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WO2016047027A1 - Procédé de détection de fluorescence et cellule échantillon pour la détection - Google Patents

Procédé de détection de fluorescence et cellule échantillon pour la détection Download PDF

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Publication number
WO2016047027A1
WO2016047027A1 PCT/JP2015/004059 JP2015004059W WO2016047027A1 WO 2016047027 A1 WO2016047027 A1 WO 2016047027A1 JP 2015004059 W JP2015004059 W JP 2015004059W WO 2016047027 A1 WO2016047027 A1 WO 2016047027A1
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Prior art keywords
sample
mixed phase
flow path
fluorescence
channel
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PCT/JP2015/004059
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English (en)
Japanese (ja)
Inventor
大塚　尚
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富士フイルム株式会社
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Priority to JP2016549906A priority Critical patent/JP6386062B2/ja
Publication of WO2016047027A1 publication Critical patent/WO2016047027A1/fr

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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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • 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
    • G01N21/62—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

Definitions

  • the present invention relates to a fluorescence detection method and a detection sample cell. More specifically, the target substance that specifically binds to the fluorescent label is caused to flow down to the microchannel, captured by the detection unit provided in the microchannel, and the fluorescence emitted by the fluorescent label is detected by irradiating excitation light.
  • the present invention relates to a fluorescence detection method and a detection sample cell for detecting the abundance of a substance to be detected based on the amount of fluorescence detected.
  • Fluorescence is widely used as a biochemical test using microchannels.
  • a sample liquid containing a fluorescent label that is excited by light of a specific wavelength and emits fluorescence, and a target substance that specifically binds to the fluorescent label is allowed to flow down into a microchannel, and a part of the microchannel It is made to capture by the detection part which is the provided solid surface (solid phase).
  • the excitation light is irradiated toward the detection unit that captures the target substance that specifically binds to the fluorescent label, the fluorescence emitted by the fluorescent label is detected, and the presence of the target substance is detected based on the detected amount of fluorescence. It is a method of detecting the quantity.
  • Patent Document 1 A technique for feeding a sample solution in a direction orthogonal to the surface of the detection unit as shown in FIG. 7A and FIG. 7B, and a sample solution as shown in FIG.
  • Patent Document 2 A liquid feeding technique
  • a reaction channel in which the reactant is fixed a sample injection channel that guides the sample solution to the reaction channel, and another channel that guides fluid other than the sample solution to the reaction channel are provided.
  • Patent Document 3 a technique in which a sample solution is caused to flow down along one wall surface on the reactant side by a fluid other than the sample solution.
  • Non-patent Document 1 A method for separating and classifying fine particles using a microchannel has been proposed (Non-Patent Documents 2 and 3).
  • FIG. 10 is a schematic diagram showing the feeding of the sample solution flowing down the microchannel.
  • the substance to be detected (antigen) flows down toward the center in the microchannel, and most of the substance to be detected does not pass through the region near the detection unit. Therefore, the amount of the detection object that reaches the vicinity of the detection unit due to the dispersion is only about 0.001% with respect to all the detection target substances in the sample liquid. That is, most of the substance to be detected is not captured by the detection unit, and it may be difficult to perform highly sensitive detection.
  • the width of the microchannel is about 100 ⁇ m, which is about 1000 times wider than the neighboring region, from the viewpoint of manufacturing, and it takes time for the substance to be detected located near the center to reach the vicinity of the detecting unit due to dispersion. There is also a possibility that detection in a short time becomes difficult.
  • Non-Patent Documents 1 to 3 do not make any suggestion about increasing the amount of the substance to be detected captured by the detection unit in the fluorescence method.
  • the present invention provides a fluorescence detection method and a detection sample that increase the amount of an object to be detected that is specifically bound to a fluorescent label, which is captured by a detection unit, and realizes high sensitivity and shortened detection time.
  • the purpose is to provide a cell.
