させた状態において標識されたリガンドに生じる光学的変化を検出する。従って、当該検体に別途の操作を行うことなしに被検表面に導入することのみによって、当該標識されたリガンドに光学的変化を生じさせることができる。その結果、微量の検体から検査対象成分についての光学測定を精確に行うことができる。 According to the detection method of the present invention, an analyte that reacts with a ligand is introduced into an analysis chip having a test surface on which the labeled ligand is immobilized, and the optical generated in the labeled ligand in a state where they are bound. Change is detected. Therefore, an optical change can be caused in the labeled ligand only by introducing the sample into the test surface without performing a separate operation. As a result, it is possible to accurately perform optical measurement of a test target component from a very small amount of sample.
本発明の分析チップの構成の一例を検出装置に配置された状態で示す平面図である。It is a top view which shows an example of a structure of the analysis chip of this invention in the state arrange | positioned at the detection apparatus. 図１に示す分析チップの構成のＡ－Ａ断面端面図である。FIG. 2 is an AA cross-sectional end view of the configuration of the analysis chip shown in FIG. 1. 図１に示す分析チップの要部を示す説明図であり、（ａ）は、第一流路、マイクロ流路および貯まり部を拡大して示す平面図、（ｂ）は、（ａ）のＢ－Ｂ断面端面図である。FIG. 2 is an explanatory view showing a main part of the analysis chip shown in FIG. 1, wherein (a) is an enlarged plan view showing a first flow path, a micro flow path and a reservoir, and (b) is a B- It is B sectional end view. 本発明の分析チップの構成の別の一例を検出装置に配置された状態で示す平面図である。It is a top view which shows another example of a structure of the analysis chip of this invention in the state arrange | positioned at the detection apparatus. 図４に示す分析チップの構成のＣ－Ｃ断面端面図である。FIG. 5 is a cross-sectional end view taken along the line CC of the structure of the analysis chip shown in FIG. 4. 本発明の分析チップの構成のさらに別の一例を検出装置に配置された状態で示す平面図である。It is a top view which shows another example of a structure of the analysis chip of this invention in the state arrange | positioned at the detection apparatus. 図６に示す分析チップの構成のＤ－Ｄ断面端面図である。FIG. 7 is a DD cross-sectional end view of the configuration of the analysis chip shown in FIG. 6.
　以下、本発明の実施の形態について詳細に説明する。
　本発明の検出方法は、標識されたリガンドが固定された被検表面を有する分析チップの当該被検表面にこのリガンドと反応するアナライトを導入し、標識されたリガンドにアナライトを結合させることによって当該標識されたリガンドに生じる光学的変化を検出する方法である。具体的には、標識されたリガンドが、蛍光色素によって標識された消光状態のものであってアナライトを結合させることにより蛍光を放射するものであり、この標識されたリガンドに生じる蛍光強度の変化を測定することにより、アナライトの濃度を算出する方法である。 Hereinafter, embodiments of the present invention will be described in detail.
In the detection method of the present invention, an analyte that reacts with the ligand is introduced to the test surface of the analysis chip having the test surface on which the labeled ligand is immobilized, and the analyte is bound to the labeled ligand. To detect an optical change occurring in the labeled ligand. Specifically, the labeled ligand is in a quenching state labeled with a fluorescent dye, and emits fluorescence when bound to an analyte, and a change in fluorescence intensity generated in the labeled ligand This is a method for calculating the concentration of the analyte by measuring.
　まず、本発明の検出方法に用いられる分析チップについて説明する。
　図１は、本発明の分析チップの構成の一例を示す平面図、図２は、図１に示す分析チップの構成のＡ－Ａ断面端面図、図３は、図１に示す分析チップの要部を示す説明図であり、（ａ）は、第一流路、マイクロ流路および貯まり部を拡大して示す平面図、（ｂ）は、（ａ）のＢ－Ｂ断面端面図である。
　この分析チップ１０は、血液などの液状の検体から、検査対象成分（アナライト）を含む血漿などの特定の成分を分離して当該アナライトの濃度などを光学測定により測定する検出方法に用いるバイオセンサチップである。 First, the analysis chip used in the detection method of the present invention will be described.
1 is a plan view showing an example of the configuration of the analysis chip of the present invention, FIG. 2 is an end view taken along the line AA of the configuration of the analysis chip shown in FIG. 1, and FIG. 3 is a schematic diagram of the analysis chip shown in FIG. 4A is an explanatory plan view showing an enlarged view of the first flow path, the micro flow path, and the reservoir, and FIG. 4B is an end view taken along the line BB in FIG.
This analysis chip 10 is a bio used in a detection method in which a specific component such as plasma containing a test target component (analyte) is separated from a liquid sample such as blood and the concentration of the analyte is measured by optical measurement. It is a sensor chip.
