WO2006016617A1 - Procédé de quantification de substances et dispositif de quantification de substances - Google Patents

Procédé de quantification de substances et dispositif de quantification de substances Download PDF

Info

Publication number
WO2006016617A1
WO2006016617A1 PCT/JP2005/014682 JP2005014682W WO2006016617A1 WO 2006016617 A1 WO2006016617 A1 WO 2006016617A1 JP 2005014682 W JP2005014682 W JP 2005014682W WO 2006016617 A1 WO2006016617 A1 WO 2006016617A1
Authority
WO
WIPO (PCT)
Prior art keywords
substance
solution
microchannel
substrate
bead
Prior art date
Application number
PCT/JP2005/014682
Other languages
English (en)
Japanese (ja)
Inventor
Eiichi Tamiya
Tatsurou Endou
Yoshihiro Murakami
Original Assignee
Japan Science And Technology Agency
Japan Advanced Institute Of Science And Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency, Japan Advanced Institute Of Science And Technology filed Critical Japan Science And Technology Agency
Priority to JP2006531695A priority Critical patent/JP4253695B2/ja
Publication of WO2006016617A1 publication Critical patent/WO2006016617A1/fr

Links

Classifications

    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence

Definitions

  • the present invention relates to a method of quantifying a substance for quantifying a measurement target substance in a sample using an enzyme immunoassay, and a quantification device of the substance used in the method of quantification.
  • ELISA enzyme-linked immunosorbent assay
  • GC-MS gas chromatography-mass spectrometry
  • HPLC high performance liquid chromatography
  • a conventional ELISA for example, a direct competition ELISA, which is a type of competitive method, comprises an antibody 102 immobilized on the surface of a carrier 101 such as a microplate well or test tube as shown in FIG.
  • a sample or standard substance) 103 and an enzyme-labeled antigen 104 are added to cause a competition reaction, and after washing away the target substance 103 or labeled antigen 104 not bound to the antibody 102, an enzyme substrate 105 is added.
  • fluorescent substance 106 is produced by enzyme reaction, that is, it is made to fluoresce, then the fluorescence intensity is measured with a colorimeter etc., and the concentration of the measurement object is measured by comparing with the fluorescence intensity of the standard of the measurement object It is a method to measure.
  • the calibration characteristic by the direct competition method is a decay curve.
  • the fluorescence labeling hormone power that is bound to the receptor is used to detect fluorescence.
  • the fluorescence molecule of hormone bound to the receptor There is a problem that it is possible to obtain only low light emission intensity because the light emission intensity is extremely weak.
  • the present invention has been proposed in view of such conventional circumstances, and provides a method of quantifying a substance capable of quantifying a substance to be measured with extremely high sensitivity and a device for quantifying a substance.
  • the purpose is to
  • the method for quantifying a substance according to the present invention is completed based on such findings, and is a method for quantifying a substance to be measured in a sample solution by enzyme immunoassay, which comprises: The bead carrier on which the specifically binding antibody or antigen is immobilized is arranged in the microchannel so as to be arranged in a row or in substantially one row in the width direction of the microchannel, and the sample solution and the enzyme labeled After sending a mixed solution in which the solution containing the substance to be measured is mixed, the luminescence or coloring in the vicinity of the bead carrier is detected while feeding the substrate solution containing the substrate.
  • a substrate is included when detecting the degree of luminescence or color development.
  • the luminescent material or the chromogenic substance continuously produced by the reaction of the enzyme and the substrate is rapidly removed from the bead carrier surface and allowed to flow downstream and stay on the bead carrier surface. I'm preventing. For this reason, the generation rate of the light emitting material or the coloring material which hardly receives the influence of the unnecessary light emitting material or the coloring material is almost exactly reflected on the light emission intensity or the coloring intensity.
  • the number of beads arranged in the microchannel is small, such as arranging one bead or approximately one row in the width direction of the microchannel.
  • the effect of luminescent or chromogenic substances generated in the vicinity of other beads is reduced as much as possible, and detection sensitivity is improved.
  • the luminescent material or the coloring material is continuously generated by continuously supplying the enzyme substrate to the bead surface at the time of detection, the light number is sufficiently high even if the number of beads is small.
  • the intensity or coloring intensity is secured to realize high sensitivity detection.
  • the device for quantifying a substance is a device for quantifying a substance to be measured in a sample solution by enzyme immunoassay, and is an antibody or an antigen that specifically binds to the substance to be measured.
  • a narrow portion provided in the middle of the minute channel for flowing the liquid and preventing the movement of the bead carrier downstream, in the narrow portion While the bead carrier is arranged in one or in substantially one row in the width direction of the microchannel, a mixed solution in which the sample solution and a solution containing the substance to be measured which is enzyme-labeled are mixed is delivered to the microchannel. After being liquidated, it is characterized in that luminescence or coloring in the vicinity of the bead carrier is detected while a substrate solution containing the substrate is fed.
  • the measuring device of the present invention sequentially sends various solutions to the micro channel, it can be determined easily and easily by any convenient operation, which eliminates the need for complicated operations and measurement in a short time. Is possible! /, It also has the advantage. Effect of the invention
  • the present invention by arranging one bead carrier or approximately one row in the width direction of the microchannel, it is possible to keep the substrate solution flowing on the bead carrier surface at the time of detection of luminescence or color development. It is possible to provide a method of quantifying a substance that can be quantified by sensitivity. Further, according to the present invention, it is possible to provide a quantitative device capable of easily and easily realizing high sensitivity measurement of a substance to be measured.
  • FIG. 1 is a schematic view for explaining an example of a quantification method to which the present invention is applied.
  • FIG. 2 is a photograph showing a state in which a substrate supplied on the surface of a bead reacts with an enzyme and emits light according to the quantification method to which the present invention is applied.