  • a fluorescence detection method provides a fluorescent label that specifically binds to a substance to be detected, and causes a sample liquid containing the substance to be detected and the fluorescent label to flow down into the sample liquid channel.
  • the mixed phase liquid not containing the target substance and the fluorescent label is allowed to flow down to the mixed phase liquid flow path, and the sample liquid flown down from the sample liquid flow path and the mixed phase liquid flowed down from the multiphase liquid flow path into the sample liquid flow path and
  • the two-phase flow channel is narrower than the liquid channel and a two-phase flow of the sample liquid and the mixed phase liquid is generated in the two-phase flow channel.
  • the detection unit provided on one side wall captures the target substance specifically bound to the fluorescent label, irradiates the detection unit with excitation light, detects the fluorescence emitted by the fluorescent label by irradiation, and detects the detected fluorescence.
  • the abundance of the substance to be detected is detected based on the amount of.
  • fluorescence generated by a fluorescent label upon irradiation means fluorescence generated by exciting the fluorescent label directly or indirectly by irradiation with excitation light.
  • detecting the abundance of the substance to be detected means that the abundance of the substance to be detected is quantitatively detected including the presence or absence of the substance to be detected.
  • the evanescent light is oozed out from the detection unit by irradiation with excitation light, and the fluorescent label generates fluorescence by the evanescent light.
  • the detection unit includes a metal film, and the surface plasmon that enhances the electric field on the metal film by irradiating the metal film with excitation light at an incident angle greater than or equal to total reflection. It is desirable to generate
  • fluorescent beads in which fluorescent dye molecules are encapsulated in a translucent dielectric material that transmits excitation light and fluorescence.
  • the fluorescent beads are hydrophilic and the mixed phase solution is physiological saline having a concentration of 1.8% or more.
  • concentration means the concentration of sodium chloride.
  • the fluorescent beads are hydrophilic, and it is desirable to use an organic solvent as the mixed phase liquid.
  • the surface of the fluorescent beads is modified with an acidic or basic functional group
  • the pH of the sample solution is set to a value at which the functional group can undergo ionization decomposition
  • the pH of the mixed phase liquid is ionized by the functional group. It is desirable to make the value impossible.
  • the mixed phase solution contains beads not having a fluorescent dye, and the number of fluorescent beads per unit volume of the mixed phase solution is greater than the number of fluorescent beads per unit volume of the sample solution. It is desirable to increase it.
  • the detection sample cell according to the present invention is a detection sample cell used in the fluorescence detection method according to the present invention, and includes a sample liquid flow path for flowing down the sample liquid and a mixed phase liquid flow for flowing down the mixed phase liquid.
  • a two-phase flow channel communicating with the downstream end of the channel, the downstream end of the sample channel and the downstream end of the mixed phase liquid channel, and having a width narrower than any of the sample liquid channel and the detection liquid channel; It may be provided with a detection part provided in a part of the side wall of the two-phase flow channel that captures the specifically detected substance to be detected.
  • the width of the mixed phase liquid channel is equal to or larger than the width of the sample liquid channel.
  • the width of the two-phase flow channel is in the range of 5 ⁇ m or more and less than 100 ⁇ m.
  • the downstream end of the sample liquid channel, the downstream end of the mixed phase liquid channel, and the upstream end of the two-phase flow channel communicate with each other.
  • the angle formed between the sample liquid flow path and the two-phase flow path is equal to the angle formed between the mixed-phase liquid flow path and the two-phase flow flow path.
  • a two-phase flow of a sample solution and a mixed phase solution is generated in a two-phase flow channel, and the sample solution is caused to flow down along one side wall of the two-phase flow channel.
  • the detection unit provided in the apparatus captures the target substance specifically bound to the fluorescent label, irradiates the detection unit with excitation light, detects the fluorescence emitted by the fluorescent label by irradiation, and detects the target based on the detected amount of fluorescence.