　分析チップ１０は、第一基板１２および第二基板１５よりなるチップ基体１１を有し、第一基板１２と第二基板１５とが接合された板状体によって構成されている。
　第一基板１２および第二基板１５の各々の厚みは、特に限定されるものではないが、例えば０．１ｍｍ以上５．０ｍｍ以下である。
　チップ基体１１の第一基板１２を構成する材料としては、光吸収性および自己粘着性を有するもの、例えば、黒色樹脂材料などを用いることができる。
　また、チップ基体１１の第二基板１５を構成する材料としては、光源３０からの光を透過し、さらに、自家蛍光が低い、例えばガラスやシクロオレフィンコポリマーなどの光透過性材料を用いることができる。 The analysis chip 10 includes a chip base 11 including a first substrate 12 and a second substrate 15, and is configured by a plate-like body in which the first substrate 12 and the second substrate 15 are joined.
Although the thickness of each of the 1st board | substrate 12 and the 2nd board | substrate 15 is not specifically limited, For example, they are 0.1 mm or more and 5.0 mm or less.
As a material constituting the first substrate 12 of the chip base 11, a material having light absorption and self-adhesiveness, for example, a black resin material can be used.
Moreover, as a material which comprises the 2nd board | substrate 15 of the chip | tip base | substrate 11, light transmissive materials, such as glass and a cycloolefin copolymer, which permeate | transmit the light from the light source 30 and have low self-fluorescence can be used. .
　そして、この分析チップ１０は、液状の検体を流通させる第一流路２０と、この第一流路２０に連通する、検体から分離された特定の成分が流入される複数の第二流路（以下、「マイクロ流路」ともいう。）２５とを有する。マイクロ流路２５の各々は、第一流路２０から分岐して当該第一流路２０に対して垂直方向に伸び、等間隔で離間して配列された状態で形成されている。
　第一流路２０における上流側端は、検体導入部２１から導入された検体を貯留する検体貯留部２２に接続されている。第一流路２０の下流側端は、第一排出部２３に接続されている。また、マイクロ流路２５の各々の下流側端は、貯まり部２６に接続されている。この貯まり部２６の一端は、第二排出部２８に接続されている。 The analysis chip 10 includes a first flow path 20 through which a liquid sample is circulated, and a plurality of second flow paths (hereinafter referred to as “the first flow path 20”) into which a specific component separated from the sample flows. 25). Each of the micro flow paths 25 is formed in a state where the micro flow paths 25 are branched from the first flow path 20 and extend in the vertical direction with respect to the first flow paths 20 and are spaced apart at equal intervals.
The upstream end of the first flow path 20 is connected to a sample storage unit 22 that stores the sample introduced from the sample introduction unit 21. The downstream end of the first flow path 20 is connected to the first discharge part 23. In addition, the downstream end of each of the micro flow paths 25 is connected to the accumulation portion 26. One end of the storage part 26 is connected to the second discharge part 28.
　図示の例では、第一流路２０は、第一基板１２に形成された第一流路用溝１３ａの内壁面と、第二基板１５とによって区画されることにより形成されている。また、貯まり部２６は、第一基板１２に形成された貯まり部用凹所１３ｂの内壁面と、第二基板１５とによって区画されることにより形成されている。また、マイクロ流路２５は、第一基板１２に、第一流路用溝１３ａおよび貯まり部用凹所１３ｂを連通する状態に、かつ、これらの深さよりも浅く形成されたマイクロ流路用溝１６の内壁面と、第一基板１２とによって区画されることにより形成されている。 In the illustrated example, the first flow path 20 is formed by being partitioned by the inner wall surface of the first flow path groove 13 a formed in the first substrate 12 and the second substrate 15. The reservoir 26 is formed by being partitioned by the inner wall surface of the reservoir recess 13 b formed in the first substrate 12 and the second substrate 15. Further, the microchannel 25 is formed in a state where the first channel 12 and the reservoir recess 13b are communicated with the first substrate 12 and shallower than these depths. It is formed by being partitioned by the inner wall surface of the first substrate 12 and the first substrate 12.