  • FIG. 3 is a schematic perspective view showing an example of a micro flow chip to which the present invention is applied.
  • FIG. 4 is a schematic cross-sectional view along the length direction of the microchannel of the microflow chip shown in FIG.
  • FIG. 5 is a schematic cross-sectional view along the width direction of the microchannel of the microflow chip shown in FIG.
  • FIG. 6 is an enlarged photograph of the area of the narrow portion of the micro flow chip shown in Fig. 3 as viewed in plane.
  • FIG. 7 is a schematic plan view showing an example provided with a plurality of microchannels, which is a micro flow chip to which the present invention is applied.
  • Fig. 8 is a schematic diagram for explaining the measurement principle by o-ferie diamine (OPD).
  • OPD o-ferie diamine
  • FIG. 9 is a schematic view for explaining the principle of detection of fluorescence using 10-acetonitrile 3 and 7-dihydroxydienoxazine.
  • FIG. 10 is a characteristic diagram for confirmation of antibody immobilization and determination of optimal FK-506 POD concentration.
  • FIG. 11 is a characteristic diagram showing a calibration curve when about 100 beads are used.
  • FIG. 12 is a characteristic diagram showing a change in calibration curve depending on the number of beads.
  • FIG. 13 is a characteristic diagram showing a calibration curve when 10 beads are used.
  • FIG. 14 is a photograph showing the luminescence of beads when 10 beads were used and a microflow antibody-type chip was used.
  • FIG. 15 is a characteristic diagram showing a calibration curve when using 10 beads and using a microflow antibody-type chip.
  • FIG. 16 is a characteristic diagram showing a calibration line when using one bead and using a microflow antibody type chip.
  • FIG. 17 is a photograph showing the luminescence of the bead when using one microbead and using a microflow antibody-type chip.
  • FIG. 18 is a diagram for explaining the principle of direct competition ELISA.
  • the quantification method to which the present invention is applied is a method of quantifying a measurement target substance in a sample by applying an enzyme-linked immunosorbent assay (ELISA), which comprises one bead carrier or one microflow in a minute flow path. While arranging so as to be arranged in approximately one row in the width direction of the passage, while flowing a substrate solution containing a substrate onto the surface of the bead carrier to which the enzyme is bound, the surface of each bead carrier is in the vicinity of the surface. For example, the degree of fluorescence is detected by the reaction between the enzyme and the substrate.
  • ELISA enzyme-linked immunosorbent assay
  • a direct competition ELISA when using a direct competition ELISA, first, a predetermined number of bead carriers on which an antibody that specifically binds to a substance to be measured is immobilized are arranged in a microchannel, and the target substance and the substance to be measured are placed on the bead carrier surface. An enzyme-labeled substance to be measured at a known concentration is supplied and reacted competitively. Next, while the substrate solution containing the substrate is continuously supplied to the bead carrier surface, the fluorescence near the bead carrier surface due to the reaction between the enzyme and the substrate is detected and measured based on the degree of the detected fluorescence. Determine the target substance.
  • the substance to be measured is not particularly limited, and any substance can be adopted such as, for example, a drug such as an immunosuppressant and various compounds such as a carcinogen such as dioxin.
  • a bead carrier for immobilizing an antibody that specifically binds to a substance to be measured has a large surface area, can immobilize a large amount of antibody etc., has a large contact area with a sample solution, and can be easily arranged in a microchannel. Certain things are also effective.
  • the bead carrier is not particularly limited as long as it can immobilize an antibody or the like that specifically binds to the substance to be measured, and for example, glass, polystyrene or the like can be used.
  • the bead carrier can also be reused by removing the immobilized antibody after measurement and washing.
  • the number of bead carriers can be generated in the vicinity of other beads by setting the number of bead carriers to one or a small number such as arranged in approximately one row in the width direction of the microchannel. Can reduce the influence of the Furthermore, by using a small number of bead carriers, the desired number of bead carriers can be simply and accurately disposed in the microchannel. Having one bead carrier is advantageous in that the error in analysis can be reduced.
  • the enzyme used to label the substance to be measured is not particularly limited, and enzymes such as peroxidase, galactosidase, and the like that generate a fluorescent substance by reacting with a substrate can be used. Further, as the substrate contained in the substrate solution, a fluorescent substrate which becomes a fluorescent substance such as, for example, resorufin by enzymatic reaction can be used.
  • a fluorescent substrate which becomes a fluorescent substance such as, for example, resorufin by enzymatic reaction can be used.
  • the use of fluorescent substrates is more sensitive than other luminescent or chromogenic substrates, such as luciferin, for example. It is preferable in the possible point.
  • the concentration of the substance to be measured is measured using a direct competitive ELISA as an example is an antibody as a substance that specifically binds to the substance to be measured.
  • a bead 2 on which an antibody 1 that specifically binds to a substance to be measured is immobilized is prepared, and a minute flow such that the flow of the liquid interferes with the flow of the bead 2 Place beads 2 in the street.
  • the number of beads 2 should be such that one bead or one bead or two beads will be arranged in substantially one row in the width direction of the microchannel.
  • a solution containing a sample solution containing measurement target substance 3 of unknown concentration, and a solution containing labeled substance 4 (known concentration) obtained by labeling the measurement target substance (antigen) with an enzyme The mixed solution thereof is supplied to the surface of the beads 1 for 5 minutes, for example, to cause the substance 3 to be measured and the labeled substance 4 to compete with each other.
  • the flow rate of the mixed solution should be 10 1 Z min or less from the viewpoint of reliably advancing the antigen-antibody reaction. preferable. However, if the flow rate of the mixed solution is too small, nonspecific adsorption may occur on the bead surface, so for example, 1 ⁇ 1 Z min or more is preferable.
  • buffer 1 etc. is supplied for 5 minutes, for example, and the substance to be measured 3 and the labeled substance 4 nonspecifically adsorbed on the surface of the beads 1 are washed and removed.