  • the detected substance that specifically binds to the fluorescent label flows down the vicinity of the detection section along the side wall on the detection section side, and the amount captured by the detection section increases, resulting in high sensitivity.
  • the detection time can be shortened.
  • FIG. 1 is a diagram showing sample liquid feeding according to the prior art (part 1).
  • Fig. 2 is a diagram showing sample solution feeding according to the prior art (No. 2)
  • FIG. 3 is a diagram showing sample liquid feeding by the prior art (No. 3) Image of fluorescent particles flowing down the microchannel Schematic diagram showing sample liquid flow down the microchannel
  • FIG. 1 is a schematic diagram of a fluorescence detection apparatus 1 used in an embodiment of the present invention.
  • FIG. 2 is a block diagram of the fluorescence detection apparatus 1.
  • the fluorescence detection device 1 is a device using a surface plasmon enhanced fluorescence method.
  • the fluorescence detection apparatus 1 is equipped with a sample container KB for storing a sample liquid containing the antigen A, a detection sample cell 100 for capturing the antigen A, and a nozzle chip NC.
  • the sample liquid is, for example, serum, plasma, urine, and the like, and examples of the antigen A include hCG.
  • the fluorescence detection apparatus 1 includes a sample processing unit 11 that drives a nozzle chip NC and the like, a light irradiation unit 12 that irradiates the detection sample cell 100 with excitation light, a fluorescence detection unit 13 that detects fluorescence generated by the excitation light irradiation, A data analysis unit 14 for measuring the number of detected fluorescence and analyzing the abundance of the antigen A is provided.
  • the detection sample cell 100 injects a base material 100a on which a microchannel 110 is formed, a reagent cell 120 that contains a reagent containing a fluorescent label F, a mixed phase liquid cell 130 that contains a mixed phase liquid G, and a sample liquid S.
  • a resin such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), or amorphous polyolefin (APO) containing cycloolefin for the substrate 100a.
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methacrylate
  • PC polycarbonate
  • APO amorphous polyolefin
  • Fluorescent label F specifically binds to antigen A and emits fluorescence when irradiated with excitation light.
  • the mixed phase solution G is a solution that does not contain the antigen A and the fluorescent label F.
  • the sample solution S is derived from a body fluid, such as serum, plasma, urine, or runny nose
  • the mixed phase solution G is at least twice the normal concentration of saline (about 0.9 percent) (1.8 percent or more). Examples thereof include physiological saline having a concentration, and specifically, phosphate buffered saline (PBS) is preferably used.
  • PBS phosphate buffered saline
  • the sample liquid S and the mixed phase liquid G are flowed down into the microchannel 110 by the specimen processing unit 11.
  • the sample liquid S is prepared by extracting the sample liquid from the sample container KB by the nozzle tip NC, and mixing and stirring in the reagent cell 120.
  • the sample solution S contains the antigen A specifically bound to the fluorescent label F, the unbound antigen A, and the fluorescent label F.
  • FIG. 3 is a schematic diagram of the vicinity of the detection unit 200 in the microchannel 110.
  • the detection unit 200 includes a metal film 201 formed on a part of the inner wall of the microchannel and a primary antibody B fixed to the metal film 201.
  • the detection unit 200 captures the antigen A when the primary antibody B specifically binds to the antigen A by the antigen-antibody reaction.
  • Primary antibody B can be appropriately adjusted according to the type of antigen A.
  • antigen A which is hCG
  • an anti-hCG monoclonal antibody (Anti-hCGC5008 SP-5, manufactured by Medix Biochemical) can be used.
  • the primary antibody B is immobilized on the metal film 201 by subjecting the terminal to a carboxyl group and performing an amino coupling method.
  • the fluorescent label F is composed of a fluorescent bead FB and a secondary antibody C fixed to the fluorescent bead FB.
  • Secondary antibody C also specifically binds to antigen A by an antigen-antibody reaction.