　第一流路２０は、液状の検体（例えば血液）を流通させることが可能な幅を有する。本発明において、流路の「幅」とは、流路における当該流路が伸びる方向に垂直な断面において、当該流路の最も小さい幅を意味する。図示の例の第一流路２０およびマイクロ流路２５においては、分析チップ１０の厚み方向の幅が最も小さい幅である。
　このような第一流路２０の幅は、１０μｍ以上１０００μｍ以下であることが好ましく、より好ましくは５０μｍ以上３００μｍ以下であり、特に好ましくは１００μｍである。第一流路２０の幅が過小である場合には、マイクロ流路２５に供給することができる特定の成分（例えば血漿）の量が少なくなり、特定の成分を抽出するために相当に長い時間を要する虞がある。一方、第一流路２０の幅が過大である場合には、毛細管力が小さくなるために検体が流れるのが遅くなり、マイクロ流路２５に到達するのに相当に長い時間を要するおそれがある。
　また、第一流路２０の上流側端からマイクロ流路２５との分岐点までの長さは、特に限定されるものではないが、例えば１０μｍ以上１００ｍｍ以下である。 The first flow path 20 has a width that allows a liquid specimen (for example, blood) to circulate. In the present invention, the “width” of the flow channel means the smallest width of the flow channel in a cross section perpendicular to the direction in which the flow channel extends. In the first flow path 20 and the micro flow path 25 in the illustrated example, the width in the thickness direction of the analysis chip 10 is the smallest width.
The width of the first flow path 20 is preferably 10 μm or more and 1000 μm or less, more preferably 50 μm or more and 300 μm or less, and particularly preferably 100 μm. When the width of the first flow path 20 is too small, the amount of a specific component (for example, plasma) that can be supplied to the micro flow path 25 is reduced, and a considerably long time is required for extracting the specific component. It may take. On the other hand, when the width of the first flow path 20 is excessive, the capillary force becomes small, so that the specimen flows slowly and may take a considerably long time to reach the micro flow path 25.
Further, the length from the upstream end of the first flow path 20 to the branch point with the micro flow path 25 is not particularly limited, but is, for example, 10 μm or more and 100 mm or less.
　マイクロ流路２５は、第一流路２０を流通する検体（血液）から検査対象成分（アナライト）を含む特定の成分（血漿）を分離することが可能な幅を有する。
　このようなマイクロ流路２５の幅は、１μｍ以上５μｍ以下であることが好ましく、より好ましくは１μｍ以上２μｍ以下である。マイクロ流路２５の幅が過小である場合には、抽出することができる特定の成分の量が少なくなるおそれがある。一方、マイクロ流路２５の幅が過大である場合には、特定の成分（例えば血漿）以外の成分（例えば赤血球などの血球成分）が混入するおそれがある。
　また、マイクロ流路２５の長さは、特に限定されるものではないが、例えば１ｍｍ以上１０ｍｍ以下である。
　また、マイクロ流路２５の数は、例えば１０本以上１０００本以下である。 The microchannel 25 has a width capable of separating a specific component (plasma) including a test target component (analyte) from a specimen (blood) flowing through the first channel 20.
The width of such a microchannel 25 is preferably 1 μm or more and 5 μm or less, more preferably 1 μm or more and 2 μm or less. When the width of the microchannel 25 is too small, the amount of a specific component that can be extracted may be reduced. On the other hand, when the width of the microchannel 25 is excessive, components (for example, blood cell components such as red blood cells) other than a specific component (for example, plasma) may be mixed.
The length of the microchannel 25 is not particularly limited, but is, for example, 1 mm or more and 10 mm or less.
Moreover, the number of the micro flow paths 25 is 10 or more and 1000 or less, for example.
　また、貯まり部２６における厚み方向の幅が１０μｍ以上１０００μｍ以下であることが好ましく、より好ましくは１００μｍ以上５００μｍ以下である。
　また、貯まり部２６における面方向の幅は、それぞれ０．１μｍ以上１．０ｍｍ以下であることが好ましい。 Moreover, it is preferable that the width | variety of the thickness direction in the storage part 26 is 10 micrometers or more and 1000 micrometers or less, More preferably, they are 100 micrometers or more and 500 micrometers or less.
Moreover, it is preferable that the width | variety of the surface direction in the storage part 26 is 0.1 to 1.0 mm, respectively.
　この分析チップ１０においては、第一基板１２内における光源３０とマイクロ流路２５との間の位置に、被検表面に光を導入するための空洞２９が形成されている。空洞２９は、光源３０からの光を、第二基板１５内において被検表面まで導光するときに、その下面において全反射させるためのものである。 In this analysis chip 10, a cavity 29 for introducing light to the surface to be examined is formed at a position between the light source 30 and the microchannel 25 in the first substrate 12. The cavity 29 is for totally reflecting the light from the light source 30 on the lower surface when the light is guided to the test surface in the second substrate 15.
　そして、この分析チップ１０においては、検査対象成分（アナライト）と特異的に反応する標識されたリガンドが表面に固定された被検表面が形成されている。
　被検表面は、具体的には、検体が供給される第一流路２０に連通する測定用空間であるマイクロ流路２５を区画する部材の表面、具体的には、第二基板１５におけるマイクロ流路用溝１６と対向するマイクロ流路用領域面２５Ａからなる。 And in this analysis chip 10, the test surface by which the labeled ligand which reacts specifically with a test object component (analyte) was fixed on the surface is formed.