  • a substrate solution containing a substrate 5 that specifically reacts with the enzyme is supplied, and while flowing through the substrate solution, the fluorescence of the fluorescent substance generated by the enzyme reaction is measured. To detect. Then, the concentration of the substance to be measured 3 in the sample solution is determined based on the calibration curve prepared separately.
  • the flow rate of the substrate solution is preferably set so that the substrate supply rate is higher than the reaction rate of the enzyme on the surface of the bead 1. If the substrate supply rate is smaller than the reaction rate of the enzyme, the reaction rate of the enzyme may be saturated, and accurate fluorescence intensity may not be measured.
  • FIG. 2 A state in which the substrate supplied on the surface of the bead carrier is reacted with the enzyme by the above-described quantitative method is shown in FIG. As shown in FIG. 2, fluorescence due to the fluorescent substance generated by the enzyme reaction is observed along the surface of the bead carrier.
  • the fluorescent substance continuously generated by the reaction between the enzyme and the substrate is rapidly flowed downstream, and the retention of the fluorescent substance in the vicinity of the bead carrier is prevented. This makes it possible to reduce the background while securing a sufficient fluorescence intensity.
  • the number of bead carriers to one or a small number such that they are arranged in approximately one row in the width direction of the microchannel, fluorescent substances generated in the vicinity of other bead carriers interact with each other. Can be minimized. Therefore, according to the present invention, it is possible to accurately know the fluorescence intensity (generation rate of the fluorescent substance) in the vicinity of the surface of each bead carrier while securing a sufficient fluorescence intensity, and highly sensitive quantification of the measurement target substance is possible. It becomes possible.
  • the fluorescence intensity when the fluorescence intensity is measured while the flow of the substrate solution is stopped, as in the conventional ELISA, for example, the fluorescence intensity significantly increases due to the retention of the fluorescent substance around the beads, and the fluorescence intensity increases. Accurate measurement of strength becomes difficult.
  • the bead carriers are arranged in two or more rows in the width direction of the microchannel, the fluorescence in the vicinity of the bead carriers mutually affects each other, again making accurate measurement of the fluorescence intensity difficult. Become.
  • a method for quantifying a substance to be measured by flowing a sample solution on the surface of a bead carrier by a flow is described in the above-mentioned Japanese Patent Application Laid-Open No. 2001-116753.
  • the invention described in Japanese Patent Application Publication No. hei8-99 is to directly attach a fluorescent labeling substance to a substance (hormone receptor) on the surface of a bead carrier and observe fluorescence in this state, and after binding an enzyme to the surface of the bead carrier, the substrate is It is fundamentally different from the technique of the present invention which is introduced and lighted by the enzyme.
  • the bead carrier since the fluorescence directly bound to the bead carrier is detected, the bead carrier may be, for example, a small number such as arranged in a single row in the width direction of the flow path. In that case, weak fluorescence can not be obtained.
  • the present invention As described above, by using the enzyme and keeping the substrate solution flowing, fluorescence can be obtained even if the number of bead carriers used is small.
  • the method for quantifying the substance as described above is, for example, a quantitation device (microflow chip) in which the flow of the solution around the bead carrier is finely controlled by having an extremely narrow microchannel as described below. Can be performed reliably and simply.
  • 3 is a perspective view of the microflow-type chip 11
  • FIG. 4 is a cross-sectional view along the microchannel of the chip 11
  • FIG. 5 is a cross-sectional view of the microchannel in the width direction near the narrow portion.
  • 6 is an enlarged photograph of the narrow area seen from the plane.
  • a narrow micro channel 13 in the form of a groove is formed on the surface of a substrate 12 made of a transparent resin material such as polydimethyl siloxane (PDMS), for example, in the micro flow type chip 11.
  • PDMS polydimethyl siloxane
  • beads 14 in which an antibody, an antigen, etc. are bound to a bead carrier.
  • a lid 17 on which a hole for sample introduction (sample introduction part) 15 and a hole for sample discharge (sample discharge part) 16 are formed.
  • a convex portion 18 is provided in the middle of the length direction of the microchannel 13 of the lid 17 to allow the flow of the solution in the microchannel 13 while preventing the downstream flow of the beads 14.
  • a detector 20 such as a colorimeter is disposed around the chip 11 so as to detect the fluorescence in the vicinity of the surface of the bead 14 staying on the upstream side of the narrowing portion 19.
  • the optimum shape and size of the microchannel 13 vary depending on the shape and size of the bead 14 to be placed, but the groove depth of the microchannel 13 is set in the microchannel 13
  • the diameter of the bead 14 is greater than or equal to twice the diameter of the bead 14.
  • the beads 14 are completely immersed in the solution, and only one bead 14 is arranged in the height direction, that is, the beads 14 are in the height direction. (The direction of detection) will not overlap.
  • the fluorescence in the vicinity of the bead surface can be easily spatially distinguished from other fluorescence and measured, and the intensity can be measured more accurately. Therefore, the S / N ratio is improved, and the sensitivity can be dramatically improved.
  • a plurality of beads 14 may be micro flow channels 13. When arranged inside, these beads 14 are arranged in rows along the width direction of the microchannel 13.
  • the arrangement of the beads 14 in the minute channel 3 may be carried out manually with tweezers or the like, or may be carried out, for example, by liquid transfer using a syringe pump!
  • the number of beads 14 arranged in the micro channel 13 is set to a small number from the viewpoint of high sensitivity measurement, and in the present invention, the number of beads 14 is set to one or approximately 1 row in the width direction of the micro channel. .
  • beads 14 to which an antibody that specifically binds to the substance to be measured is immobilized are prepared, and a predetermined number of the beads 14 are arranged on the upstream side of the narrowing portion 19 of the microchannel 13.
  • the non-bound or non-specifically bound measurement target substance and enzyme labeled substance are washed.