  • the detection unit 200 captures the antigen A specifically bound to the secondary antibody C fixed to the fluorescent beads FB by specifically binding to the primary antibody B (sandwich method).
  • the fluorescent beads FB are desirably beads that encapsulate fluorescent dye molecules with a material that transmits fluorescence generated from the fluorescent dye molecules.
  • the fluorescent beads FB are prepared by preparing a polystyrene solution prepared by mixing polystyrene particles and a fluorescent dye solution of a fluorescent dye, impregnating them while mixing and evaporating, and then performing centrifugation.
  • the particle size of the produced fluorescent beads FB is the same as that of polystyrene particles, and the particle size of each fluorescent bead FB is uniform.
  • the particle size of the fluorescent beads FB is preferably 1 ⁇ m or more and less than 5 ⁇ m, and more preferably 1 ⁇ m or more and less than 3 ⁇ m.
  • the fluorescent beads FB for example, beads manufactured by Bangs Laboratories, Inc. with a particle size of 1 ⁇ m, an excitation wavelength of 660 nm, and a fluorescence wavelength of 690 nm can be used.
  • Secondary antibody C can also be appropriately adjusted according to antigen A.
  • anti-hCG monoclonal antibody Anti-Alpha subunit 6601 SPR-5, manufactured by Medix Biochemical
  • the secondary antibody C is fixed to the fluorescent bead FB by surface-modifying the fluorescent bead FB with a carboxyl group, aminoating the end of the secondary antibody C, and performing an amino coupling method.
  • the light irradiation unit 12 applies the excitation light L from the back side of the detection unit 200 to the total reflection condition.
  • the metal film 201 is irradiated through the prism at an incident angle as follows.
  • the evanescent wave E oozes out on the metal film 201.
  • the velocities of the evanescent wave E and the free electrons of the metal film 201 become equal, plasmon resonance is excited on the metal film 201, and light absorption / surface plasmon enhanced electric field is generated.
  • the fluorescent beads FB are excited by the evanescent wave E to emit enhanced fluorescence.
  • the surface plasmon enhanced electric field is generated in the range of about 200 nm from the surface of the metal film 201.
  • the fluorescence detector 13 detects the fluorescence as a fluorescence signal.
  • the fluorescence detection unit 13 for example, a photodiode, CCD, CMOS, or the like can be used.
  • the fluorescence detection unit 13 detects only the fluorescence signal using a filter (not shown) that blocks the excitation light.
  • the data analysis unit 14 creates a detection image based on the fluorescence signal, measures the amount of fluorescence per predetermined area in the detection image, and analyzes the number of antigens A present. .
  • the data analysis unit 14 may cause the sample solution S and the mixed phase solution G to flow down and analyze the number of antigens A after a predetermined time (for example, 1 to 20 minutes) has passed. By sampling the fluorescence signal at regular intervals (for example, 5 minutes) while flowing down G, the number of antigens A present may be analyzed based on the temporal change rate of the fluorescence intensity (rate method).
  • FIG. 4 is a diagram showing a partially enlarged view of the microchannel 110.
  • the micro flow channel 110 communicates with the injection well 140 (see FIG. 1) to allow the sample solution flow channel 113 to flow down, and the micro flow channel 110 communicates with the injection well 150 (see FIG. 1) to mix the mixed phase solution G.
  • the flow path 114, the downstream end of the sample liquid flow path 113 and the downstream end of the mixed phase liquid flow path 114, the downstream end of the two phase flow path 115, and the discharge well 160 (see FIG. 1) It is comprised from the discharge flow path 116 which connects.
  • the sample liquid channel 113 and the two-phase flow channel 115 communicate with each other at an angle ⁇ 1, and the mixed phase liquid channel 114 and the two-phase flow channel 115 communicate with each other at an angle ⁇ 2. Further, the downstream end of the sample liquid channel 113, the downstream end of the mixed phase liquid channel 114, and the upstream end of the two-phase flow channel 115 are in communication with each other.