The test surface is specifically the surface of a member that defines the micro flow path 25 that is a measurement space communicating with the first flow path 20 to which the specimen is supplied, specifically, the micro flow in the second substrate 15. It consists of a microchannel region surface 25A facing the channel groove 16.
　リガンドおよびアナライトとしては、アナライトが抗原であり、かつ、リガンドが当該抗原に対する抗体とされる。
　アナライトとしては、例えばＣ反応性タンパク（ＣＲＰ）や前立腺特異抗原（ＰＳＡ）などのタンパク、薬物検査時のメタンフェタミンなどの低分子化合物など、血漿中に含まれているものが挙げられ、好適にその濃度を測定することができる。 As the ligand and the analyte, the analyte is an antigen, and the ligand is an antibody against the antigen.
Examples of the analyte include those contained in plasma, such as proteins such as C-reactive protein (CRP) and prostate specific antigen (PSA), and low molecular weight compounds such as methamphetamine at the time of drug testing. Its concentration can be measured.
　標識されたリガンドは、当該リガンドと特異的に反応するアナライトを結合させたときに光学的変化を生じるものである。
　標識されたリガンドは、蛍光色素によって標識された、アナライトが結合されていない状態において消光状態のものであり、アナライトを結合させることにより蛍光を放射するもの（Ｑ－ｂｏｄｙ（登録商標））であることが好ましい。ここに、「消光状態のもの」とは、光が完全に放射しない状態を意味するものではなく、所望の波長の蛍光を放射しない状態のものを意味する。
　また、標識されたリガンドは、アクセプター色素を標識した第１のリガンドとドナー色素を標識した第２のリガンドとからなる、蛍光共鳴エネルギー移動（ＦＲＥＴ）を利用した系のものであってもよい。ＦＲＥＴを利用した系のものは、アクセプター色素を標識した第１のリガンドとドナー色素を標識した第２のリガンドとを一定の間隔を介して固定し、両者にアナライトが結合したときのみにＦＲＥＴが生じて、ドナー色素からの蛍光が観測されるものである。
　また、標識されたリガンドは、アナライトを結合させることにより、アナライトが結合されていない状態よりも蛍光強度が小さくなるものであってもよい。 A labeled ligand is one that produces an optical change when an analyte that specifically reacts with the ligand is bound.
The labeled ligand is labeled with a fluorescent dye and is in a quenching state when no analyte is bound, and emits fluorescence when bound to the analyte (Q-body (registered trademark)). It is preferable that Here, “in a quenching state” does not mean a state in which light is not completely emitted, but means a state in which fluorescence of a desired wavelength is not emitted.
Further, the labeled ligand may be a system using fluorescence resonance energy transfer (FRET), which includes a first ligand labeled with an acceptor dye and a second ligand labeled with a donor dye. In the system using FRET, the first ligand labeled with the acceptor dye and the second ligand labeled with the donor dye are fixed at a predetermined interval, and only when the analyte binds to the FRET. And fluorescence from the donor dye is observed.
Further, the labeled ligand may have a lower fluorescence intensity by binding the analyte than in a state where the analyte is not bound.
　標識されたリガンドの光学的変化は、被検表面におけるエバネッセント光成分により生じるものであることが好ましい。これにより、例えば特定の成分に、アナライト以外に蛍光を放射する物質が含有されている場合であっても、これらに係る蛍光を除外してアナライトに係る蛍光強度のみを精確に測定することができる。
　また、標識されたリガンドの光学的変化は、アナライトの濃度依存的に光学強度が変化するものであることが好ましい。 The optical change of the labeled ligand is preferably caused by an evanescent light component on the test surface. Thus, for example, even if a specific component contains a substance that emits fluorescence in addition to the analyte, only the fluorescence intensity related to the analyte is accurately measured by excluding the fluorescence related to these. Can do.
The optical change of the labeled ligand is preferably such that the optical intensity changes depending on the concentration of the analyte.
　リガンドを標識するための物質としては、例えばＴＡＭＲＡ（励起波長５５５ｎｍ、蛍光波長５８０ｎｍ）、Ｃｙ５（励起波長６４３ｎｍ、蛍光波長６６７ｎｍ）、ＡＴＴＯ６５５（励起波長６６３ｎｍ、蛍光波長６８４ｎｍ）などの蛍光色素を用いることができる。 As a substance for labeling the ligand, for example, fluorescent dyes such as TAMRA (excitation wavelength 555 nm, fluorescence wavelength 580 nm), Cy5 (excitation wavelength 643 nm, fluorescence wavelength 667 nm), ATTO 655 (excitation wavelength 663 nm, fluorescence wavelength 684 nm) are used. Can do.