  • the substrate solution is supplied from the solution introducing tube (not shown) connected to the sample introducing unit 15 to the microchannel using a syringe pump. Then, while supplying the substrate solution, the detector 20 detects fluorescence near the surface of the bead 14. At this time, since the substrate is kept flowing by the syringe pump, at the same time as the substrate is continuously supplied to the enzyme bound to the beads 14, the fluorescent substance generated by the enzyme reaction flows in the sample discharge portion by the flow of the solution. Continue to be discharged from 16. As a result, the fluorescent substance does not stagnate in the vicinity of the beads 14, and the influence of knock ground and the like is reduced, so that accurate fluorescence intensity can be measured. Therefore, by using the chip 1 of the present invention, the SN ratio can be improved, the detection sensitivity can be dramatically improved, and accurate determination can be performed.
  • the chip 1 of the present invention since the chip 1 of the present invention is small, it has an advantage of being excellent in portability.
  • the microchannel formed in the chip 1 is a very minute space, it is possible to suppress an increase in the amount of use of various solutions and shorten the measurement time.
  • a chip used in the quantification method of the present invention is a single chip as shown in FIGS.
  • the present invention is not limited to the one in which the microchannel 13 is formed, but may be one in which a plurality of microchannels are formed.
  • An example of such a multi-flow chip is shown in FIG.
  • eleven microchannels 13 are arranged in parallel in a comb shape on the surface of a substrate 12 which is also a transparent material.
  • the sample introduction unit 15 is shared, and a sample discharge unit 16 for discharging the liquid after liquid feeding is individually formed in each of the microchannels 13.
  • Ten of the microchannels 13 are microchannels for preparing a calibration curve, and the remaining one is a microchannel for sample solution.
  • Indirect competition E LISA is prepared by adding the substance to be measured and the primary antibody to the one obtained by immobilizing a hapten bound to the antigen used for the immunogen or protein different from the immunogen on the surface of the carrier such as beads, After washing away the substance to be measured and the primary antibody which did not bind to the immobilized antigen, the secondary antibody is added to bind to the primary antibody, and then the substrate is added to cause fluorescence reaction by the enzyme reaction.
  • the present invention can also be applied to so-called sandwich ELISA.
  • the above-mentioned competition method is suitable to be applied when the substance to be measured is a small molecule, whereas the sandwich method is suitable to be applied to cases where the molecular weight of the substance to be measured is relatively large.
  • a substrate solution is prepared using a luminescent substrate other than a fluorescent substrate, specifically, a luminescent substrate such as luciferase, and an optimum enzyme is appropriately selected according to the substrate, and bioluminescence associated with the enzyme reaction is generated. ( ⁇ ⁇ ⁇ ⁇ ) can be used for detection.
  • color development may be used for detection.
  • an optimum enzyme may be appropriately selected depending on the substrate, and color development associated with the enzyme reaction may be detected.
  • the chromogenic substrate include o-phenylenediamine (OPD) and tetramethylbenzidine (TMB).
  • the drug FK 506 which is used as an immunosuppressant at the time of organ transplantation, was used as the measurement target substance, and quantification was performed by ELISA using a short micro flow path microflow antibody-type chip in order to simplify the system.
  • a quantitative method an FK506 antibody immobilized on polyethylene beads is placed in a micro channel, and a mixed solution of FK 506 and FK 506-peroxidase (POD) (horseradish peroxidase (HRP) conjugated FK 506) is added to the micro channel. The reaction was allowed to proceed, and the fluorescent substance produced by peroxidase and the substrate was measured using an optical detection system.
  • a microflow antibody-type chip produced as follows was used.
  • anti-FK506 mouse monoclonal antibody (Funakoshi Co., Ltd.), FK506, FK506-POD (provided by Fujisawa Pharmaceutical Co., Ltd.), o-Felen-diamine (OPD) ) (Manufactured by SIGMA CHEMICALS), Amplex (TM) re d ELIS A kit (Molecular probe), hydrogen peroxide (30% aqueous solution, for biochemistry, Wako Pure Chemical Industries), ushi serum albumin (SIGMA CHEMICALS), polydimethylsiloxane (PDMS: polydimethyl siloxane) 'Manufactured by Asia, Silpot 184 Silicon Elastmer kit', FEP tube (manufactured by BAS), polystyrene beads (manufactured by Polyscience, diameter 90 ⁇ m), glass plate (manufactured by MATSUNAMI), immuno module (strip and frame, F16, manufactured by NU
  • the apparatus includes a stereo fluorescent microscope (manufactured by Leica, MZFL III), a CCD camera (manufactured by Hamamatsu Photonitas, ORCA-ER), a syringe pump (manufactured by HARVARD APPARATUS), a spectrophotometer (manufactured by JASCO, V530).
  • a stereo fluorescent microscope manufactured by Leica, MZFL III
  • CCD camera manufactured by Hamamatsu Photonitas, ORCA-ER
  • a syringe pump manufactured by HARVARD APPARATUS
  • a spectrophotometer manufactured by JASCO, V530.
  • Direct competition ELISA is a method in which an antibody is coated (immobilized) on a microplate or test tube, etc., and a substance to be measured (sample or standard substance) and an antigen labeled with an enzyme are added for competition reaction to bind to the antibody. After washing away the substance to be measured and the labeled antigen, the enzyme substrate is added to make the enzyme react with light, and then the degree of luminescence is measured with a colorimeter etc. The luminescence degree of the standard substance of the substance to be measured This is a method of measuring the concentration of the substance to be measured by comparison.
  • the antibody was immobilized on the surface of a polystyrene bead, and placed in the microchannel of the microflow antibody type chip for measurement.
  • Antibody immobilized beads were prepared by the following method.
  • the immobilized beads are washed with PBS, soaked in PBS (BT-PBS) containing 0.05% Tween 20, 0.5% sushi serum albumin (BSA) and soaked overnight. Blocking of the bead surface was performed. The obtained beads are washed three times with PBS containing 0.05% Tween 20 (T-PBS), and the T-PBS The medium was stored until use at a temperature of 4 ° C.
  • a 10 / z 1 Z ml methanol solution of FK506 was prepared, and this was diluted with methanol to make a 0.1 ng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml, 100 ng / ml solution, 100 ng / ml solution. Each one of them was placed in a glass tube and concentrated to dryness under a stream of nitrogen. FK506-POD was diluted 10-fold with BT-PBS, and 2001 each was added for dissolution.
  • the obtained sample solution was put into an Epppen tube in 180 ⁇ l aliquots, 101 of bead solution (about 100 beads) was added thereto, and an antigen-antibody reaction was carried out at room temperature for 2 hours.
  • the enzyme reaction was then stopped by adding 2 M sulfuric acid (501).