  • the sample liquid channel 113, the mixed phase liquid channel 114, and the two-phase flow channel 115 are channels having a constant width, but the width d3 of the two-phase flow channel 115 is equal to the width d1 of the sample liquid channel 113 and the mixed phase.
  • the liquid channel 114 is narrower than any of the widths d2.
  • the discharge channel 116 has a widened portion 116a whose width increases toward the downstream direction at an angle ⁇ 3 and a constant width portion 116b that communicates with the widened portion 116a.
  • the sample liquid flow path 113, the mixed phase liquid flow path 114, the two-phase flow flow path 115, and the discharge flow path 116 constitute a so-called pinched flow path structure.
  • the detection unit 200 is provided on a part of the side wall 115a of the two-phase flow channel 115 on the sample solution channel 113 side.
  • the sample processing unit 11 injects the sample liquid S into the injection well 140 and the mixed phase liquid G into the injection well 150 and applies a negative pressure to the discharge well 160, whereby the sample liquid S and the mixed phase liquid G are sampled at the same flow rate.
  • the liquid flow path 113 and the mixed phase liquid flow path 114 flow down, and are simultaneously guided to the two-phase flow flow path 115.
  • the sample liquid S and the mixed phase liquid G guided to the two-phase flow channel 115 are mixed with each other in the two-phase flow channel 115 by the action of the pinched channel structure. It flows down as a two-phase flow along the opposite side wall 115b side.
  • the antigen A, the fluorescent label F, and the antigen A specifically bound to the fluorescent label contained in the sample liquid S flow down toward the central portion due to laminar flow in the sample liquid flow path 113, but two phases
  • the antigen A, the fluorescent label F, and the antigen A specifically bound to the fluorescent label F are caused to flow down while being pressed against the side wall 115a according to the principle of the so-called pinched flow fractionation method. .
  • the antigen A specifically bound to the fluorescent label F in the sample solution S passes through the vicinity of the metal film 201, and the amount of the antigen A captured by the detection unit 200 can be increased. Note that after the unbound antigen A is captured by the detection unit 200, the amount of the antigen A specifically bound to the unbound fluorescent label F flowing down along the side wall 115a also increases.
  • the antigen A specifically bound to the fluorescent label F not captured by the detection unit 200, the unbound antigen A, and the fluorescent label F are guided to the discharge channel 116.
  • the unbound fluorescent label F and the group consisting of the antigen A specifically bound to the fluorescent label F and the group consisting of the unbound antigen A flow different from each other. It flows down in the direction and is discharged from the discharge well 160.
  • the flow rate of the mixed phase liquid G guided to the two-phase flow channel 115 is larger than the amount of the sample liquid S guided to the two-phase flow channel 115.
  • the width of the sample liquid S is narrower than the width of the mixed phase liquid G, and the probability that the antigen A specifically bound to the fluorescent label F is close to the detection unit 200 increases.
  • the amount of antigen A captured by the detection unit 200 can be increased.
  • the width d2 of the mixed phase liquid flow path 114 is wider than the width d1 of the sample liquid flow path 113.
  • each flow rate can be calculated from the flow velocity distribution using the Hagen-Poiseuille flow equation.
  • the angle ⁇ 1 formed by the sample liquid path 113 and the two-phase flow path 115 and the mixed phase liquid are determined. It is desirable that the angle ⁇ 2 formed by the channel 114 and the two-phase flow channel 115 is equal. Further, it is desirable that the angle ⁇ 1 and the angle ⁇ 2 are in the range of 110 to 160 degrees. Further, the angle ⁇ 3 of the widened portion 116a is preferably in the range of 90 degrees to 130 degrees.