　標識されたリガンドは、マイクロ流路２５を構成する第二基板１５におけるマイクロ流路用溝１６と対向するマイクロ流路用領域面２５Ａに、以下のように固定することができる。
　すなわち、まず、マイクロ流路用領域面２５Ａをシランカップリング剤によって表面処理してカルボキシル基を付与する。シランカップリング剤としては、例えば（ＣＨ3 ）2 ＳｉＣｌ－（ＣＨ2 ）m －ＣＯＮＨＳ（アルテック社製）などを使用することができる。次いで、標識されたリガンドにおけるアミノ基をマイクロ流路用領域面２５Ａに固定されたカルボキシル基にアミンカップリングすることにより、標識されたリガンドをマイクロ流路用領域面２５Ａに固定することができる。 The labeled ligand can be fixed to the microchannel region surface 25A facing the microchannel groove 16 in the second substrate 15 constituting the microchannel 25 as follows.
That is, first, the microchannel region surface 25A is surface-treated with a silane coupling agent to give a carboxyl group. As the silane coupling agent, for example, (CH 3 ) 2 SiCl— (CH 2 ) m —CONHS (manufactured by Altec) can be used. Next, the labeled ligand can be fixed to the microchannel region surface 25A by amine coupling the amino group in the labeled ligand to the carboxyl group fixed to the microchannel region surface 25A.
　本発明の検出方法を実施する検出装置は、分析チップ１０のマイクロ流路２５の被検表面に対して当該分析チップ１０の平面方向に伸びるシート状の光を照射する光源３０と、マイクロ流路２５の被検表面の標識されたリガンドから放射された光を受光する、第二基板１５の端面１５Ａに対向する位置に配置された検出器４０とを備えてなる。 The detection apparatus for carrying out the detection method of the present invention includes a light source 30 that irradiates a test surface of the microchannel 25 of the analysis chip 10 with a sheet-like light extending in the plane direction of the analysis chip 10, and a microchannel. And a detector 40 disposed at a position facing the end face 15A of the second substrate 15 for receiving light emitted from the labeled ligands on the 25 test surfaces.
　光源３０からの光の光路と、分析チップ１０から検出器４０に至る光の光路との交点位置には、分析チップ１０からの光を透過すると共に光源３０からの光を反射するダイクロイックミラー３４が、光源３０からの光の光路および分析チップ１０から検出器４０に至る光の光路の各々に対して４５°に傾斜した状態で配置されている。また、光源３０とダイクロイックミラー３４との間には、入射される光を平行化するレンズ３８が配置されている。
　また、分析チップ１０から検出器４０に至る光の光路におけるダイクロイックミラー３４と検出器４０との間の光路上には、透過する光の波長域を制限する光学フィルタ３５が配置されている。 A dichroic mirror 34 that transmits light from the analysis chip 10 and reflects light from the light source 30 is located at the intersection of the optical path of light from the light source 30 and the optical path of light from the analysis chip 10 to the detector 40. The optical path of the light from the light source 30 and the optical path of the light from the analysis chip 10 to the detector 40 are arranged in a state inclined at 45 °. A lens 38 for collimating incident light is disposed between the light source 30 and the dichroic mirror 34.
An optical filter 35 that restricts the wavelength range of the transmitted light is disposed on the optical path between the dichroic mirror 34 and the detector 40 in the optical path from the analysis chip 10 to the detector 40.
　光源３０としては、特に限定されず、例えば半導体レーザ、Ｈｅ－Ｎｅレーザ、ＬＥＤ、ハロゲンランプなどを用いることができる。
　例えばリガンドを標識するための蛍光色素としてＴＡＭＲＡを用いる場合は、光源３０としてピーク波長が５２０ｎｍのものを用い、Ｃｙ５やＡＴＴＯ６５５を用いる場合は、光源３０としてピーク波長が６３５ｎｍのものを用いることができる。 The light source 30 is not particularly limited, and for example, a semiconductor laser, a He—Ne laser, an LED, a halogen lamp, or the like can be used.
For example, when TAMRA is used as a fluorescent dye for labeling a ligand, a light source 30 having a peak wavelength of 520 nm can be used, and when Cy5 or ATTO655 is used, a light source 30 having a peak wavelength of 635 nm can be used. .
　光学フィルタ３５としては、透過する光の波長域を、検査対象成分の濃度を測定するために必要な波長域に制限するバンドパスフィルタまたはロングパスフィルタが用いられている。
　例えばリガンドを標識するための蛍光色素としてＴＡＭＲＡを用いる場合は、ロングパスフィルタとして５７０ｎｍ以上の波長範囲の光を透過するものを用い、Ｃｙ５やＡＴＴＯ６５５を用いる場合は、ロングパスフィルタとして６７０ｎｍ以上の波長範囲の光を透過するものを用いることができる。 As the optical filter 35, a band-pass filter or a long-pass filter that limits the wavelength range of transmitted light to a wavelength range necessary for measuring the concentration of the component to be inspected is used.
For example, when TAMRA is used as a fluorescent dye for labeling a ligand, a long-pass filter that transmits light in a wavelength range of 570 nm or more is used. When Cy5 or ATTO655 is used, a long-pass filter has a wavelength range of 670 nm or more. Those that transmit light can be used.
　検出器４０としては、アナライトの濃度を測定するために必要な波長域を含む光を検出することが可能なものが用いられ、具体的には、例えば光電子増倍管（フォトマル）、Ｓｉフォトダイオードなどの高感度光検出器を用いることができる。 As the detector 40, a detector capable of detecting light including a wavelength region necessary for measuring the concentration of the analyte is used. Specifically, for example, a photomultiplier tube (photomultiplier), Si A highly sensitive photodetector such as a photodiode can be used.
　本発明の検出方法において、蛍光色素によって標識されたリガンドが固定された分析チップ１０を用いると共に血液を検体として用いた場合について説明する。
　先ず、液状の検体（血液）が分析チップ１０の検体導入部２１から導入されて検体貯留部２２に貯留される。検体貯留部２２に貯留された検体は、毛細管現象によって第一流路２０を流通し、マイクロ流路２５との分岐点に到達する。そして、この分岐点においては、検体中におけるマイクロ流路２５の幅より大きいサイズの成分（血球）は、マイクロ流路２５に進入することができないため、第一流路２０を下流側に向かって流通する。一方、検体中におけるマイクロ流路２５の幅より小さいサイズの特定の成分（検査対象成分であるアナライトを含む血漿）は、マイクロ流路２５に進入することができるため、マイクロ流路２５を流通し、貯まり部２６に流入される。
　このとき、マイクロ流路２５の被検表面においては、当該被検表面に固定された標識されたリガンドと特定の成分に含まれるアナライトとが結合し、特定の成分におけるアナライト以外の成分が貯まり部２６に流入される。 In the detection method of the present invention, a case will be described in which an analysis chip 10 to which a ligand labeled with a fluorescent dye is immobilized and blood is used as a specimen are described.
First, a liquid sample (blood) is introduced from the sample introduction unit 21 of the analysis chip 10 and stored in the sample storage unit 22. The sample stored in the sample storage unit 22 flows through the first flow path 20 by capillary action and reaches the branch point with the micro flow path 25. At this branch point, a component (blood cell) having a size larger than the width of the microchannel 25 in the sample cannot enter the microchannel 25, and therefore flows through the first channel 20 toward the downstream side. To do. On the other hand, a specific component having a size smaller than the width of the microchannel 25 in the sample (plasma containing the analyte, which is the analyte), can enter the microchannel 25, and thus flows through the microchannel 25. Then, it flows into the storage part 26.
At this time, on the test surface of the microchannel 25, the labeled ligand immobilized on the test surface binds to the analyte contained in the specific component, and components other than the analyte in the specific component are present. It flows into the storage part 26.
　一方、光源３０から放射された光が、レンズ３８およびダイクロイックミラー３４を介して、分析チップ１０のチップ基体１１の第二基板１５の端面１５Ａから入射され、第二基板１５内を全反射しながら導光される。そして、マイクロ流路２５の被検表面において生じたエバネッセント光成分によって、アナライトが結合された標識されたリガンドの蛍光色素が励起され、蛍光が放射される。放射された蛍光は、第二基板１５内を全反射されながら導光され、端面１５Ａから出射されてダイクロイックミラー３４および光学フィルタ３５を介して、検出器４０によって出射された蛍光の強度が検出される。そして、検出器４０によって検出された蛍光強度に係るデータに基づいて検査対象成分であるアナライトの濃度が演算される。
　第二基板１５内を全反射しながら導光された光のうちエバネッセント光成分以外の成分は、第一基板１２に照射されて当該第一基板１２に吸収される。 On the other hand, light emitted from the light source 30 is incident on the end surface 15A of the second substrate 15 of the chip base 11 of the analysis chip 10 via the lens 38 and the dichroic mirror 34, and is totally reflected in the second substrate 15. Light is guided. Then, the fluorescent dye of the labeled ligand to which the analyte is bound is excited by the evanescent light component generated on the test surface of the microchannel 25, and fluorescence is emitted. The emitted fluorescence is guided through the second substrate 15 while being totally reflected, and is emitted from the end face 15A, and the intensity of the fluorescence emitted by the detector 40 is detected via the dichroic mirror 34 and the optical filter 35. The Then, based on the data relating to the fluorescence intensity detected by the detector 40, the concentration of the analyte, which is the component to be examined, is calculated.
Components other than the evanescent light component in the light guided while totally reflecting in the second substrate 15 are irradiated onto the first substrate 12 and absorbed by the first substrate 12.
　以上の検出方法によれば、標識されたリガンドが固定された被検表面を有する分析チップ１０にこのリガンドと反応するアナライトを導入し、これらを結合させた状態において標識されたリガンドに生じる光学的変化を検出する。従って、当該検体に別途の操作を行うことなしに被検表面に導入することのみによって、当該標識されたリガンドに光学的変化を生じさせることができる。その結果、微量の検体から検査対象成分についての光学測定を精確に行うことができる。 According to the above detection method, an analyte that reacts with the ligand is introduced into the analysis chip 10 having the test surface on which the labeled ligand is immobilized, and the optical generated in the labeled ligand in a state where they are bound. Change is detected. Therefore, an optical change can be caused in the labeled ligand only by introducing the sample into the test surface without performing a separate operation. As a result, it is possible to accurately perform optical measurement of a test target component from a very small amount of sample.
　以上、本発明の実施の形態について説明したが、本発明は上記の実施の形態に限定されるものではなく、種々の変更を加えることができる。
　例えば、本発明の検出方法に用いられる検出装置は、図４および図５に示されるように、検出器４０は、第二基板１５の端面１５Ａに対向する位置に設けられることに限定されず、第二基板１５の下面（図５において下面）のマイクロ流路２５に対向する位置に設けられていてもよい。この例において、光源３０は、第二基板１５の端面１５Ａに対向する位置に設けられている。
　光源３０と第二基板１５との間の光路上には、入射される光を平行化するレンズ３８が配置され、第二基板１５と検出器４０との間の光路上には、入射される光を平行化するレンズ３９、および、透過する光の波長域を制限する光学フィルタ３５が配置されている。図４および図５に示す検出装置におけるその他の構成は、図１および図２に示す検出装置における構成と同様である。 As mentioned above, although embodiment of this invention was described, this invention is not limited to said embodiment, A various change can be added.
For example, as shown in FIGS. 4 and 5, the detection device used in the detection method of the present invention is not limited to the detector 40 provided at a position facing the end surface 15 </ b> A of the second substrate 15. It may be provided at a position facing the micro flow path 25 on the lower surface of the second substrate 15 (lower surface in FIG. 5). In this example, the light source 30 is provided at a position facing the end surface 15 </ b> A of the second substrate 15.
A lens 38 that collimates incident light is disposed on the optical path between the light source 30 and the second substrate 15, and is incident on the optical path between the second substrate 15 and the detector 40. A lens 39 for collimating the light and an optical filter 35 for limiting the wavelength range of the transmitted light are arranged. Other configurations of the detection device shown in FIGS. 4 and 5 are the same as those of the detection device shown in FIGS. 1 and 2.
　また、分析チップ１０は、例えば、図６および図７に示されるように、検体導入部２１から伸びる第一流路２０が同等の幅の分岐路２０Ａ，２０Ｂに分岐し、各分岐路２０Ａ，２０Ｂから複数のマイクロ流路２５に分岐した構成を有するものであってもよい。この例において、各マイクロ流路２５は、シート状の光の幅方向と同方向に伸びる状態に、かつ、当該シート状の光の第二基板１５内の上面における反射位置に、それぞれ形成されている。 In the analysis chip 10, for example, as shown in FIGS. 6 and 7, the first flow path 20 extending from the sample introduction part 21 branches into branch paths 20 </ b> A and 20 </ b> B having the same width, and the respective branch paths 20 </ b> A and 20 </ b> B. To a plurality of microchannels 25 may be used. In this example, each microchannel 25 is formed in a state extending in the same direction as the width direction of the sheet-like light and at a reflection position on the upper surface in the second substrate 15 of the sheet-like light. Yes.
　本発明の検出方法は、血液中の血漿に含まれる特定の成分の濃度を分析する方法として有効に用いることができる。 The detection method of the present invention can be effectively used as a method for analyzing the concentration of a specific component contained in plasma in blood.
１０　分析チップ
１１　チップ基体
１２　第一基板
１３ａ　第一流路用溝
１３ｂ　貯まり部用凹所
１５　第二基板
１５Ａ　端面
１６　マイクロ流路用溝
２０　第一流路
２０Ａ，２０Ｂ　分岐路
２１　検体導入部
２２　検体貯留部
２３　第一排出部
２５　マイクロ流路
２５Ａ　マイクロ流路用領域面
２６　貯まり部
２８　第二排出部
２９　空洞
３０　光源
３４　ダイクロイックミラー
３５　光学フィルタ
３８，３９　レンズ
４０　検出器 DESCRIPTION OF SYMBOLS 10 Analysis chip 11 Chip base | substrate 12 1st board | substrate 13a 1st groove | channel 13b Reservoir part recess 15 2nd board | substrate 15A End surface 16 Microchannel groove | channel 20 1st flow path 20A, 20B Branch path 21 Sample introduction part 22 Sample storage Section 23 First discharge section 25 Micro flow path 25A Micro flow path area surface 26 Storage section 28 Second discharge section 29 Cavity 30 Light source 34 Dichroic mirror 35 Optical filter 38, 39 Lens 40 Detector

Claims (6)

  1. 　標識されたリガンドが固定された被検表面を有する分析チップの当該被検表面に、前記リガンドと反応するアナライトを導入し、
    　前記標識されたリガンドにアナライトを結合させることによって当該標識されたリガンドに生じる光学的変化の程度を測定することにより、前記アナライトの濃度を検出する検出方法であって、
    　前記アナライトが抗原であり、前記リガンドが前記抗原に対する抗体であり、
    　前記標識されたリガンドは、蛍光色素によって標識された消光状態のものであり、アナライトを結合させることにより蛍光を放射するものであることを特徴とする検出方法。     An analyte that reacts with the ligand is introduced to the test surface of the analysis chip having the test surface to which the labeled ligand is fixed;
    A detection method for detecting the concentration of the analyte by measuring the degree of optical change that occurs in the labeled ligand by binding the analyte to the labeled ligand,
    The analyte is an antigen and the ligand is an antibody against the antigen;
    The detection method, wherein the labeled ligand is in a quenching state labeled with a fluorescent dye, and emits fluorescence by binding an analyte.
  2. 　前記アナライトは、血漿中に含まれているものであることを特徴とする請求項１に記載の検出方法。 The detection method according to claim 1, wherein the analyte is contained in plasma.
  3. 　請求項１または請求項２に記載の検出方法に使用される分析チップであって、
    　標識されたリガンドが固定された被検表面を有し、
    　前記標識されたリガンドは、当該リガンドと反応するアナライトを結合させたときに光学的変化を生じるものであることを特徴とする分析チップ。     An analysis chip used in the detection method according to claim 1 or 2,
    Having a test surface to which a labeled ligand is immobilized;
    The analysis chip according to claim 1, wherein the labeled ligand generates an optical change when an analyte that reacts with the ligand is bound.
  4. 　前記標識されたリガンドが固定された被検表面を有する、光透過性材料からなる基板を有し、
    　前記光学的変化が、前記被検表面におけるエバネッセント光成分により生じるものであることを特徴とする請求項３に記載の分析チップ。     A substrate made of a light transmissive material having a test surface on which the labeled ligand is fixed;
    4. The analysis chip according to claim 3, wherein the optical change is caused by an evanescent light component on the test surface.
  5. 　前記被検表面が、前記基板における、検体が供給される流路に連通する測定用空間を区画する部材の表面であることを特徴とする請求項６に記載の分析チップ。 The analysis chip according to claim 6, wherein the test surface is a surface of a member defining a measurement space communicating with a flow path to which a specimen is supplied on the substrate.
  6. 　液状の検体を流通させる第一流路と、この第一流路から分岐して形成された、当該第一流路に連通する第二流路とを内部に有する板状体よりなり、
    　前記第二流路は、前記第一流路を流通する検体から検査対象成分を分離することが可能な幅を有し、
    　当該第二流路の内壁面が被検表面とされることを特徴とする請求項３～請求項５のいずれかに記載の分析チップ。     A plate-like body having a first flow path for circulating a liquid specimen and a second flow path formed by branching from the first flow path and communicating with the first flow path,
    The second flow path has a width capable of separating the test target component from the sample flowing through the first flow path,
    The analysis chip according to any one of claims 3 to 5, wherein an inner wall surface of the second flow path is a test surface.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010210378A (en) * 2009-03-10 2010-09-24 Fujifilm Corp Sensing method and sensing kit to be used of the same
JP2011013208A (en) * 2009-06-05 2011-01-20 Advance Co Ltd Biological operation system and industrial operation system
JP2015152404A (en) * 2014-02-13 2015-08-24 ウシオ電機株式会社 Method of measuring cannabis components using antigen-antibody reaction

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010210378A (en) * 2009-03-10 2010-09-24 Fujifilm Corp Sensing method and sensing kit to be used of the same
JP2011013208A (en) * 2009-06-05 2011-01-20 Advance Co Ltd Biological operation system and industrial operation system
JP2015152404A (en) * 2014-02-13 2015-08-24 ウシオ電機株式会社 Method of measuring cannabis components using antigen-antibody reaction

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