  • the reaction solution was dispensed in 200 1 aliquots into 96-well plates, and the absorbance at 490 nm was measured.
  • a solution of FK506 in ⁇ / z lml was prepared, and these were diluted with methanol to prepare a solution of 0.1 ngz ml, 0.1 lng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml, 100 ng / ml.
  • Each 50 1 was added to a glass tube and concentrated to dryness under a nitrogen stream.
  • FK 506-POD was diluted 3-fold with BT-PBS, and each 1001 was added and dissolved.
  • the resulting sample solution was placed in an aliquot of 90 ⁇ l each into an Eppendorf tube, and 5 ⁇ l of the bead solution was added thereto to carry out an antigen-antibody reaction for 2 hours at room temperature.
  • the resulting reaction solution was washed nine times with T-PBS. After that, 1 bead, 3 beads and 10 beads were placed in duplicate on a 96-well plate.
  • 2% sulfuric acid 50 ⁇ l was added to stop the enzyme reaction.
  • the reaction solution was divided into 200 ⁇ l aliquots in a 96-well plate, and the absorbance at 490 nm was measured.
  • the resulting sample solution was placed in an aliquot of 90 ⁇ l per well, and the bead solution 51 was added thereto to carry out an antigen-antibody reaction for 2 hours at room temperature.
  • the resulting reaction solution was washed 9 times with T-PBS, and then 10 beads were placed on a 96-well plate, respectively.
  • 200 1 was added and reacted at room temperature for 20 minutes.
  • the enzyme reaction was then stopped by adding 2 M sulfuric acid (501).
  • the reaction solution was divided into 200 aliquots in a 96-well plate, and the absorbance at 490 nm was measured.
  • the preparation procedure of the micro flow type antibody chip used in this research is as follows.
  • the silicon wafer was cut into 40 mm ⁇ 30 mm with a diamond cutter and cleaned. This cleaning was performed by ultrasonic cleaning with pure water and boiling in acetone, and then immersed in a mixed solution of hydrofluoric acid and ammonium (1: 6) for 5 minutes to remove the natural oxide film.
  • SU-8 manufactured by MicroChem, NANO XP SU-850
  • Pre-bake was performed under the following conditions.
  • the spin coating conditions were changed so that the film thickness of SU-8 became 100 m, and coating was performed. Then, the pre-baked silicon substrate is irradiated with ultraviolet light by a mask alignment device through a mask pattern (manufactured by Yamada Photographic Engraving Co., Ltd.), and again, the temperature is 65 ° C .: 3 minutes, 95 ° C .: 10 on a hot plate. I did post-baking in minutes. This substrate The substrate was immersed in a SU-8 developer for about 10 minutes, and upon irradiation with ultraviolet light by a mask alignment device, SU-8 on a non-hardened substrate was removed and dried to obtain a target mold.
  • a chip using PDMS as a substrate was produced.
  • the PDMS prepolymer and the catalyst were mixed (10: 1) and thoroughly mixed, and then the pressure was reduced with a rotary vacuum pump in a bell jar to degas the bubbles present in the mixture.
  • PDMS was poured on a mold and heated and cured in an oven at 80 ° C. for 1 hour.
  • the silicon rubber sheet provided a frame on the mold so that the PDMS would not spill.
  • PDMS was carefully removed from the mold, and a PDMS chip to which the mold was transferred was produced.
  • the transferred chip was immersed overnight in a 1 N hydrochloric acid solution to make the chip surface hydrophilic, washed with ultrapure water, and then immersed in BT-PBS to perform blocking in the microchannel.
  • microchannel formed in PDMS has a groove shape with a length of 30 mm, a width of 1000 ⁇ m, and a depth of 100 ⁇ m, and in the middle of the microchannel, the bottom force height of the microchannel is 50 A narrow portion opened by m is provided.
  • the operation procedure in the characterization of the microflow antibody chip basically conforms to Figure 1.
  • a mixed solution of FK506 diluted with each concentration and horseradish peroxidase (POD) -labeled FK506 (FK506-POD) in the microchannels of the chip on which the antibody-immobilized beads were placed using a syringe pump.
  • the reaction was introduced for 5 minutes at a flow rate of 1 ⁇ l Z minutes, and the antigen-antibody reaction was performed by the competition method on the surface of the antibody immobilized beads.
  • BT-PBS was introduced for 5 minutes at a flow rate of 10 / z lZ minutes and washing operation was performed.
  • Resolvin generated by Amplex (trademark) r ed was excited with excitation light at 546 nm, and a fluorescence image of fluorescence 590 nm was taken.
  • the principle of detection using 10-asacetinole 3,7-dihydroxydienoxazine is shown in FIG.
  • the obtained fluorescence image was analyzed by analysis software AQACOS MOS (manufactured by Hamamatsu Photonitas Co., Ltd.).
  • ROI (Region of Interest) was taken around the bead, and fluorescence intensity was calculated as an average value per unit pixel in ROI.
  • the concentration of FK506 was measured in the same manner as in the above-described operation 1 for evaluating the calibration characteristics of the above-mentioned microfuge type 1 antibody chip, where the number of beads to be arranged on the microflow antibody chip is one. Also, blood samples were prepared assuming actual blood samples. Likewise, the concentration of FK506 was measured with a microflow antibody chip for blood samples.
  • the assay was performed using FK506 dilution.
  • approximately 100 beads are included by dispensing a suspension of beads of a predetermined concentration.
  • I was supposed to be The results are shown in FIG. As shown in Fig. 11, although the variation is large, the correlation was obtained up to the concentration of 0. IngZml. The cause of this variation was considered to be the variation in the number of beads. When the bead solution was dispensed in suspension, the number of beads was actually counted, and an error of about 10% occurred.
  • the sensitivity (0. IngZml) of the conventional ELISA may be sufficient in the actual medical field, for example. Further simplification of the examination and improvement of the examination time in terms of high speed can be said to be crucial. So, in the next experiment, the number of beads was reduced and the same experiment was performed (with one bead). In the case of one bead, the sensitivity is expected to decrease according to the result of absorbance measurement using a 96-well plate. Ten beads have a sensitivity 1000 times that of the conventional ELISA method. Also in the case of individual, sensitivity equal to or higher than ELISA can be expected.
  • the sensitivity of this experiment was found to be about 100 times smaller than that obtained using 10 beads. Even with this method, the sensitivity was about 10 times that of the conventional ELISA method.
  • the arrangement of the beads changes (changes with each flow) every measurement, but there is a disadvantage that an analytical error is likely to occur, but in the case of one bead, R OI
  • the achievement can not be achieved by the conventional ELISA or MEIA (microparticle enzyme-mediated immunoassay).
  • MEIA microparticle enzyme-mediated immunoassay