  • the width d3 of the two-phase flow channel 115 is preferably in the range of 5 ⁇ m or more and less than 100 ⁇ m, and more preferably 5 ⁇ m or more and less than 10 ⁇ m. Further, it is desirable that the width d1 of the sample liquid channel 113 and the width d2 of the mixed phase liquid channel 114 are 10 ⁇ m or more and less than 2000 ⁇ m.
  • the sample liquid channel 113, the mixed phase liquid channel 114, the two-phase flow channel 115, and the discharge channel 116 have uniform depths, and the depths are equal to each other.
  • FIG. 5A shows a detection image after the sample solution S having a molar concentration of antigen A of 90 pM is allowed to flow down to a normal detection sample cell for 15 minutes.
  • FIG. 5B shows a detection image after the sample solution S having a molar concentration of antigen A of 0 pM is allowed to flow down to the detection sample cell in the normal channel for 15 minutes.
  • the number of fluorescence measured using a fluorescence microscope was about 63 per unit area (1 mm 2 ) in FIG. 5A.
  • the detection image in FIG. 5B is an image as a signal indicating the number of fluorescence correlated with the abundance of the antigen A. In FIG. 5B, the number was about 25 per unit area (1 mm 2 ).
  • the detection image in FIG. 5B shows the number of fluorescence emitted by the fluorescent label F nonspecifically adsorbed on the metal film 201, and is an image showing noise.
  • the amount of the sample solution S is about 100 ⁇ L.
  • FIG. 6A is a detection image after the sample solution S and the mixed phase solution G having the antigen A molar concentration of 90 pM are allowed to flow down to the detection sample cell 100 for 2 minutes.
  • FIG. 6B shows a detection image after the sample solution S and the mixed phase solution G having the antigen A molar concentration of 0 pM are allowed to flow down to the detection sample cell 100 for 2 minutes.
  • the amount of the sample liquid S in the two-phase flow channel 115 is about 100 ⁇ L, and the amount of the mixed phase liquid G is about 3000 ⁇ L.
  • the measured number of fluorescence was about 8125 per unit area (1 mm 2 ) in FIG. 6A and about 156 per unit area (1 mm 2 ) in FIG. 6B. Therefore, the S / N ratio when using the normal detection sample cell is 63/25, whereas the S / N ratio when using the detection sample cell 100 is 8125/156, which is the normal flow rate.
  • the sensitivity has been improved by about 20 times compared to the case of using a road.
  • the antigen A specifically bound to the fluorescent label F flows down along the side wall 115a. There is no need to wait until the vicinity of 201 is distributed. Therefore, the flow rate of the sample liquid S can be increased, and a detection image can be obtained after the sample liquid S and the mixed phase liquid G have flowed down for 2 minutes, and the detection time can be shortened.
  • the image after the sample liquid S and the mixed phase liquid G have flowed down for 15 minutes is 100 as compared with the case of the normal flow path. An S / N exceeding double was confirmed.
  • suppressing the dispersion of the antigen A specifically bound to the fluorescent label F in the mixed phase liquid G in the two-phase flow channel 115 also improves the probability of being captured by the detection unit 200.
  • first to third modifications of the present embodiment that can suppress the dispersion of the antigen A specifically bound to the fluorescent label F into the mixed phase solution G will be described.
  • the first modification is a method of incorporating polystyrene beads having no fluorescent dye in the mixed phase liquid G.
  • the number of polystyrene beads per unit volume in the mixed phase liquid G is made larger than the number of fluorescent beads FB per unit volume in the sample liquid.
  • the bead concentration of the mixed phase solution G that is, the bead concentration contained per unit volume becomes higher than the bead concentration of the sample solution S. Therefore, the difference in the bead concentration between the sample solution S and the mixed phase solution G makes it difficult for the fluorescent beads FB to be dispersed in the high concentration mixed phase solution G. As a result, the antigen A specifically bound to the fluorescent label F Dispersion in the mixed phase liquid G is also suppressed.
  • the data analysis unit 14 is more efficient than the rate method described above. It is preferable to analyze the sample liquid S and the mixed phase liquid G after a predetermined time has elapsed.
  • the second modification will be described.
  • the second modification is a method using an organic solvent as the mixed phase liquid G.
  • the surface of the fluorescent bead FB is modified with a functional group. Since the functional group is ionized in the sample solution S, the hydrophilicity of the fluorescent beads FB is increased.
  • a carboxyl group (—COOH) that is an acidic functional group is used, and this carboxyl group is ionized into COO— in the sample solution S.
  • a sulfonic acid group or the like is used as the acidic functional group, the ionized state is similarly obtained.
  • the hydrophilicity of the fluorescent beads FB is increased.
  • an amino group —NH 2
  • the sample solution S is ionized to NH 3 +.
  • a quaternary ammonium group or the like is used as a basic functional group, the ionized state is similarly obtained.
  • the mixed phase solution G becomes hydrophobic for the fluorescent beads FB, and as a result, dispersion of the antigen A specifically bound to the fluorescent label F into the mixed phase solution G is suppressed.
  • ethanol, methanol, dimethyl sulfoxide (DMSO) can be used as the organic solvent.
  • the third modification is a method of adjusting the pH of the mixed phase liquid G according to the type of functional group.
  • the functional group is ionized in the sample solution S.
  • the fluorescent bead FB is negatively charged.
  • the fluorescent bead FB is positively charged.
  • the pH of the sample solution S is a value at which functional groups are ionized, and specifically the pH is about 7.4.
  • the pH value of the mixed phase liquid G is set to a pH at which the functional group is not ionized. Specifically, when the surface of the fluorescent beads FB is modified with an acidic functional group, the pH of the mixed phase solution G is made lower than the pH of the sample solution S, and the surface of the fluorescent beads FB is modified with a basic functional group. The pH of the mixed phase solution G is made larger than the pH of the sample solution.
  • the charge concentration of the mixed phase solution G becomes higher than the charge concentration of the sample solution S. Therefore, due to the difference in charge concentration between the sample solution S and the mixed phase solution G, the functional group is difficult to separate in the mixed phase solution G, and as a result, the mixed phase solution G of the antigen A specifically bound to the fluorescent label F is obtained. Dispersion into is suppressed. Specifically, when the fluorescent bead FB is modified with an acidic functional group, the pH of the mixed phase solution is set to less than 4, and when the fluorescent bead FB is modified with a basic functional group, the pH of the mixed phase solution is set to exceed 10. desirable.
  • the present invention increases the amount of antigen A captured by the detection unit and realizes high sensitivity of the fluorescence method. Therefore, in addition to the surface plasmon enhanced fluorescence method, the evanescent fluorescence method and the optical waveguide are used.
  • the present invention can be applied to a fluorescence detection method and various fluorescence detection methods.

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Abstract

Le problème décrit par la présente invention est, dans un procédé de détection de fluorescence pour capturer une substance à détecter qui se lie spécifiquement à un marqueur fluorescent, et une cellule d'échantillon pour la détection de celle-ci, d'augmenter la quantité capturée de la substance à détecter, et d'atteindre une sensibilité élevée et un temps de détection plus court. La solution de l'invention porte sur une solution échantillon (S) contenant un antigène (A) se liant spécifiquement à un marqueur fluorescent (F) et autorisé à s'écouler vers le bas d'un canal (113) de solution échantillon. Une solution à phase mixte (G) ne contenant pas l'antigène (A) ni le marqueur fluorescent (F) est autorisée à s'écouler vers le bas d'un canal (114) de solution à phase mixte. La solution échantillon (S) et la solution à phase mixte (G) sont guidées vers un canal (115) d'écoulement à deux phases qui est plus étroit que les largeurs à la fois du canal (113) de solution échantillon et du canal (114) de solution à phase mixte. Dans le canal (115) d'écoulement à deux phases, un écoulement à deux phases de la solution échantillon (S) et de la solution à phase mixte (G) se produit, et la solution échantillon (S) est autorisée à s'écouler vers le bas le long d'une paroi latérale (115a). Une unité de détection (200) située sur la paroi latérale (115a) capture l'antigène (A) lié de manière spécifique au marqueur fluorescent (F), une lumière d'excitation (L) est dirigée sur l'unité de détection (200) et la fluorescence est détectée, et la quantité de l'antigène (A) présent est détectée sur la base de la fluorescence détectée.
PCT/JP2015/004059 2014-09-24 2015-08-17 Procédé de détection de fluorescence et cellule échantillon pour la détection WO2016047027A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018054495A (ja) * 2016-09-29 2018-04-05 コニカミノルタ株式会社 血液検査用センサチップ、血液検査装置及び血液検査方法
CN111417853A (zh) * 2018-04-06 2020-07-14 松下知识产权经营株式会社 病原体检测装置以及病原体检测方法
JP2020159901A (ja) * 2019-03-27 2020-10-01 日本無線株式会社 弾性表面波センサ及び弾性表面波センサを用いる抗体抗原混和検出方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009109245A (ja) * 2007-10-26 2009-05-21 Canon Inc 検出方法および検出装置
JP2012143205A (ja) * 2011-01-14 2012-08-02 Hitachi High-Technologies Corp 核酸分析装置
JP2013083632A (ja) * 2011-09-28 2013-05-09 Fujifilm Corp 蛍光粒子を用いた検出対象物質の検出方法
JP2013083631A (ja) * 2011-09-26 2013-05-09 Fujifilm Corp 蛍光粒子を用いたコルチゾール免疫アッセイ

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8507208B2 (en) * 2009-07-23 2013-08-13 University Of Wyoming Methods and compositions for detection of biological materials using microfluidic devices
JP5666277B2 (ja) * 2010-12-10 2015-02-12 富士フイルム株式会社 分析チップ
JP5797427B2 (ja) * 2011-03-10 2015-10-21 富士フイルム株式会社 検出方法および検出装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009109245A (ja) * 2007-10-26 2009-05-21 Canon Inc 検出方法および検出装置
JP2012143205A (ja) * 2011-01-14 2012-08-02 Hitachi High-Technologies Corp 核酸分析装置
JP2013083631A (ja) * 2011-09-26 2013-05-09 Fujifilm Corp 蛍光粒子を用いたコルチゾール免疫アッセイ
JP2013083632A (ja) * 2011-09-28 2013-05-09 Fujifilm Corp 蛍光粒子を用いた検出対象物質の検出方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MASUMI YAMADA ET AL.: "Classification of Particles Using Microfluidic Channels", JOURNAL OF THE VACUUM SOCIETY OF JAPAN, vol. 49, no. 7, 2006, pages 404 - 408 *
NAKASHIMA M ET AL.: "PINCHED FLOW FRACTIONATION (PFF) FOR CONTINUOUS PARTICLE SEPARATION IN A MICROFLUIDIC DEVICE", MEMS 2004 TECHNICAL DIGEST, January 2004 (2004-01-01), pages 33 - 36, XP010767819, DOI: doi:10.1109/MEMS.2004.1290515 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018054495A (ja) * 2016-09-29 2018-04-05 コニカミノルタ株式会社 血液検査用センサチップ、血液検査装置及び血液検査方法
CN111417853A (zh) * 2018-04-06 2020-07-14 松下知识产权经营株式会社 病原体检测装置以及病原体检测方法
JP2020159901A (ja) * 2019-03-27 2020-10-01 日本無線株式会社 弾性表面波センサ及び弾性表面波センサを用いる抗体抗原混和検出方法
JP7251901B2 (ja) 2019-03-27 2023-04-04 日本無線株式会社 弾性表面波センサ及び弾性表面波センサを用いる抗体抗原混和検出方法

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