JPWO2016047027A1 - Fluorescence detection method and detection sample cell - Google Patents

Abstract

In a fluorescence detection method for capturing a substance to be detected that specifically binds to a fluorescent label and a sample cell for the detection, the amount of the substance to be detected to be captured is increased to achieve high sensitivity and a reduction in detection time. A sample solution (S) containing an antigen (A) that specifically binds to a fluorescent label (F) is caused to flow down to a sample solution channel (113). The mixed phase liquid (G) not containing the antigen (A) and the fluorescent label (F) is caused to flow down to the mixed phase liquid channel (114). The sample liquid (S) and the mixed phase liquid (G) are guided to the two-phase flow path (115) narrower than any of the sample liquid flow path (113) and the mixed phase liquid flow path (114), and the two-phase flow path In (115), a two-phase flow of the sample liquid (S) and the mixed phase liquid (G) is generated, and the sample liquid (S) flows down along the side wall (115a). The detection unit (200) provided on the side wall (115a) captures the antigen (A) specifically bound to the fluorescent label (F), and the detection unit (200) is irradiated with excitation light (L) to emit fluorescence. And detecting the abundance of the antigen (A) based on the detected fluorescence. [Selection] Figure 4

Description

  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.

  In recent years, a POCT (Point Of Care Testing) test that enables a test in a hospital room or at home has attracted attention. In order to realize this POCT inspection, research on biochemical inspection using a detection sample cell provided with a microchannel has been actively conducted.

  The fluorescence method is widely used as a biochemical test using a microchannel. In the fluorescence method, 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). Then, 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.

  In the fluorescence method, surface plasmon using electric field enhancement by plasmon resonance is used to further improve the sensitivity of the evanescent fluorescence method using the evanescent wave that oozes from the surface of the detection unit and the evanescent fluorescence method to improve the sensitivity. An enhanced fluorescence method and the like are known.

  In order to improve the sensitivity, it is also important to increase the amount of the substance to be detected captured by the detection unit. A technique (Patent Document 1) 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. A liquid feeding technique (Patent Document 2) is proposed.

  In addition, 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. There has also been proposed a technique (Patent Document 3) 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.

  However, the fluorescent particles flowing down in the microchannel tend to flow down toward the central portion as shown in FIG. 9 (Non-Patent Document 1). Therefore, it is desired that the fluorescent particles flow down so as to pass through the vicinity of the detection unit, but this is not technically established. A method for separating and classifying fine particles using a microchannel has been proposed (Non-Patent Documents 2 and 3).

JP 2011-257266 A International Publication WO2011 / 027851 JP 2008-203158 A

D. Ross et al., Anal. Chem. 2001, 73, 2509 Masumi Yamada, Minoru Seki Multiphase flow Vol.19 No.2 (2005) pp.102-107 “Continuous separation and classification of fine particles using microfluidics” Masami Yamada, Minoru Seki Vacuum Vol.49 No.7, 2006 pp.404-408 “Particle Classification Using Microchannels”

  In order for the detection part to capture the substance to be detected that specifically binds to the fluorescent label, it is necessary to bring the substance to be detected close to the region of about 100 nm from the surface of the detection part. FIG. 10 is a schematic diagram showing the feeding of the sample solution flowing down the microchannel.

  However, as can be seen from FIG. 10, the substance to be detected (antigen) flows down toward the center of 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.

  In addition, the width of the microchannel is about 100 μm, 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.

  For the sample liquid flowing down as a laminar flow, a technique using a technique of simply feeding the sample liquid toward the detection unit, such as Patent Documents 1 and 2, or another fluid such as Patent Document 3 is used. In the technique for guiding the liquid in a desired direction, it is difficult to move the detection object to the vicinity of the detection unit in a short time. In addition, Non-Patent Documents 1 to 3 do not make any proposal about increasing the amount of the substance to be detected that is captured by the detection unit in the fluorescence method.

  In view of the above circumstances, 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.

  In order to solve the above problems, a fluorescence detection method according to the present invention 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.

  In addition, the above-mentioned “fluorescence emitted from a fluorescent label by irradiation” means fluorescence generated by exciting the fluorescent label directly or indirectly by irradiation with excitation light. Further, the above-mentioned “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.