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Plasma & Fusion (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

[PROBLÈMES] Rendre possible la quantification extrêmement sensible d’une substance cible. [MOYEN DE RÉSOLUTION DES PROBLÈMES] Un procédé de quantification d’une substance cible dans un extrait de solution utilisant un procédé de dosage immuno-enzymatique qui comprend la fourniture d’un ou de plusieurs micro-éléments porteurs, sur lesquels un anticorps ou un antigène spécifiquement lié à la substance cible a été immobilisé, dans un microcanal (en cas d’utilisation de deux micro-éléments ou plus, les micro-éléments sont disposés presque en ligne dans le sens de la largeur du microcanal), alimentant ainsi un mélange d’extrait de solution avec une solution contenant la substance enzymatique cible, et, pendant l’alimentation d’une solution de substrat contenant un substrat, détectant la luminescence ou la coloration autour des micro-éléments porteurs. Après alimentation du mélange de solution, par exemple, une lessive est alimentée, puis la solution de substrat est ensuite alimentée à son tour. Il est préférable d’alimenter le mélange de solution, la lessive et la solution de substrat en continu dans le microcanal. Il est également préférable de placer les micro-éléments porteurs dans le microcanal pendant l’évacuation du surplus dans le sens de la hauteur.
PCT/JP2005/014682 2004-08-11 2005-08-10 Procédé de quantification de substances et dispositif de quantification de substances WO2006016617A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006531695A JP4253695B2 (ja) 2004-08-11 2005-08-10 物質の定量方法及び物質の定量デバイス

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004234846 2004-08-11
JP2004-234846 2004-08-11

Publications (1)

Publication Number Publication Date
WO2006016617A1 true WO2006016617A1 (fr) 2006-02-16

Family

ID=35839383

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/014682 WO2006016617A1 (fr) 2004-08-11 2005-08-10 Procédé de quantification de substances et dispositif de quantification de substances

Country Status (2)

Country Link
JP (1) JP4253695B2 (fr)
WO (1) WO2006016617A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011215023A (ja) * 2010-03-31 2011-10-27 Central Res Inst Of Electric Power Ind 測定用部材、並びに標的検出装置及び標的検出方法
JP2012211960A (ja) * 2011-03-30 2012-11-01 Shimane Univ 物質相互作用をリアルタイムに可視化する技術
WO2014106881A1 (fr) * 2013-01-07 2014-07-10 パナソニック株式会社 Dispositif de conduit
WO2014168020A1 (fr) 2013-04-09 2014-10-16 株式会社Ｊｖｃケンウッド Dispositif pour l'analyse d'échantillon et procédé pour la capture d'exosome
JP2014219424A (ja) * 2014-08-26 2014-11-20 ローム株式会社 回転式分析チップおよびそれを用いた測定システム
WO2017171293A1 (fr) * 2016-03-31 2017-10-05 (주) 인텍플러스 Procédé de quantification hautement sensible de biomarqueurs par amplification de photo-oxydation