  In the fluorescence detection method according to the present invention, it is desirable to use a flow channel having a width equal to or larger than the width of the sample liquid flow channel as the mixed phase liquid flow channel.

  Further, in the fluorescence detection method according to the present invention, it is desirable that the evanescent light oozes out from the detection unit by irradiation of excitation light, and the fluorescent label generates fluorescence by the evanescent light.

  Further, in the fluorescence detection method according to the present invention, 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

  In the fluorescence detection method according to the present invention, it is desirable to use, as a fluorescent label, fluorescent beads in which fluorescent dye molecules are encapsulated in a translucent dielectric material that transmits excitation light and fluorescence.

  In the fluorescence detection method according to the present invention, it is desirable that the fluorescent beads are hydrophilic and the mixed phase solution is physiological saline having a concentration of 1.8% or more. Here, “concentration” means the concentration of sodium chloride.

  In the fluorescence detection method according to the present invention, the fluorescent beads are hydrophilic, and it is desirable to use an organic solvent as the mixed phase liquid.

  Further, in the fluorescence detection method according to the present invention, 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, and the pH of the mixed phase liquid is ionized by the functional group. It is desirable to make the value impossible.

  Further, in the fluorescence detection method according to the present invention, 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.

  In addition, 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.

  In the sample cell for detection according to the present invention, it is desirable that the width of the mixed phase liquid channel is equal to or larger than the width of the sample liquid channel.

  In the detection sample cell according to the present invention, it is desirable that the width of the two-phase flow channel is in the range of 5 μm or more and less than 100 μm.

  In the detection sample cell according to the present invention, it is desirable that the downstream end of the sample liquid flow channel, the downstream end of the mixed phase liquid flow channel, and the upstream end of the two-phase flow flow channel communicate with each other.

  In the sample cell for detection according to the present invention, it is desirable that the angle formed between the sample liquid channel and the two-phase flow channel is equal to the angle formed between the mixed phase liquid channel and the two-phase flow channel.

  According to the fluorescence detection method of the present invention, 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. In order to detect the abundance of the detection substance, 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. In addition, the detection time can be shortened.

Schematic diagram of fluorescence detector Block diagram of fluorescence detector Schematic diagram inside the microchannel Partial enlarged view of microchannel Detection image using a normal detection sample cell (Part 1) Detection image using a normal detection sample cell (Part 2) Detection image using a detection sample cell having a two-phase flow channel (Part 1) Detection image using detection sample cell with two-phase flow channel (Part 2) 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

  Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. This embodiment is a method for detecting antigen A as a substance to be detected. 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. As an example, the fluorescence detection device 1 is a device using a surface plasmon enhanced fluorescence method.

  The fluorescence detection apparatus 1 is equipped with a specimen container KB that contains a specimen liquid containing the antigen A, a detection sample cell 100 that captures 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. The injection well 140, the injection well 150 for injecting the mixed phase solution G, the lid member 100b in which the discharge well 160 for discharging the sample solution S and the mixed phase solution G is formed, and the antigen A provided in a part of the microchannel 110 A detection unit 200 for capturing is provided.

  As the base material 100a, it is desirable to use a resin such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), or amorphous polyolefin (APO) containing cycloolefin.

  The fluorescent label F specifically binds to the 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. When 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.

  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.

  The primary antibody B can be appropriately adjusted according to the type of the antigen A. For example, for antigen A which is hCG, an anti-hCG monoclonal antibody (Anti-hCG 5008 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 includes 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. Specifically, 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 μm or more and less than 5 μm, and more preferably 1 μm or more and less than 3 μm. As the fluorescent beads FB, for example, beads manufactured by Bangs Laboratories, Inc. having a particle diameter of 1 μm, an excitation wavelength of 660 nm, and a fluorescence wavelength of 690 nm can be used.

  The secondary antibody C can also be appropriately adjusted according to the antigen A. For example, for antigen A which is hCG, an anti-hCG monoclonal antibody (Anti-Alpha subunit 6601 SPR-5, manufactured by Medix Biochemical) can be used. 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.

  After the detection unit 200 captures the antigen A specifically bound to the secondary antibody C to which the fluorescent beads FB are immobilized, 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.

  When the excitation light L is irradiated, the evanescent wave E oozes out on the metal film 201. When 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.

  When the fluorescent beads FB emit fluorescence, the fluorescence detection unit 13 detects the fluorescence as a fluorescence signal. As 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.

  When the fluorescence detection unit 13 detects the fluorescence signal, 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).

  The structure of the micro flow path 110 of the detection sample cell 100 and its operation will be described. 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 θ1, and the mixed phase liquid channel 114 and the two-phase flow channel 115 communicate with each other at an angle θ2. 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.

  Further, the discharge flow path 116 has a widened portion 116a whose width increases toward the downstream direction at an angle θ3 and a constant width portion 116b that communicates with the widened portion 116a. As described above, 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 When guided to the flow channel 115, 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. .

  Therefore, 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 that has not been captured by the detection unit 200, the unbound antigen A, and the fluorescent label F are guided to the discharge channel 116. Depending on the size of 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.

  Further, it is desirable that the flow rate of the mixed phase liquid G led to the two-phase flow channel 115 is larger than the amount of the sample liquid S led to the two-phase flow channel 115. Thereby, in 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. Specifically, it is desirable that the width d2 of the mixed phase liquid flow path 114 is wider than the width d1 of the sample liquid flow path 113. Note that it is desirable to adjust the flow rates of the sample liquid S and the mixed phase liquid G in order to form a laminar flow throughout the mixed phase liquid path 114 and the two-phase flow path 115. Specifically, each flow rate can be calculated from the flow velocity distribution using the Hagen-Poiseuille flow equation.

  Further, in order to facilitate the generation of a two-phase flow composed of the sample liquid S and the mixed phase liquid G in the two-phase flow path 115, the angle θ1 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 θ2 formed by the channel 114 and the two-phase flow channel 115 is equal. Further, it is desirable that the angle θ1 and the angle θ2 are in the range of 110 to 160 degrees. The angle θ3 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 μm or more and less than 100 μm, and more preferably 5 μm or more and less than 10 μm. 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 μm or more and less than 2000 μm. 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.

  The superiority of this embodiment will be described in detail. First, an analysis result using a normal detection sample cell in which a microchannel having no two-phase channel structure is formed will be described. 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 μL.

  Next, an analysis result using the detection sample cell 100 of the present embodiment will be described. 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 μL, and the amount of the mixed phase liquid G is about 3000 μL.

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.

  In addition, when the detection sample cell 100 is used, 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. In the method using the detection sample cell 100, as in the method using the normal flow path, 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.

  Here, 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. . Below, the 1st-3rd modification of this embodiment which can suppress that the antigen A specifically couple | bonded with the fluorescent label F disperse | distributes to the mixed phase liquid G is demonstrated.

  The first modification is a method of incorporating polystyrene beads having no fluorescent dye in the mixed phase liquid G. In the first modification, 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.

  Thereby, the bead concentration of the mixed phase liquid G, that is, the bead concentration contained per unit volume, becomes higher than the bead concentration of the sample liquid 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.

  In the first modified example, since the polystyrene beads that do not have the fluorescent dye in the mixed phase liquid G scatter the irradiated excitation light L, 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.

  A second modification will be described. The second modification is a method using an organic solvent as the mixed phase liquid G. As described above, 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.

  In this embodiment, as described above, 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. Of course, even if a sulfonic acid group or the like is used as the acidic functional group, the ionized state is similarly obtained.

Further, when the fluorescent beads FB are surface-modified with a basic functional group, the hydrophilicity of the fluorescent beads FB is increased. For example, when an amino group (—NH ₂ ) is used, the sample liquid S is ionized to NH ₃ +. Of course, even when a quaternary ammonium group or the like is used as a basic functional group, the ionized state is similarly obtained.

  Therefore, when an organic solvent is used as the mixed phase solution G, 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. . Specifically, ethanol, methanol, dimethyl sulfoxide (DMSO) can be used as the organic solvent.

  A third modification will be described. The third modification is a method of adjusting the pH of the mixed phase liquid G according to the type of functional group. As described above, when the surface of the fluorescent beads FB is modified with a functional group, the functional group is ionized in the sample solution S. When the acidic functional group is used, the fluorescent bead FB is negatively charged. When the basic functional group is used, 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.

  In the third modification, 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.

  As a result, 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.

  As mentioned above, although preferable embodiment and its modification of this invention were described, this invention is not limited to these embodiment and its modification. That is, 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.

Claims (14)

Prepare a fluorescent label that specifically binds to the target substance,
Let the sample liquid containing the substance to be detected and the fluorescent label flow down to the sample liquid flow path;
Let the mixed phase liquid not containing the substance to be detected and the fluorescent label flow down to the mixed phase liquid flow path;
The sample liquid flowing down from the sample liquid flow path and the mixed phase liquid flowing down from the mixed phase liquid flow path are guided to a two-phase flow path narrower than the sample liquid flow path and the mixed phase liquid flow path. In the two-phase flow channel, a two-phase flow of the sample liquid and the mixed phase liquid is generated, and the sample liquid is caused to flow down along one side wall of the two-phase flow channel,
The detection unit provided on the one side wall captures the substance to be detected specifically bound to the fluorescent label;
Irradiating the detection unit with excitation light,
Detecting the fluorescence emitted by the fluorescent label upon irradiation with the excitation light;
A fluorescence detection method for detecting the abundance of the substance to be detected based on the detected amount of fluorescence.   The fluorescence detection method according to claim 1, wherein a flow path having a width equal to or larger than the width of the sample liquid flow path is used as the mixed phase liquid flow path.   3. The fluorescence detection method according to claim 1, wherein, in the step of irradiating the excitation light, evanescent light is oozed out of the detection unit by irradiation of the excitation light, and the fluorescent label emits the fluorescence by the evanescent light. The detector comprises a metal film;
The step of irradiating the excitation light generates surface plasmons that enhance an electric field on the metal film by irradiating the metal film with the excitation light at an incident angle greater than or equal to total reflection. The fluorescence detection method as described.   The fluorescence detection method according to any one of claims 1 to 4, wherein a fluorescent bead in which a fluorescent dye molecule is included in a translucent dielectric material that transmits the excitation light and the fluorescence is used as the fluorescent label. .   The fluorescence detection method according to claim 5, wherein the fluorescent beads are hydrophilic, and physiological saline having a concentration of 1.8% or more is used for the mixed phase solution.   The fluorescence detection method according to claim 5, wherein the fluorescent beads have hydrophilicity, and an organic solvent is used 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 be ionized, and the pH of the mixed phase solution is set to a value at which the functional group cannot be ionized. 5. The fluorescence detection method according to 5.   6. The mixed phase solution includes beads not having a fluorescent dye, and the number of the beads per unit volume of the mixed phase solution is larger than the number of the fluorescent beads per unit volume of the sample solution. Fluorescence detection method. A detection sample cell used in the fluorescence detection method according to any one of claims 1 to 9,
A sample liquid flow path for flowing down the sample liquid;
A mixed phase liquid flow path for flowing down the mixed phase liquid;
A two-phase flow channel that communicates with the downstream end of the sample channel and the downstream end of the mixed-phase channel and has a width narrower than any of the sample solution channel and the detection solution channel;
A detection sample cell comprising: a detection unit provided on a part of a side wall of the two-phase flow channel that captures the substance to be detected specifically bound to the fluorescent label.   The sample cell for detection according to claim 10, wherein the width of the mixed phase liquid channel is equal to or larger than the width of the sample liquid channel.   The sample cell for detection according to claim 10 or 11, wherein a width of the two-phase flow channel is in a range of 5 µm or more and less than 100 µm.   The detection sample according to any one of claims 10 to 12, wherein a downstream end of the sample liquid flow path, a downstream end of the mixed phase liquid flow path, and an upstream end of the two-phase flow flow path communicate with each other. cell.   The sample cell for detection according to claim 13, wherein an angle formed by the sample liquid flow path and the two-phase flow flow path is equal to an angle formed by the mixed-phase liquid flow path and the two-phase flow flow path.

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