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7376583B1 (en) 1999-08-10 2008-05-20 Gofigure, L.L.C. Device for making a transaction via a communications link
US8055526B2 (en) 2006-09-08 2011-11-08 Varec, Inc. Method for the automated dispatch of fueling operations
KR102435668B1 (ko) * 2015-10-20 2022-08-24 주식회사 퀀타매트릭스 다중 분석 칩 및 이를 이용한 분석 장치

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001004628A (ja) * 1999-06-18 2001-01-12 Kanagawa Acad Of Sci & Technol 免疫分析装置と免疫分析方法
WO2003062823A1 (fr) * 2002-01-24 2003-07-31 Kanagawa Academy Of Science And Technology Puce et procede d'analyse de l'activite enzymatique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001004628A (ja) * 1999-06-18 2001-01-12 Kanagawa Acad Of Sci & Technol 免疫分析装置と免疫分析方法
WO2003062823A1 (fr) * 2002-01-24 2003-07-31 Kanagawa Academy Of Science And Technology Puce et procede d'analyse de l'activite enzymatique

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011215023A (ja) * 2010-03-31 2011-10-27 Central Res Inst Of Electric Power Ind 測定用部材、並びに標的検出装置及び標的検出方法
JP2012211960A (ja) * 2011-03-30 2012-11-01 Shimane Univ 物質相互作用をリアルタイムに可視化する技術
WO2014106881A1 (fr) * 2013-01-07 2014-07-10 パナソニック株式会社 Dispositif de conduit
WO2014168020A1 (fr) 2013-04-09 2014-10-16 株式会社Ｊｖｃケンウッド Dispositif pour l'analyse d'échantillon et procédé pour la capture d'exosome
JP2014219424A (ja) * 2014-08-26 2014-11-20 ローム株式会社 回転式分析チップおよびそれを用いた測定システム
WO2017171293A1 (fr) * 2016-03-31 2017-10-05 (주) 인텍플러스 Procédé de quantification hautement sensible de biomarqueurs par amplification de photo-oxydation
US11054427B2 (en) 2016-03-31 2021-07-06 Absology Co., Ltd Method of quantifying biomarker with high sensitivity using photo-oxidation induced amplification

Also Published As

Publication number Publication date
JP4253695B2 (ja) 2009-04-15
JPWO2006016617A1 (ja) 2008-07-31

Similar Documents

Publication Publication Date Title
WO2006016617A1 (fr) Procédé de quantification de substances et dispositif de quantification de substances
KR101394221B1 (ko) 한 번의 시료 주입으로 순차적인 반응 조건의 변화가 가능한 멤브레인 센서
KR20170099786A (ko) 항체 제공 키트, 항체 저장 패치, 이를 이용하는 면역 진단 방법 및 장치
US20180120310A1 (en) Method of and System for Printing In-Well Calibration Features
US10191037B2 (en) Methods of and systems for improved detection sensitivity of assays
US20210072237A1 (en) Method and device for determining biological analytes
CN102460127A (zh) 用于识别和定量分析被分析物，特别是真菌毒素的装置和方法
Guo et al. Immunoassays on thiol-ene synthetic paper generate a superior fluorescence signal
EP1512012B1 (fr) Nouveau procede pour surveiller des interactions biomoleculaires
JP2013543981A5 (fr)
JP4519794B2 (ja) マイクロ流路素子
JP5419775B2 (ja) 測定用基板、並びに、これを用いた生化学的結合形成および生化学的結合量の測定方法
US11130136B2 (en) Systems and methods to enhance consistency of assay performance
DK2603325T3 (en) METHOD AND SYSTEM FOR APPLICATION OF BLOCKING MATERIAL ON sample substrates
JP4600787B2 (ja) クロマトデバイス
Klapproth et al. Development of a multi-analyte CMOS sensor for point-of-care testing
JP2008209403A (ja) 要素上での複数の免疫化学アッセイ
KR102306097B1 (ko) 고감도 면역접합체, 이의 제조방법, 이를 포함한 체외 진단시약 및 체외 진단키트
WO2007016665A2 (fr) Dosages par fluorescence a usage unique servant a determiner des substances a analyser
WO2008018631A1 (fr) Complexe de liposome, réseau de liposome, et procédé de détection d'un analyte
WO2010106992A1 (fr) Procédé de dosage immunologique et disque d'inspection utilisé dans ledit procédé de dosage immunologique
WO2008044179A1 (fr) Biocapteurs et leur préparation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006531695

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase