Kit for detecting glycoproteins

Abstract

Provided is a kit for detecting a glycoprotein contained in a sample using an optical condensation system, the kit comprising microparticles respectively modified by host molecules and a dilution solution for diluting the sample, in which each of the host molecules binds specifically to the glycoprotein, the dilution solution comprises a blocking agent and a buffering agent, the pH value of the dilution solution is higher than the isoelectric point of the glycoprotein, the concentration of the blocking agent is lower than a concentration at which the non-specific adsorption between the host molecules is inhibited in an environment where a photo-induced force does not act on the host particles, and the salt concentration in the dilution solution is a concentration at which the microparticles modifying the host molecules cannot be precipitated by salting out.

Description

[Technical Field]

[0001] This invention relates to a kit for detecting glycoproteins. [Background technology]

[0002] Various techniques have been put into practical use to detect substances that may be present in a sample. Examples of detectable substances include allergens, proteins derived from cancer cells (e.g., glycoproteins derived from cancer cells), nucleic acids, and vesicles. For example, known protein detection techniques include the ELISA (Enzyme-Linked Immuno Sorbent Assay) method and the SPR (Surface Plasmon Resonance) method. The minimum concentration of a detectable substance (detection limit) that can be detected by the ELISA method is said to be around 0.3 ng / mL. The detection limit for the SPR method is said to be around 1 μg / mL. In addition, detection of a detectable substance by either method takes several hours. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2014 / 192937 [Patent Document 2] International Publication No. 2021 / 040021 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] In recent years, technologies that utilize the effects of light, such as photo-induced forces, to detect substances have attracted attention. For example, International Publication No. 2014 / 192937 (Patent Document 1) discloses a detection device for detecting substances that may be contained in a sample, comprising: a plurality of metal nanoparticles, each modified with a host molecule capable of specifically adhering to the substance to be detected; a first light source that emits polarized light for assembling the plurality of metal nanoparticles; an objective lens that focuses the polarized light and introduces the focused polarized light into a liquid containing the sample and the plurality of metal nanoparticles; a photodetector that receives light from the liquid; and a detector that detects the substance to be detected based on a signal from the photodetector.

[0005] Furthermore, International Publication No. 2021 / 040021 (Patent Document 2) discloses a method for detecting a substance to be detected, which includes the steps of: circulating a liquid sample containing a plurality of fine particles, each modified with a host molecule that specifically binds to the substance to be detected, through a microchannel using a pump; irradiating the liquid sample with non-resonant light, which is light outside the wavelength range of the electronic resonance of the plurality of fine particles; and detecting the substance to be detected based on a signal from a photodetector that receives light from the liquid sample.

[0006] There is always a demand for technologies that can increase the detection sensitivity of a substance to be detected or shorten the detection time for that substance; in other words, technologies that can rapidly detect trace amounts of a substance to be detected. In particular, from the perspective of early cancer diagnosis, health checkups, and the development of in vitro diagnostic medical devices (e.g., over-the-counter diagnostic reagents, medical diagnostic reagents, etc.), there is a need to shorten detection time and improve detection sensitivity in technologies for detecting glycoproteins.

[0007] This invention has been made in view of the above circumstances, and aims to provide a kit for rapidly and sensitively detecting trace amounts of glycoproteins contained in a sample using a photoconcentration system. [Means for solving the problem]

[0008] The inventors of this invention conducted diligent research to solve the above problems and, as a result, discovered that by optimizing the diluent for diluting the sample, it is possible to rapidly and sensitively detect trace amounts of glycoproteins contained in a sample using a photoconcentration system, thereby completing the present invention. That is, the present invention is as follows.

[0009] [1] The kit according to the present invention is a kit for detecting glycoproteins contained in a sample using a photoconcentration system, Microparticles with modified host molecules, A diluent for diluting the above sample, The above host molecule specifically binds to the above glycoprotein, The above diluted solution contains a blocking agent and a buffering agent. The pH of the above dilution is higher than the isoelectric point of the above glycoprotein. The concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. The salt concentration of the above dilution is such that the fine particles modified with the host molecule do not settle by salting out.

[0010] [2] In the above [1], it is preferable that the diluent is neutral.

[0011] [3] In the above [1] or [2], it is preferable that the salt concentration of the diluent is such that the fine particles modified with the host molecule do not settle by salting out, and that the thickness of the electrical double layer in the fine particles decreases.

[0012] [4] In any of the above [1] to [3], it is preferable that the fine particles further contain an additive that enhances electrostatic repulsion.

[0013] [5] In any of the above [1] to [4], it is preferable that the fine particles include two or more types of fine particles of different sizes.

[0014] [6] In any one of the above [1] to [5], it is preferable that the host molecule contains at least one selected from the group consisting of an antibody, a Fab fragment, an F(ab’)2 fragment, an Fv fragment, and a scFv.

[0015] [7] In any one of the above [1] to [6], it is preferable that the blocking agent contains at least one selected from the group consisting of albumin, gelatin, casein, and goat serum.

[0016] [8] In any one of the above [1] to [7], it is preferable that the concentration of the blocking agent is 0.000001% by mass or more and less than 0.001% by mass with respect to the diluent.

[0017] [9] In any one of the above [1] to [8], it is preferable that the buffer contains at least one selected from the group consisting of a phosphate compound, tris(hydroxymethyl)aminomethane, HEPES, and MES.

[0018]

[10] In any one of the above [1] to [9], the kit further contains a dispersion liquid for dispersing the microparticles modified with the host molecule. The dispersion liquid contains the blocking agent and the buffer. It is preferable that the concentration of the blocking agent is 0.00000001% by mass or more and 0.001% by mass or less with respect to the dispersion liquid.

[0019]

[11] In any one of the above [1] to

[10] , it is preferable that the glycoprotein is a cancer marker protein.

[0020]

[12] In

[11] above, it is preferable that the cancer marker protein contains at least one selected from the group consisting of carcinoembryonic antigen (CEA) and carbohydrate antigen (such as CA19-9).

[0021]

[13] In any of the above [1] to

[12] , the sample is a sample that has been frozen after collection, or refrigerated for a specified period and then frozen, It is preferable that the glycoprotein contained in the above sample aggregates to form a polymer or nanoparticles.

[0022]

[14] In any of the above [1] to

[13] , the kit preferably further includes a microfluidic chip.

[0023]

[15] Another kit according to the present invention is a kit for detecting glycoproteins contained in a sample using a photoconcentration system, Microparticles with modified host molecules, A diluent for diluting the above sample, A dispersion for dispersing fine particles modified with the above-mentioned host molecule, The above host molecule specifically binds to the above glycoprotein, The above dilution contains a buffering agent, The above dispersion contains a blocking agent and the above buffering agent. The pH of the above dilution is higher than the isoelectric point of the above glycoprotein. The concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. The salt concentration of the above dilution is such that the microparticles modified with the host molecule do not precipitate by salting out, according to the kit. [Effects of the Invention]

[0024] Based on the above, it is possible to provide a kit for rapidly and sensitively detecting trace amounts of glycoproteins contained in a sample using a photoconcentration system. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 is a schematic diagram illustrating the mechanism of the photoconcentration system. [Figure 2]Figure 2 is a schematic cross-sectional view of the microfluidic chip 90 along the line II-II in Figure 1. [Figure 3] Figure 3 illustrates the aggregation mechanism of latex beads (microparticles modified with host molecules) when a measurement sample is irradiated with laser light. [Figure 4] Figure 4 is a conceptual diagram illustrating the detection principle of a certain glycoprotein. [Figure 5] Figure 5 is a conceptual diagram illustrating other detection principles for glycoproteins. [Figure 6] Figure 6 illustrates the aggregation mechanism of latex beads under defocusing conditions. [Figure 7] Figure 7 is a graph showing the correlation between the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when the sample is measured using a photoconcentration system. [Figure 8] Figure 8 is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when the sample is measured using a photoconcentration system. [Figure 9] Figure 9 is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when the sample is measured using a photoconcentration system. [Figure 10] Figure 10 is a photograph showing the vicinity of the laser spot when a sample is measured using a light concentration system. [Figure 11A] Figure 11A is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the measured sample (horizontal axis) when the sample was measured using a photoconcentration system, with a BSA concentration of 1.65 × 10⁻⁸% in the antibody B-modified bead dispersion and a BSA concentration of 2.16 × 10⁻⁴% in the antibody A-modified bead dispersion. [Figure 11B]Figure 11B is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the measured sample (horizontal axis) when the BSA concentration in the antibody B-modified bead dispersion and the antibody A-modified bead dispersion is 3.6 × 10⁻⁵%, and the sample was measured using a photoconcentration system. [Figure 12] Figure 12 is a graph showing the correlation between the aggregate area of ​​the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when the sample is measured using a photoconcentration system. [Figure 13] Figure 13 is a schematic diagram illustrating the state of glycoproteins in a sample after it has been frozen and stored. [Figure 14] Figure 14 is a graph showing the correlation between light intensity (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when the sample is measured using the ELISA method. [Figure 15] Figure 15 shows a graph (left) illustrating the aggregate area (vertical axis) of the multilayer portion when the sample (plasma sample, horizontal axis) is measured using a photoconcentration system, and a graph (right) illustrating the concentration of glycoproteins (vertical axis) when the sample (plasma sample, horizontal axis) is measured using the ELISA method. [Figure 16] Figure 16 is a graph showing the aggregate area (vertical axis) of the multilayer portion when the measurement sample (plasma sample, horizontal axis) is measured using a photoconcentration system. [Figure 17] Figure 17 is a graph showing the relationship between the distance from the particle surface and the electric potential. [Figure 18] Figure 18 is a graph showing the pH dependence and antibody modification dependence of the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when fluorescent beads are used as probe particles and the sample is measured using a photoconcentration system. [Figure 19]Figure 19 is a graph showing the laser power dependence of the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when fluorescent beads are used as probe particles and the sample is measured using a photoconcentration system. [Figure 20] Figure 20 is a graph showing the total aggregate area, the aggregate area of ​​the multilayer portion, or the aggregate area of ​​the multilayer portion (vertical axis) when a measurement sample (plasma sample, horizontal axis) is measured using a photoconcentration system with fluorescent beads as probe particles (laser irradiation time: 5 minutes). [Figure 21] Figure 21 is a graph showing the total aggregate area, the aggregate area of ​​the multilayer portion, or the aggregate area of ​​the multilayer portion (vertical axis) when a measurement sample (plasma sample, horizontal axis) is measured using a photoconcentration system with fluorescent beads as probe particles (laser irradiation time: 4 minutes). [Figure 22] Figure 22 is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins and sodium chloride in the sample (horizontal axis) when fluorescent beads are used as probe particles, the salt concentration in the diluent is adjusted, and the sample is measured using a photoconcentration system. [Figure 23] Figure 23 is a schematic diagram illustrating a blocking method within a microchannel. [Figure 24] Figure 24 is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoproteins in the sample (horizontal axis) when a sample is measured using a photoconcentration system with fluorescent beads as probe particles and a microfluidic channel blocked. [Figure 25] Figure 25 is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoprotein in the sample (horizontal axis) when two types of fluorescent beads with diameters of 2 μm and 1 μm are mixed in a volume ratio of 1:0.05 to form probe particles, and the sample is measured using a photoconcentration system. [Figure 26]Figure 26 is a conceptual diagram of the mechanism of stabilization of the aggregated structure when measurement samples prepared by varying the mixing ratio of two types of fluorescent beads of different sizes were measured using a photoconcentration system, and a graph showing the correlation between the proportion of the multilayer portion (vertical axis) and the concentration of glycoprotein in the measurement sample (horizontal axis). [Modes for carrying out the invention]

[0026] Hereinafter, one embodiment of the present invention (hereinafter sometimes referred to as "this embodiment") will be described in detail with reference to the drawings. However, this embodiment is not limited thereto. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and their descriptions will not be repeated. In this specification, the notation in the form of "A~Z" means the upper and lower limits of a range (i.e., A or more and Z or less). If no unit is specified for A, but a unit is specified only for Z, the unit for A and the unit for Z are the same.

[0027] ≪A kit for detecting glycoproteins in a sample using a light concentration system≫ The kit according to this embodiment is a kit for detecting glycoproteins contained in a sample using a photoconcentration system, Microparticles with modified host molecules, A diluent for diluting the above sample, The above host molecule specifically binds to the above glycoprotein, The above diluted solution contains a blocking agent and a buffering agent. The pH of the above dilution is higher than the isoelectric point of the above glycoprotein. The concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. The salt concentration of the above dilution is such that the microparticles modified with host molecules do not settle by salting out.

[0028] <Photoconcentration System> In this embodiment, the term "photoconcentration system" refers to a technology that utilizes the effects of light on a substance, such as photoinduced force and photoinduced convection, and the effect of causing convection in a liquid due to photothermal effects, to concentrate or accumulate a target substance in a predetermined area. In one aspect of this embodiment, if the target substance is present in a liquid, the photoconcentration system can also be interpreted as being able to concentrate the target substance in a predetermined area of ​​the liquid (the area irradiated with light). In this embodiment, the target substance may be a glycoprotein, host molecule, or fine particle that is the substance to be detected. This will be explained in detail below.

[0029] Figure 1 is a schematic diagram illustrating the mechanism of the photoconcentration system. Figure 2 is a schematic cross-sectional view of the microfluidic chip 90 along the line II-II in Figure 1. In Figure 1, the measurement sample SP is introduced into the microfluidic channel 92 formed in the substrate 91 of the microfluidic chip 90. In the example shown in Figure 1, the measurement sample SP is introduced from the inlet 921, passes through the microfluidic channel 92, and is discharged from the outlet 922. The flow direction of the sample SP in the microfluidic channel 92 is defined as the x-direction. The measurement sample SP is, for example, a solution obtained by diluting the sample with the diluent. The measurement sample SP contains fine particles modified with a host molecule.

[0030] The measurement sample SP introduced into the microchannel 92 is irradiated with laser light L1 in a predetermined area. The laser light L1 captures the fine particles in the measurement sample SP by generating a photo-induced force. Here, "photo-induced force" is used as a general term for dissipative force, gradient force, and intermaterial photo-induced force. Dissipative force is a force generated when the momentum of light is transferred to a substance in a dissipative process such as light scattering or light absorption. Gradient force is a force that moves a substance to a stable point of its electromagnetic potential when a substance that has undergone photo-induced polarization is placed in a non-uniform electromagnetic field. Intermaterial photo-induced force is the sum of the force due to the longitudinal electric field and the force due to the transverse electric field (radiation field) arising from the induced polarization in multiple photo-excited substances.

[0031] Figure 3 illustrates the aggregation mechanism of latex beads (microparticles modified with host molecules) when a measurement sample is irradiated with laser light. By adjusting the beam waist of the laser beam L1 to be located within the measurement sample SP and then irradiating the microfluidic chip 90 with the laser beam L1, beads B1 and B2 gather near the beam waist due to photo-induced forces (more specifically, intermaterial photo-induced forces and gradient forces). As a result, the density of beads B1 and B2 near the beam waist becomes locally higher compared to the density of beads B1 and B2 at other locations (positions sufficiently far from the beam waist).

[0032] When the substance to be detected X (glycoprotein) is present around the beam waist, antigen-antibody reactions occur between the first antibody B11 (host molecule) modified on the surface of bead B1 and the substance to be detected X, and between the second antibody B21 (host molecule) modified on the surface of bead B2 and the substance to be detected X, causing beads B1 and B2 to bind via the substance to be detected X (see Figures 4 and 5). The antigen-antibody reaction will be described later. By introducing a new sample SP from inlet 921, new substances to be detected X are successively supplied around the beam waist. Therefore, antigen-antibody reactions occur more easily compared to in a stationary liquid. As the antigen-antibody reaction is repeated, aggregates of beads B1 and B2 are formed.

[0033] As the size of the aggregates of beads B1 and B2 increases, the probability of the substance to be detected X, which is present around the aggregates, encountering the aggregates increases, thus increasing the frequency of antigen-antibody reactions. In other words, "photo-induced acceleration" can be achieved by irradiating the measurement sample SP with laser light L1 to accelerate the aggregation of beads B1 and B2. As a result, aggregates of beads B1 and B2 with high density are formed in a short time. Then, by optically detecting the formed aggregates, it can be quickly determined that the measurement sample SP contains the substance to be detected X.

[0034] In addition to the photo-induced intermaterial force and gradient force, beads B1 and B2 are subjected to a dissipative force acting in the same direction as the laser beam L1 irradiation. In the case of downward irradiation, beads B1 and B2 are pressed against the bottom surface of the microchannel 92 by the dissipative force acting from above to below (Figure 3).

[0035] Figure 4 is a conceptual diagram illustrating the detection principle of a certain glycoprotein. In this embodiment, the glycoprotein to be detected is detected using the so-called latex agglutination method. More specifically, in the example shown in Figure 4, two types of beads, B1 and B2, are prepared.

[0036] Each of beads B1 and B2 contains a common bead body B0. The bead body B0 may be fine particles, as described later. The bead body B0 is, for example, a resin bead (latex bead) made of polystyrene. The bead body B0 has a size on the order of micrometers (typically about 1 μm to 5 μm in diameter), similar to typical latex beads. The material of the bead body B0 may also be other resins such as acrylic, polyolefin, polyethylene, or polypropylene.

[0037] In bead B1, the bead body B0 is modified by the first antibody B11 (the first host molecule). Avidin B12 and biotin B13 are used to modify the first antibody B11. Avidin B12 is immobilized on the surface of the bead body B0 through interaction between avidin B12 and the bead body B0. Biotin B13 labels the first antibody B11 by binding to it. The first antibody B11 is modified on the surface of the bead body B0 due to the strong affinity between avidin B12 and biotin B13.

[0038] In bead B2, the bead body B0 is modified by the second antibody B21 (second host molecule). Similar to the first antibody B11, the surface of the bead body B0 is also modified by avidin B22 and biotin B23.

[0039] In this embodiment, the substance to be detected X in the example shown in Figure 4 is a glycoprotein. Specifically, cancer marker proteins such as CEA and CA19-9 can be used as the substance to be detected X. The substance to be detected X has a site to which it is specifically bound by the host molecule. When the host molecule is an antibody, this site is called an "epitope," "antigen determinant," or "antibody recognition site." The substance to be detected X may be a glycoprotein having multiple epitopes. Examples of such glycoproteins include CEACAM-5, as described in the [Examples] section below.

[0040] The substance X to be detected undergoes an antigen-antibody reaction with the first antibody B11 and also with the second antibody B21. Therefore, in the presence of the substance X, beads B1 and B2 bind to each other via the substance X. Figure 4 shows an example where bead B1 is modified with only one first antibody B11. However, in reality, bead B1 is modified with many first antibodies B11. The same is true for bead B2. Therefore, when multiple beads B1 and B2 are introduced into a sample containing the substance X to be detected, the multiple beads B1 and B2 aggregate through antigen-antibody reactions, forming aggregates of beads B1 and B2.

[0041] Figure 5 is a conceptual diagram illustrating other detection principles for glycoproteins. Depending on the type of glycoprotein to be detected, two types of beads may not always be necessary. In the example shown in Figure 5, only one type of bead, B3, is prepared. Bead B3 consists of the bead body B0 and the antibody B31 that modifies the bead body B0.

[0042] In this example, the detected substance Y is also a glycoprotein, specifically, for example, the aggregates of CEACAM-5 in the [Examples] described later. When multiple beads B3 are combined with the detected substance Y, the multiple beads B3 aggregate through an antigen-antibody reaction with the detected substance Y, forming aggregates of beads B3.

[0043] In this embodiment, bead aggregates are formed using the principle described above, and by observing these aggregates, the substance to be detected (i.e., glycoprotein) can be detected and quantified.

[0044] Figure 6 illustrates the aggregation mechanism of latex beads (beads B1 and B2) under defocus conditions. More specifically, defocus conditions refer to the condition when the beam waist of the laser beam L1 is located behind the microchannel 92 in the direction of laser beam L1 irradiation. In Figure 6, the laser beam L1 (laser spot) at the bottom surface of the microchannel 92 is shown as a black circle.

[0045] When beads B1 and B2 are irradiated with laser light L1, they are pressed against the bottom surface of the microchannel 92 by the photo-induced force (especially the dissipative force) of the laser light L1. As a result, beads B1 and B2 are arranged in a single layer on the bottom surface of the microchannel 92 (see the upper diagram in Figure 6). Other beads B1 and B2 are then pressed further onto this single layer, forming a multilayer structure of beads B1 and B2 (see the middle diagram in Figure 6). However, beads B1 and B2 that are not bound via the substance to be detected X are pushed in the direction of flow of the measurement sample SP (see the lower diagram in Figure 6). As a result of this so-called "cleaning effect," regions remain where the multilayer structure of beads B1 and B2 bound via the substance to be detected X has been formed. For example, the dark-colored region shown in Figure 10 is thought to be a region where the multilayer structure of beads B1 and B2 was maintained as a result of the antigen-antibody reaction.

[0046] Thus, the dark-colored region is a region that depends on the amount of beads B1 and B2 bound via the substance X to be detected. In contrast, the light-colored region is basically determined by the size of the laser spot, regardless of whether or not beads B1 and B2 are bound via the substance X to be detected. Therefore, under defocus conditions, by using the area of ​​the dark-colored region as the evaluation target, it becomes possible to quantify the concentration of the substance X to be detected with high accuracy.

[0047] In this embodiment, for convenience, the area of ​​the dark-colored region is normalized. Specifically, the ratio of the area of ​​the dark-colored region to the area of ​​the light-colored region (the total area within the light-colored contour of the image, hereinafter sometimes simply referred to as "total area") is defined as the "ratio of the multi-layered portion." Proportion of multi-layered area = Area of ​​dark-colored region / Total area

[0048] Here, the area of ​​the light-colored region and the area of ​​the dark-colored region are both determined by automatically measuring the area using NIS Elements, image analysis software manufactured by Nikon Corporation.

[0049] <Sample> In this embodiment, "sample" means a substance containing the substance to be detected or a substance that may contain the substance to be detected. In this embodiment, the substance to be detected includes glycoproteins. That is, the sample may contain glycoproteins. The sample may be a biological sample from an animal (for example, a human, a cow, a horse, a pig, a goat, a chicken, a rat, a mouse, etc.). A biological sample may include, for example, blood, tissue, cells, secretions, body fluids, etc. The "sample" may also include dilutions or separations thereof (serum, plasma, etc.). "Liquid sample" is a liquid containing the sample.

[0050] In one aspect of this embodiment, it is preferable that the sample is frozen after collection, or refrigerated for a predetermined period and then frozen. This allows the glycoproteins contained in the sample to form polymers or nanoparticles, enabling efficient detection or quantification of the glycoproteins even when using only one type of host molecule. The refrigerated storage period of the sample before freezing may be, for example, 1 day to 10 days.

[0051] (Glycoprotein) In this embodiment, "glycoprotein" refers to a substance in which a sugar chain is attached to some of the amino acid residues that make up a protein. The glycoprotein is not particularly limited, but examples include CEA, CA19-9, CD99, AFP, immunoglobulin, collagen, mucin, follicular-stimulating hormone, erythropoietin, transferrin, lectin, etc. In one aspect of this embodiment, the glycoprotein is preferably a cancer marker protein. The cancer marker protein preferably includes at least one selected from the group consisting of CEA, CD99, AFP, and CA19-9.

[0052] <Fine particles> In this embodiment, "fine particles" refers to substances having a size ranging from the nanometer order to the micrometer order. In this embodiment, the fine particles are modified with a host molecule, which will be described later. The shape of the fine particles is not limited to a spherical shape, but may also be ellipsoidal or rod-shaped. If the fine particles are ellipsoidal, at least one of the length along the major axis and the length along the minor axis of the ellipsoid should be within the range of nanometers to micrometers. If the fine particles are rod-shaped, at least one of the width and length of the rod should be within the range of nanometers to micrometers.

[0053] Examples of fine particles include metal nanoparticles, metal nanoparticle aggregates, metal nanoparticle aggregate structures, semiconductor nanoparticles, organic nanoparticles, resin beads, magnetic beads, and particulate matter (PM). "Metal nanoparticles" are metal particles having a size on the order of nanometers. "Metal nanoparticle aggregates" are aggregates formed by the aggregation of multiple metal nanoparticles. "Metal nanoparticle aggregate structures" are structures in which, for example, multiple metal nanoparticles are fixed to the surface of a bead via interaction sites, with gaps between them, and are arranged at intervals smaller than the diameter of the metal nanoparticles. "Semiconductor nanoparticles" are semiconductor particles having a size on the order of nanometers. "Organic nanoparticles" are particles made of organic compounds having a size on the order of nanometers. "Resin beads" are particles made of resin having a size ranging from nanometers to micrometers. Examples of resin beads include resin beads made of polystyrene. "Magnetic beads" are magnetic particles (polymer fine particles with magnetic material dispersed or embedded inside) having a size ranging from nanometers to micrometers. "PM" refers to particulate matter having a size on the order of micrometers.

[0054] In one aspect of this embodiment, the fine particles may further contain an additive that enhances electrostatic repulsion. Examples of the "additive that enhances electrostatic repulsion" include fluorescent dyes and surface modification reagents. Examples of functional groups that can be added by the surface modification reagent include carboxyl groups and amino groups. If the fine particles are resin beads, the resin beads may contain a fluorescent dye. As shown in the examples described later, resin beads containing a fluorescent dye (hereinafter sometimes referred to as "fluorescent beads") have a strongly negative surface potential (zeta potential). Therefore, they tend to suppress nonspecific adsorption between host molecules (e.g., antibodies) modified on the fine particles, and consequently tend to improve measurement accuracy. The inventors believe that a similar mechanism is at work when other "additives that enhance electrostatic repulsion" other than fluorescent beads are used.

[0055] In this embodiment, "nanometer order" includes the range from 1 nm to 1000 nm (= 1 μm). "Micrometer order" includes the range from 1 μm to 1000 μm (= 1 mm). Therefore, "from nanometer order to micrometer order" includes the range from 1 nm to 1000 μm. The term "from nanometer order to micrometer order" typically means a range of a few nm to several hundred μm, preferably a range of 100 nm to 100 μm, and more preferably a range of 1 μm to several tens of μm.

[0056] In one aspect of this embodiment, the size of the fine particles (e.g., diameter, major axis, minor axis) is preferably 100 nm to 100 μm, and more preferably 1 μm to 10 μm. When the wavelength of the laser light irradiated onto the fine particles is 1064 nm, setting the size of the fine particles within the above range tends to facilitate Mie scattering, and consequently, to generate a strong photoinduced force. The size of the fine particles can be measured, for example, by dynamic light scattering.

[0057] In one aspect of this embodiment, the fine particles may include two or more types of fine particles of different sizes, or two types of fine particles of different sizes. For example, fluorescent beads with a size of 2 μm and fluorescent beads with a size of 1 μm may be mixed and used. As for the mixing ratio, for example, the ratio of the number of 2 μm beads to 1 μm beads can be in the range of 1:8 to 1:0, and a range of 1:1 to 1:0.01 is preferred. By using such a range, nonspecific adsorption between beads is reduced, and a stable multilayer structure can be formed in the photoconcentration system.

[0058] (Host molecule) In this embodiment, "host molecule" refers to a substance capable of specifically binding (or specifically adhering) to the substance to be detected. Examples of combinations of host molecules and substances to be detected include antigens and antibodies, glycans and proteins, lipids and proteins, small molecule compounds (ligands) and proteins, proteins and proteins, and single-stranded DNA and single-stranded DNA. When either of these two substances with specific affinity is the substance to be detected, the other can be used as the host molecule. That is, for example, if the antigen is the substance to be detected, the antibody can be used as the host molecule. Conversely, if the antibody is the substance to be detected, the antigen can be used as the host molecule. In DNA hybridization, the substance to be detected is the target DNA, and the host molecule is the probe DNA. Anti-DNA antibodies that specifically bind to DNA (for example, anti-dsDNA antibodies that specifically bind to double-stranded DNA, anti-ssDNA antibodies that specifically bind to single-stranded DNA, etc.) can also be used as host molecules. Antigens may include cancer marker proteins, allergens, microorganisms (bacteria, fungi, etc.), viruses, vesicles, etc. Furthermore, by changing the type of antibody, it is possible to change the types of cancer marker proteins, allergens, microorganisms, or viruses that can be detected.

[0059] Host molecules are immobilized (sometimes described as "modified") on the surface of microparticles through interactions between the host molecules and the microparticles. The type of interaction used to immobilize the host molecules on the surface of the microparticles depends on the type of microparticles (e.g., the material of the microparticles). These interactions include, for example, covalent bonds, ionic bonds, metallic bonds, van der Waals forces, electrostatic interactions, hydrophobic interactions, intermolecular forces (e.g., hydrogen bonds), and adsorption forces. There are no particular limitations on the method of immobilizing the host molecules on the surface of microparticles, and known methods can be employed. When immobilizing host molecules on the surface of microparticles in a liquid, it is preferable to do so in a liquid containing a blocking agent, as described later, from the viewpoint of suppressing nonspecific adsorption of the host molecules.

[0060] In this embodiment, the host molecule specifically binds to the glycoprotein. Examples of the host molecule include antibodies, Fab fragments, F(ab')2 fragments, Fv fragments, and scFv. In one aspect of this embodiment, it is preferable that the host molecule includes at least one selected from the group consisting of antibodies, Fab fragments, F(ab')2 fragments, Fv fragments, and scFv. The animal from which the antibody is derived is not particularly limited and examples include mice, sheep, goats, camels, rats, and rabbits. The Fab fragments, F(ab')2 fragments, Fv fragments, and scFv can be produced by known methods (e.g., enzyme-based methods, genetic engineering methods).

[0061] <Diluent> In this embodiment, "diluent" refers to a liquid used to dilute the sample. The diluent contains a blocking agent and a buffer. The solvent constituting the diluent is usually water, such as deionized water or distilled water. The method for producing the diluent is not particularly limited and can be produced by general methods. For example, one method is to add the blocking agent and the buffer to deionized water to a predetermined concentration and dissolve them. The obtained diluent may then be subjected to post-treatment such as filter sterilization.

[0062] The pH of the above diluent is higher than the isoelectric point of the glycoprotein. Furthermore, the diluent is preferably neutral. This suppresses nonspecific adsorption between microparticles modified with host molecules. As a result, the accuracy and sensitivity of glycoprotein detection and quantification using the photoconcentration system are improved. Specifically, the pH of the above diluent is preferably between 4.8 and 9, more preferably between 6 and 8, and even more preferably between 6 and 7.2. The pH of the above diluent can be measured using a commonly used pH meter.

[0063] When the glycoprotein is a carcinoembryonic antigen (isoelectric point: 4.7), the pH of the diluent is preferably 4.7 to 7.0, and more preferably 6.3 to 6.8.

[0064] In this embodiment, the salt concentration of the diluent is such that the host molecule-modified microparticles do not precipitate by salting out. Suppressing the salting out of the host molecule-modified microparticles improves the accuracy and sensitivity of the detection and quantification of glycoproteins using the photoconcentration system. The salt concentration of the diluent may be between 0 ng / mL and 100 ng / mL, or between 1 ng / mL and 10 ng / mL. Here, if there are multiple types of salts in the diluent, the sum of the concentrations of each salt is referred to as the "salt concentration of the diluent." The salt concentration can be estimated, for example, by observing that beads form due to salting out during photoconcentration, regardless of the concentration of the substance to be detected.

[0065] In one aspect of this embodiment, the salt concentration of the diluent is preferably such that the fine particles modified with the host molecule do not settle by salting out, and the thickness of the electrical double layer in the fine particles decreases. This further improves the binding specificity of the host molecule to the glycoprotein. Here, "thickness of the electrical double layer in the fine particles" is expressed as the reciprocal of a parameter called the Debye length, which indicates the degree of attenuation of the surface potential. The Debye length is closely correlated with the surface potential and the zeta potential. Therefore, the Debye length can be estimated by measuring the zeta potential of the fine particles, and consequently, the thickness of the electrical double layer can be estimated. If the fine particles are fluorescent beads, the salt concentration of the diluent may be 80 ng / mL or more and 100 ng / mL or 90 ng / mL or more and 100 ng / mL.

[0066] In this embodiment, examples of salts that may be included in the diluent include sodium chloride and potassium chloride.

[0067] (Blocking agent) In this embodiment, the blocking agent is a substance that suppresses the nonspecific adsorption of the host molecules to each other in the sample.

[0068] Examples of the blocking agents mentioned above include albumin (such as bovine serum albumin (BSA)), gelatin, casein, and goat serum. In one aspect of this embodiment, it is preferable that the blocking agent includes at least one selected from the group consisting of albumin, gelatin, casein, and goat serum. It goes without saying that the substance used as the blocking agent is a different substance from the glycoprotein that is the substance to be detected.

[0069] In this embodiment, the concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. This suppresses the nonspecific adsorption of microparticles modified with host molecules. As a result, the accuracy and sensitivity of the detection and quantification of glycoproteins using the photoconcentration system are improved. The concentration of the blocking agent is preferably 0.000001% by mass or more and less than 0.001% by mass relative to the diluent. The concentration of the blocking agent can be determined, for example, by an ELISA method using an anti-BSA antibody or by a photoconcentration system.

[0070] (buffering agent) In this embodiment, "buffering agent" means a substance that suppresses pH fluctuations in a target liquid (for example, in a diluent) caused by changes in the environment within that liquid. Examples of changes in the environment within the liquid include changes in salt concentration, mixing with other liquids, and temperature changes.

[0071] Examples of the buffering agent include phosphate compounds, trishydroxymethylaminomethane, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and MES (2-(N-morpholino)ethanesulfonic acid). In one aspect of this embodiment, it is preferable that the buffering agent includes at least one selected from the group consisting of phosphate compounds, trishydroxymethylaminomethane, HEPES, and MES. Examples of phosphate compounds include phosphoric acid, disodium hydrogen phosphate, and potassium dihydrogen phosphate. The concentration of the buffering agent is not particularly limited, but may be, for example, 0.1 mM to 1000 mM, or 1 mM to 100 mM.

[0072] (Other ingredients) The diluent according to this embodiment may further contain other components, to the extent that the effects of the present invention are achieved.

[0073] <Dispersion> The kit according to this embodiment preferably further includes a dispersion for dispersing the fine particles modified with the host molecule. The dispersion preferably contains the blocking agent and the buffer. The blocking agent contained in the dispersion may be the same as the blocking agent contained in the diluent, or it may be a different component. Specific examples of the blocking agent contained in the dispersion are those described above. The concentration of the blocking agent is preferably 0.00000001% by mass or more and 0.001% by mass or less relative to the dispersion. By setting the concentration within this range, it is possible to suppress nonspecific adsorption of the fine particles. The method for producing the dispersion is not particularly limited and can be produced by general methods. For example, one method is to add the blocking agent and the buffer to deionized water to a predetermined concentration and dissolve them. The obtained dispersion may then be subjected to post-treatment such as filter sterilization.

[0074] The buffering agent contained in the dispersion may be the same as the buffering agent contained in the diluent, or it may be a different component. Specific examples of the buffering agent contained in the dispersion are those mentioned above. The concentration of the buffering agent is not particularly limited, but for example, it may be between 0.1 mM and 1000 mM, or between 1 mM and 100 mM.

[0075] In one aspect of this embodiment, the microparticles modified with the host molecule and the dispersion may be included separately in the kit. Alternatively, the microparticles modified with the host molecule may be included in the kit already dispersed in the dispersion. That is, the dispersion may contain the microparticles modified with the host molecule.

[0076] <Microfluidic chip> The above kit preferably further includes a microfluidic chip. An example of the above microfluidic chip is the microfluidic chip 90 shown in Figure 1. Figure 2 is a schematic cross-sectional view of the microfluidic chip 90 along the line II-II in Figure 1. As shown in Figure 2, the cross-section of the microfluidic channel 92 has, for example, a rectangular shape. For example, the width of the rectangle (channel width, length in the y direction) is 100 μm, and the height of the rectangle (length in the z direction) is 100 μm.

[0077] The microfluidic chip 90 is preferably made of a material that is transparent to the laser light L1. The material used for the microfluidic chip 90 is preferably a material that does not exhibit anisotropy with respect to the polarized laser light L1, such as glass or quartz. In one aspect of this embodiment, the surface of the microfluidic channel 92 in the microfluidic chip 90 is preferably made of a material that does not nonspecifically adsorb the substance to be detected and the host molecule.

[0078] In one aspect of this embodiment, it is preferable that the surface of the microchannel 92 in the microfluidic chip 90 is blocked with a blocking agent. The blocking agent can be one of those listed above. There are no particular limitations on the blocking method; known methods can be used. For example, the surface of the microchannel 92 can be blocked by the method described in the embodiments below.

[0079] <Other configurations> The kit according to this embodiment may further include other components to the extent that the effects of the present invention are achieved. Examples of other components include instructions describing how to use the kit, microtubes, the glycoprotein (positive control), etc.

[0080] ≪Kit Usage Instructions≫ The kit according to this embodiment can be used, for example, as follows. First, the sample is prepared by adding the diluent and the fine particles to the sample. At this time, the diluent may be added to the sample and then the fine particles may be added, or the fine particles may be added to the sample and then the diluent may be added. Alternatively, the diluent and the fine particles may be added to the sample simultaneously. Here, "sample to be measured" means the sample immediately before being subjected to measurement using the photoconcentration system.

[0081] Next, the prepared sample is measured using the above-mentioned photoconcentration system to detect or quantify the glycoproteins contained in the sample. Examples of measurement conditions at this time include those described in the [Examples] section below.

[0082] The details of the kit according to this embodiment have been described above. While it has been anticipated that a photoconcentration system can be used to detect and quantify trace amounts of substances, the specific methods for preparing and measuring the sample had not been specifically investigated. In this embodiment, by configuring the diluent used for sample dilution as described above, it has become possible to rapidly and sensitively detect trace amounts of glycoproteins contained in the sample using a photoconcentration system. More specifically, a sensitivity improvement of 10 to 100 times and a speed improvement of 60 times have been achieved compared to the conventionally used ELISA method. Such a rapid and highly sensitive detection technology using a photoconcentration system is expected to contribute to the realization of ultra-early diagnosis of cancer.

[0083] <Other Embodiments> Another kit according to this embodiment is a kit for detecting glycoproteins contained in a sample using a photoconcentration system, Microparticles with modified host molecules, A diluent for diluting the above sample, A dispersion for dispersing fine particles modified with the above-mentioned host molecule, The above host molecule specifically binds to the above glycoprotein, The above dilution contains a buffering agent, The above dispersion contains a blocking agent and the above buffering agent. The pH of the above dilution is higher than the isoelectric point of the above glycoprotein. The concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. The salt concentration of the above dilution is such that the fine particles modified with the host molecule do not settle by salting out. [Examples]

[0084] The following describes examples of the present invention, but the present invention is not limited to these examples. In the following, "CEACAM-5" may be simply referred to as "CEA".

[0085] ≪1. Preparation of antibody-modified beads≫ <Preparing the reagents> The following reagents were prepared to create antibody-modified beads (microparticles modified with host molecules). (Beads (microparticles)) Streptavidin-coated microspheres (streptavidin-modified resin beads): 2 μm Plain (Polysciences, Cat. No. 24160, Material: Polystyrene): These beads are suspended at a concentration of 1.25% in 0.02 M sodium phosphate buffer. The sodium phosphate buffer contains NaCl (8 mg / mL), bovine serum albumin (BSA) (10 mg / mL), sodium azide (0.1%), and glycerol (5%).

[0086] (Antibody (host molecule)) The following two antibodies, included in the Human CEACAM-5 / CD66e DuoSet ELISA (manufactured by R&D Systems, Cat. No. DY4128, ELISA kit), were used. Biotinylated sheep anti-human CEACAM-5 antibody (Antibody A): 36 μg / mL (dissolved in PBS containing 1% BSA, pH 7.2-7.4). The composition of the above PBS is 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4. Mouse anti-human CEACAM-5 antibody (Antibody B): 1 mg / mL (dissolved in PBS, pH 7.2-7.4), used after biotinylation using the method described later.

[0087] (buffer) 10 mM phosphate buffer (pH 6.7): Prepared by diluting 0.1 mol / L phosphate buffer pH 6.4 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog No. 164-27135) 10-fold with distilled water (DDW). 10 mM phosphate buffer (pH 6.3): Prepared by diluting 0.1 mol / L phosphate buffer pH 6.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog No. 167-27125) 10-fold in DDW. 10 mM phosphate buffer (pH 7.2): Prepared by diluting 0.1 mol / L phosphate buffer pH 7.0 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog No. 168-27155) 10-fold in DDW. 10 mM phosphate buffer (pH 7.0): Prepared by diluting 0.1 mol / L phosphate buffer pH 6.8 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., catalog No. 161-27145) 10-fold in DDW.

[0088] <Biotinylation of antibody B> Follow these steps to enable EZ-Link TM Sulfo-NHS-LC-Biotin, No-Weigh TM Format <Thermo Scientific TM Antibody B was biotinylated using Cat.No. A39257. 720 μL of antibody B, dissolved at a concentration of 1 mg / mL in 10 mM phosphate buffer pH 7.2, was mixed with 13 μL of biotinylation reagent and allowed to stand on ice for 2 hours to biotinylate. After standing on ice, excess biotinylation reagent was removed using a desalting column (Zeba Spin Desalting Columns 7K MWCO) equilibrated with 10 mM phosphate buffer pH 6.7.

[0089] Following the above procedure, biotinylated antibody B was obtained. In the following description, unless otherwise specified, "antibody B" refers to biotinylated antibody B.

[0090] <Antibody modification of beads> 18 μL of the streptavidin-modified bead suspension was taken and transferred to a microcentrifuge tube. 90 μL of 10 mM phosphate buffer (pH 6.7) was added to the microcentrifuge tube. The microcentrifuge tube was then placed in a centrifuge and centrifuged at 10,000 g for 5 minutes, allowing the beads to settle at the bottom of the tube. 83.3% of the supernatant was removed by volume from the microcentrifuge tube, and the same amount of 10 mM phosphate buffer (pH 6.7) was added to the tube. This procedure was repeated five times to wash the streptavidin-modified beads.

[0091] 10 mM phosphate buffer (pH 6.7) was added to the above microtube to dilute the suspension containing the washed streptavidin-modified beads threefold.

[0092] In the experiments described later in Study 1 to Study 4, antibody A and antibody B were diluted with 10 mM phosphate buffer (pH 6.7) to a final concentration of 20 μg / mL. In the experiment described in Study 5 below, antibody A was diluted with 10 mM phosphate buffer (pH 6.7) to a final concentration of 20 μg / mL. Antibody B was diluted with PBS containing 1% BSA (pH 7.2-7.4) to a final concentration of 20 μg / mL.

[0093] A suspension containing streptavidin-modified beads and a solution containing antibody A or antibody B were mixed in equal volumes to form a mixture, which was then allowed to stand at 42°C for 1 hour.

[0094] Subsequently, the microcentrifuge tube containing the above mixture was placed in a centrifuge and centrifuged at 10,000 g for 5 minutes, allowing the antibody-modified beads to settle at the bottom of the tube. 83.3% of the supernatant was removed from the microcentrifuge tube by volume, and then the same amount of 10 mM phosphate buffer (pH 6.7) was added to the tube. This procedure was repeated three times to wash the antibody-modified beads. For the second and third washes, 10 mM phosphate buffer (pH 6.3) was used instead of 10 mM phosphate buffer (pH 6.7). Through these steps, antibody-modified beads were obtained. Based on the above procedure, it is estimated that the suspension of antibody-modified beads contains 7.89 ng / mL of NaCl and 23.7 ng / mL of KCl.

[0095] ≪2. Investigation of detection conditions for glycoproteins using a light concentration system≫ <Study 1: pH dependence of liquid samples> (Preparation of liquid sample) Recombinant Human CEACAM-5 (glycoprotein) (concentration 100 ng / mL), included in the Human CEACAM-5 / CD66e DuoSet ELISA (manufactured by R&D Systems, Cat. No. DY4128, ELISA kit), was diluted with the diluents described below to achieve final concentrations of 7.8 pg / mL, 15.6 pg / mL, and 31.2 pg / mL, respectively, to prepare three standard samples. The above diluents were prepared using three different phosphate buffers with varying pH levels (pH: 7.2, 7.0, and 6.7). These phosphate buffers were prepared by diluting commercially available phosphate buffer (the phosphate buffer manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., mentioned above) 10-fold with DDW. In addition, the above three diluents were used as standard samples with a CEACAM-5 concentration of 0 pg / mL. In other words, since we prepared four different standard samples with varying concentrations of CEACAM-5 in each phosphate buffer, we ended up with 12 different standard samples with varying pH and CEACAM-5 concentrations.

[0096] Twelve different standard samples, each 20 μL in volume, were transferred to a microcentrifuge tube. To each tube, 20 μL of the suspension of the antibody A-modified beads (as described in "1. Preparation of antibody-modified beads") was added and thoroughly mixed. Using this procedure, twelve different measurement samples (pH 6.7-7.2, CEACAM-5 concentration 0-15.6 pg / mL) were prepared.

[0097] (Detection by a photoconcentration system) The prepared sample was injected into the microchannel of the microfluidic chip in the photoconcentration system, and the sample was irradiated with light under the following conditions. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​the multilayer portion, and the proportion of the multilayer portion (hereinafter referred to as "multilayer proportion") in the measurement field were determined by the method described above. The results are shown in Figure 7. In Figure 7, the vertical axis represents the proportion of the multilayer portion, and the horizontal axis represents the concentration of CEACAM-5 in the sample. Light irradiation conditions Microchannel: 100 μm × 100 μm Syringe: Inner diameter 2.3 mm / Capacity 0.25 mL Flow rate: 0.05 μL / min with 250 μL syringe setting. Laser intensity: 265mW for 5 minutes Beam waist position of x40 lens: 45 μm below the bottom of the channel

[0098] The results in Figure 7 show that lowering the pH can reduce the proportion of the multilayer portion at a CEACAM-5 concentration of 0 pg / mL. This result is thought to be because, when the pH of the sample was 6.7, a positive charge was imparted by hydrogen ions, and the nonspecific adsorption between antibody-modified beads was suppressed due to electrostatic repulsion. It was also found that the pH of the diluent was higher than the isoelectric point of CEACAM-5 (4.7).

[0099] <Consideration 2: Consideration of the position of the beam waist> The experiment was conducted in the same manner as in Study 1 above, except that two types of phosphate buffer A or B were used as diluents, and the beam waist position during measurement was set 65 μm below the bottom of the flow path. The aggregate area of ​​beads, the aggregate area of ​​the multilayer portion, and the proportion of the multilayer portion in the measurement field were determined by the prescribed method. The results are shown in Figure 8. In Figure 8, the vertical axis represents the aggregate area of ​​beads, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion, and the horizontal axis represents the concentration of CEACAM-5 in the liquid sample. Dilution used Phosphate buffer A (pH 6.7, does not contain NaCl or KCl) Phosphate buffer B (pH 6.7-6.8, does not contain NaCl or KCl)

[0100] The results in Figure 8 show that setting the beam waist position 65 μm below the bottom of the flow path during measurement improves sensitivity and measurement accuracy compared to when the beam waist position is 45 μm below the bottom of the flow path (see Figure 7).

[0101] <Study 3: Dependence on the concentration of the blocking agent> The experiment was conducted in the same manner as in Study 1 above, except that four types of phosphate buffers with different BSA concentrations were used as diluents, 10 μL each of suspensions of beads modified with antibody A and beads modified with antibody B were used as bead suspensions, and the flow rate was changed to 0.1 μm / min. The aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the percentage of multilayer portions in the measurement field were determined by the prescribed method. The results are shown in Figure 9. In Figure 9, the vertical axis shows the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, or the percentage of multilayer portions, and the horizontal axis shows the concentration of CEACAM-5 in the liquid sample. After modification, the final BSA concentration in the antibody-modified bead dispersion after washing (3 times) was 2.16 × 10⁻⁶ for the suspension of beads modified with antibody A. -4 The percentage is 1.65 × 10 in the suspension of antibody B-modified beads. -8 It was %. Basic composition of the diluent used 10 mM phosphate buffer (pH 6.3, does not contain NaCl or KCl) BSA concentration of the diluent used 0.00001%, 0.0001%, 0.001%, or 0.01%

[0102] The results in Figure 9 show that when the BSA concentration is 0.0001%, the accumulation of the multilayer portion at a CEACAM-5 concentration of 0 pg / mL can be kept low. This BSA concentration is an order of magnitude lower than the optimal concentration for a blocking agent in ELISA (0.001%, see Figure 14). This result is thought to be because the BSA was also concentrated by photoconcentration, allowing it to efficiently adsorb to the antibody-modified bead surface and exert a blocking effect. Note that the above BSA concentration was lower than the optimal concentration of BSA in the ELISA method described later (i.e., a concentration lower than the concentration that suppresses nonspecific adsorption between host molecules in an environment where photo-induced force is not acting on the host molecules).

[0103] Figure 10 is a photograph showing the vicinity of the laser spot when observing a sample with a BSA concentration of 0.0001%. In Figure 10, the concentration in the upper left represents the concentration of CEACAM-5. From the results in Figure 10, it was found that the dark area increases depending on the concentration of CEACAM-5.

[0104] Figure 11A is a graph showing the correlation between the total aggregate area, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion (vertical axis) and the concentration of glycoprotein in the sample (horizontal axis) when observing a sample with a BSA concentration of 0.0001%. The graph in the lower right of Figure 11A is a graph showing a model function obtained by the least squares method based on the measured experimental data, relating the proportion of the multilayer portion (vertical axis) and the concentration of glycoprotein (CEACAM-5) in the sample (horizontal axis). The coefficient of determination (R) 2 The ratio was 0.9728, suggesting that the concentration of glycoproteins in the sample (especially in the low concentration range of 0-15.6 pg / mL) can be accurately determined based on the proportion of the multilayer portion mentioned above.

[0105] <Study 4: Dependence of liquid sample on salt concentration (ionic strength)> The experiment was conducted in the same manner as in Study 1 above, except that PBS was used as the diluent, and the aggregation of beads in the measurement field of view was observed.

[0106] The experiment revealed that when PBS was used as the diluent, bead salting-out occurred regardless of the concentration of the detected substance (CEACAM-5). On the other hand, when phosphate buffer was used, as described in studies 1-3 above, no bead salting-out was observed. This result suggests that reducing the salt concentration (ionic strength) in the sample can suppress bead sedimentation due to salting-out.

[0107] From the results of Consideration 1, Consideration 3, and Consideration 4 above, it was found that the dilution solution should be as low as possible within the neutral range, the pH of the dilution solution should be higher than the isoelectric point of the glycoprotein, the concentration of the blocking agent should be lower than the concentration that suppresses the non-specific adsorption of host molecules to each other in an environment where the photoinduced force does not act on the host molecules, and the salt concentration of the dilution solution should be such that the microparticles modified with the host molecule do not precipitate by salting out. These are important for rapidly and sensitively detecting trace amounts of glycoprotein contained in a sample using a photo-concentration system.

[0108] <Consideration 5: Examination of the Concentration of the Blocking Agent in the Dispersion Liquid> In the experiment of Consideration 5, antibody A was diluted with 10 mM phosphate buffer (pH 6.7) so that the final concentration was 20 μg / mL. Antibody B was diluted with PBS (pH 7.2 - 7.4) containing 0.57% BSA so that the final concentration was 20 μg / mL. By preparing in this way, both the solution containing antibody A and the solution containing antibody B became solutions containing BSA at a final concentration of 0.56%. Furthermore, after modification, the BSA concentration in the final antibody-modified bead dispersion became (1 / 6) 4 times due to Wash (4 times), so 0.28×(1 / 6) 5 = 3.6×10 -5 %.

[0109] Regarding the subsequent operations, except that a phosphate buffer containing 0.00001% BSA was used as the dilution solution so that the BSA concentration was about the same as the BSA concentration in the above antibody-modified bead dispersion, and the suspension of beads modified with antibody A and the suspension of beads modified with antibody B were each washed 4 times with a dispersion liquid having the following composition and then used, the experiment was conducted in the same manner as in Consideration 3 above, and the aggregated area of the beads, the aggregated area of the multilayer part, and the ratio of the multilayer part in the measurement field were determined by a predetermined method. The results are shown in Fig. 11B. In Fig. 11B, the vertical axis indicates the aggregated area of the beads, the aggregated area of the multilayer part, or the ratio of the multilayer part, and the horizontal axis indicates the concentration of CEACAM-5 in the liquid sample. Composition of the Dispersion Liquid Used 10 mM phosphate buffer (pH 6.3, does not contain NaCl or KCl) BSA concentration: 1.0×10 -5 %

[0110] The results in Figure 11B show that a calibration curve with superior linearity was obtained when a suspension of beads modified with antibody A or antibody B was prepared using a dispersion containing a predetermined concentration of BSA.

[0111] ≪3. Method for storing samples containing glycoproteins≫ The following experiment investigated whether the storage method of samples containing glycoproteins affects the quantification of glycoproteins using a photoconcentration system.

[0112] First, blood samples were collected from two colorectal cancer patients, and plasma was recovered using a prescribed method. The concentration of CEACAM-5 (marker protein) in each of the obtained plasma samples was determined by ELISA, and the results were 2.4 ng / mL and 13.2 ng / mL, respectively. Hereafter, plasma with a low CEACAM-5 concentration will be referred to as "Plasma A," and plasma with a high CEACAM-5 concentration will be referred to as "Plasma D." Both Plasma A and Plasma D were refrigerated at 4°C for 1-4 hours after collection, and then stored at -80°C or below. Plasma A and Plasma D correspond to Plasma A and Plasma D in the experiments using clinical samples described later.

[0113] Next, plasma D was diluted 1,000 to 8,000 times with a diluent having the following composition to prepare four different measurement samples with varying concentrations of CEACAM-5 (1.6 pg / mL, 3.2 pg / mL, 6.6 pg / mL, and 13.2 pg / mL). For the four prepared measurement samples, experiments were conducted in the same manner as in Study 1 above, and the aggregate area of ​​beads, the aggregate area of ​​the multilayer portion, and the proportion of the multilayer portion in the measurement field were determined using a predetermined method. The results are shown in Figure 12 (left side, calibration curve α). In Figure 12, the vertical axis represents the aggregate area of ​​the multilayer portion, and the horizontal axis represents the concentration of CEACAM-5. Diluent composition: 10 mM phosphate buffer (pH 7.2)

[0114] On the other hand, for plasma A, after diluting it 1000-fold with the above diluent, Recombinant Human CEACAM-5 used in Study 1 was added to prepare five different measurement samples with varying concentrations of CEACAM-5 (2.4 pg / mL, 4.8 pg / mL, 7.2 pg / mL, 9.6 pg / mL, and 12.0 pg / mL). Experiments were conducted on the five prepared measurement samples using the same method as in Study 1, and the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the proportion of multilayer portions in the measurement field were determined using a predetermined method. The results are shown in Figure 12 (right side, calibration curve β).

[0115] The results in Figure 12 show that in the experiment using plasma D, the aggregate area of ​​the multilayer portion increased in a dependency of the CEACAM-5 concentration. On the other hand, in the experiment using plasma A, no correlation was observed between the CEACAM-5 concentration and the aggregate area of ​​the multilayer portion. In this experiment, even though only beads modified with antibody A were used, the beads aggregated efficiently in the measurement sample prepared from plasma D. Considering that there is only one epitope of CEACAM-5 for antibody A, it is thought that freezing the plasma causes CEACAM-5 to form a polymer, contributing to the aggregation of the beads (upper left figure in Figure 13). In contrast, in the measurement sample prepared from plasma A, Recombinant Human CEACAM-5, which had not been frozen, was added afterward. The added CEACAM-5 is thought to be in monomer form, and it is thought that the beads did not aggregate efficiently (upper right figure in Figure 13). From these results, it is considered preferable that samples containing glycoproteins be frozen for a predetermined period after collection.

[0116] ≪4. Comparative Example: Quantitative Analysis by ELISA Method (Low Concentration Range)≫ We investigated whether the conventional ELISA method could quantify concentrations below 15.6 pg / mL. The following ELISA kits were used.

[0117] (ELISA Kit) Human CEACAM-5 / CD66e DuoSet ELISA (R&D Systems) Duo Set Ancillary Reagent Kit2 (R&D Systems)

[0118] The Recombinant Human CEACAM-5 used in Study 1 above was dissolved in 10 mM phosphate buffer (pH 6.3). Next, the prepared CEACAM-5 solution was diluted with 10 mM phosphate buffer (pH 6.3) containing dissolved BSA to prepare the sample for measurement (2-fold dilution series, CEACAM-5 concentration 3.9 pg / mL to 250 pg / mL). At this time, the concentration of BSA in the phosphate buffer was set to 0.001%, 0.0001%, or 0.00001%. The experiment was carried out according to the manual attached to the ELISA kit, and the absorbance of each sample on the microplate was measured. The results are shown in Figure 14.

[0119] When quantifying a target protein using the ELISA method, the optimal absorbance range is considered to be 0.1 to 2.0. The results in Figure 14 show that in the CEACAM-5 concentration range of 15.6 pg / mL or less, the absorbance is below 0.1, indicating that this concentration range is unsuitable for ELISA quantification.

[0120] ≪5. Experiments using clinical samples≫ <Comparative study of photoconcentration system and conventional method (ELISA method)> The following experiment investigated whether it is possible to quantify glycoproteins using a photoconcentration system even when using plasma samples obtained from clinical settings.

[0121] First, blood samples were collected from four colorectal cancer patients, and plasma was recovered using a prescribed method. The CEACAM-5 concentration in each of the obtained plasma samples was determined by ELISA, and the results were 2.4 ng / mL, 4.7 ng / mL, 6.3 ng / mL, and 13.2 ng / mL, respectively. Hereafter, these four types of plasma will be referred to as "Plasma A," "Plasma B," "Plasma C," and "Plasma D," in order from the lowest to the highest CEACAM-5 concentration. All four types of plasma were refrigerated at 4°C for 1 to 4 hours after collection, and then stored at -80°C or below.

[0122] In the measurements using the photoconcentration system, the experiment was conducted in the same manner as in Study 1 above, except that each obtained plasma sample was diluted 1000-fold with a diluent (10 mM phosphate buffer (pH 7.2)). The proportion of the multilayer portion in the measurement field of view was determined by a predetermined method. The results are shown in Figure 15 (left side).

[0123] As a comparative experiment, the same measurements were performed using the conventional ELISA method. In the ELISA method, the experiment was conducted in the same manner as described in "4. Comparative Example: Quantitative Analysis by ELISA Method (Low Concentration Range)" above, except that each obtained plasma sample was diluted 10-fold with a diluent (10 mM phosphate buffer (pH 7.2)) before use, and the concentration of CEACAM-5 in the plasma samples was determined. The results are shown in Figure 15 (right side). From these experimental results, it was found that measurement using the photoconcentration system is possible with 100 times higher sensitivity compared to the conventional method, and that correlations between the magnitudes of CEACAM-5 concentrations among multiple plasma samples can also be confirmed.

[0124] <Measurement of clinical samples using the kit according to this embodiment> The plasma A and plasma B described above were diluted with the diluent according to this embodiment and then measured using the photoconcentration system. That is, the experiment was conducted in the same manner as in Study 3 above, except that each of the plasma A and plasma B was diluted 1000 times with the diluent (10 mM phosphate buffer, pH 6.3, 0.0001% BSA) to prepare the measurement sample, and the proportion of the multilayer portion in the measurement field was determined by a predetermined method. The results are shown in Figure 16 (upper right graph). The lower right graph of Figure 16 shows the results when the suspension of beads modified with antibody A and the suspension of beads modified with antibody B were each used in the same experiment with half the amount used in Study 3 above. The graph on the left of Figure 16 is an excerpt from the graph on the left of Figure 15.

[0125] The experimental results in Figure 16 show that by lowering the pH of the diluent used and optimizing the amount of BSA added, the detection signal (proportion of the multilayer portion) increased by more than three times, leading to an improvement in detection sensitivity.

[0126] 6. Suppression effect of nonspecific adsorption by the surface potential of beads. To investigate how the surface potential of the beads affects the nonspecific adsorption of antibodies modified on those beads, the following experiment was conducted.

[0127] <Measurement of the zeta potential of beads> First, as a preliminary experiment, two types of beads were prepared to measure the zeta potential of the beads (as shown in Figure 17, the zeta potential is an indicator of the thickness of the electrical double layer, which consists of a fixed layer and a diffusion layer, and the strength of the surface potential). The non-fluorescent beads are the same beads used in the sections "1. Preparation of antibody-modified beads" to "3. Method for storing samples containing glycoproteins" and "5. Experiments using clinical samples" above. (Beads (microparticles)) Non-fluorescent beads: Streptavidin Coated Microsphere 2μm Plain (Polysciences, Cat. No. 24160, Material: Polystyrene) Fluorescent beads: Streptavidin Fluoresbrite YG Coated Microsphere 2μm (Polysciences, Cat. No. 24159, Material: Polystyrene)

[0128] The measurement device used was the ELSZneo zeta potential, particle size, and molecular weight measurement system (product name, manufactured by Otsuka Electronics Co., Ltd.). The measurement conditions were as follows. The results are shown in Table 1. (Measurement conditions for zeta potential) Cell type: Zeta potential micro-disposable cell Solvent: Phosphate buffer (10 mM, pH 6.7) Temperature: 25℃ Applied voltage: -24.54(V)

[0129] [Table 1]

[0130] The results in Table 1 show that non-fluorescent beads have a negative zeta potential, while fluorescent beads have a strongly negative zeta potential and exhibit strong electrostatic repulsion between beads.

[0131] <Investigation of antibody modification levels when using fluorescent beads> Since fluorescent beads have a different surface potential (zeta potential) than non-fluorescent beads, we investigated how the aggregate area of ​​the beads, the aggregate area of ​​the multilayer portion, etc., are affected when the amount of antibody modification is changed (Figure 18). Antibody modification of fluorescent beads was performed in accordance with the section "1. Preparation of antibody-modified beads" described above. At this time, solutions containing antibody A and solutions containing antibody B were prepared by diluting each antibody with 10 mM phosphate buffer (pH 6.7, containing 0.00001% BSA) so that the final concentrations of each antibody were 10 μg / mL (half volume), 20 μg / mL (normal volume), and 40 μg / mL (double volume), respectively. Using this method, suspensions of beads with "half volume," "normal volume," or "double volume" of antibody modification were prepared.

[0132] (Preparation of liquid sample) Standard samples were prepared by diluting Recombinant Human CEACAM-5 (glycoprotein) (concentration 100 ng / mL), included in the Human CEACAM-5 / CD66e DuoSet ELISA (manufactured by R&D Systems, Cat. No. DY4128, ELISA kit), with the diluents described below to a final concentration of 7.8 pg / mL. Three different phosphate buffers with varying pH values ​​were used for the diluents (pH: 7.2, 6.7, and 6.3). These phosphate buffers were prepared by diluting commercially available phosphate buffer (the phosphate buffer manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. mentioned above) 10-fold with DDW. In addition, the three diluents described above were used as standard samples with a CEACAM-5 concentration of 0 pg / mL. In other words, since two standard samples with different CEACAM-5 concentrations were prepared for each phosphate buffer, a total of six standard samples with different pH values ​​and CEACAM-5 concentrations were prepared.

[0133] Six different standard samples (20 μL each) were transferred to microcentrifuge tubes. To these tubes, 10 μL of the fluorescent bead suspension modified with antibody A and 10 μL of the fluorescent bead suspension modified with antibody B were added and thoroughly mixed. Using this procedure, 18 different measurement samples were prepared (pH 6.3-7.2, CEACAM-5 concentration 0 pg / mL or 7.8 pg / mL, antibody modification amounts "half amount", "normal amount", and "double amount").

[0134] (Detection by a photoconcentration system) The prepared sample was injected into the microchannel of the microfluidic chip in the photoconcentration system, and the sample was irradiated with light under the following conditions. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​the multilayer portion, and the proportion of the multilayer portion (hereinafter referred to as "multilayer proportion") in the measurement field were determined by the method described above. The results are shown in Figure 18. In Figure 18, the vertical axis represents the aggregate area of ​​fluorescent beads, the aggregate area of ​​the multilayer portion, or the proportion of the multilayer portion, and the horizontal axis represents the concentration of CEACAM-5 in the sample. Light irradiation conditions Microchannel: 100 μm × 100 μm Syringe: Inner diameter 2.3 mm / Capacity 0.25 mL Flow rate: 0.1 μL / min with 250 μL syringe setting. Laser intensity: 265mW, measured 30 seconds after 5 minutes of irradiation. Beam waist position of x40 lens: 45 μm below the bottom of the channel

[0135] The results in Figure 18 show that, with fluorescent beads, the proportion of the multilayer portion at a CEACAM-5 concentration of 0 pg / mL was kept lowest at pH 6.7 and when the antibody modification amount was halved. This result indicates that nonspecific adsorption between fluorescent beads was sufficiently suppressed.

[0136] <Examination of calibration curves when using fluorescent beads> Except for using pH 6.7 phosphate buffer (10 mM) as the diluent, using half the amount of antibody modification for both antibody A and antibody B as the suspension of fluorescent beads, and using five types of standard samples with CEACAM-5 concentrations ranging from 0 to 15.6 pg / mL, the measurement samples were prepared in the same manner as described in the section (Preparation of Liquid Samples) in the above-mentioned <Investigation of Antibody Modification Amount When Using Fluorescent Beads>. The prepared measurement samples were injected into the microchannels of the microfluidic chip in the photoconcentration system, and the measurement samples were irradiated with light under the following conditions. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the percentage of multilayer portions in the measurement field were determined using the method described above. The results are shown in Figure 19. In Figure 19, the vertical axis shows the aggregate area of ​​fluorescent beads, the aggregate area of ​​multilayer portions, or the percentage of multilayer portions, and the horizontal axis shows the concentration of CEACAM-5 in the measurement sample. Light irradiation conditions Microchannel: 100 μm × 100 μm Syringe: Inner diameter 1.03 mm / Volume 50 μL Flow rate: 0.1 μL / min with 50 μL syringe setting. Laser intensity: 265mW or 400mW, measured 30 seconds after 5 minutes of irradiation. Beam waist position of x40 lens: 45 μm below the bottom of the channel

[0137] In Figures 11A and 11B (experimental results using non-fluorescent beads), the proportion of the multilayer portion was approximately 0.4 when the CEACAM-5 concentration was 15.6 pg / mL, but in Figure 19, the proportion of the multilayer portion reached 0.4 when the CEACAM-5 concentration was 7.8 pg / mL. These results show that detection at lower concentrations is possible by using fluorescent beads. Furthermore, it was found that increasing the laser intensity from 265 mW to 400 mW enhances the photo-induced force, allowing for more efficient pressing against the bottom of the channel, thus increasing the total aggregate area, the area of ​​the multilayer portion, and the proportion of the multilayer portion, resulting in a calibration curve with a steeper slope in the low-concentration region.

[0138] <Experiments using human CA19-9 as a glycoprotein> The following experiment investigated whether it is possible to quantify glycoproteins using a photoconcentration system when human CA19-9 is used as the glycoprotein. The experiment was conducted under the same conditions as in the above-mentioned <Investigation of Calibration Curve when Using Fluorescent Beads>, except that a biotinylated anti-human CA19-9 antibody was used as antibody A, fluorescent beads modified with antibody A were used with a "double" modification amount, human CA19-9 was used as the glycoprotein, and the laser intensity was set to 400 mW. The biotinylated anti-human CA19-9 antibody and human CA19-9 were those included in the CA19-9 ELISA Kit, Human, RayBio (1x96well strip plate) (RayBiothch, Cat. No. ELH-CA19-91). The results are shown in Table 2.

[0139] [Table 2]

[0140] The results in Table 2 show that even when human CA19-9 is used as the glycoprotein, there is a positive correlation between the glycoprotein concentration and the area and proportion of the multilayer region. In other words, it was shown that quantification of glycoprotein using the photoconcentration system is possible even when human CA19-9 is used as the glycoprotein.

[0141] <Experiments using clinical samples> The following experiments were conducted to verify whether glycoproteins in clinically obtained plasma samples could be quantified using a photoconcentration system with fluorescent beads (Figures 20 and 21). The plasma samples used were "Plasma A," "Plasma B," "Plasma C," and "Plasma D," which were used in section 5, "Experiments using Clinical Samples," above.

[0142] In measurements using the photoconcentration system, each obtained plasma sample was diluted 1000-fold with a diluent (10 mM phosphate buffer (pH 6.7, containing 0.00001% BSA)), the laser intensity was set to 400 mW, and the laser irradiation time was set to 5 minutes or 4 minutes. The experiment was conducted in the same manner as described in the section "Examination of Calibration Curves Using Fluorescent Beads" above, and the aggregate area of ​​fluorescent beads, the aggregate area of ​​multilayer portions, and the percentage of multilayer portions in the measurement field were determined by the prescribed method. The results are shown in Figures 20 and 21. In Figures 20 and 21, the vertical axis represents the aggregate area of ​​fluorescent beads, the aggregate area of ​​multilayer portions, or the percentage of multilayer portions, and the horizontal axis represents the label of the plasma sample.

[0143] The results shown in Figures 20 and 21 indicate that a correlation was observed between the CEA concentration in the plasma sample and the proportion of the multilayer portion, even when fluorescent beads were used. This correlation was particularly clear when the laser irradiation time was set to 4 minutes.

[0144] ≪7. Effect of improving specificity by salt concentration (ionic strength) of liquid samples≫ In the above study 4, it was confirmed that beads would salt out if the salt concentration of the liquid sample was too high. In this experiment, we investigated in more detail whether there is an optimal salt concentration for measurement using the photoconcentration system that does not cause bead salting out (Figure 22).

[0145] <Experiment 1> Except for using two types of phosphate buffer with different NaCl concentrations as diluents, using antibody A with "half" antibody modification and antibody B with "normal" antibody modification as the suspension of fluorescent beads, and using three types of standard samples (10 μL each) with CEACAM-5 concentrations ranging from 0 to 15.6 pg / mL, the measurement samples were prepared in the same manner as described in the section (Preparation of Liquid Samples) in the above-mentioned <Investigation of Antibody Modification Amount When Using Fluorescent Beads>. The prepared measurement samples were injected into the microchannels of the microfluidic chip in the photoconcentration system, and the measurement samples were irradiated with light under the following conditions. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the percentage of multilayer portions in the measurement field were determined using the method described above. The results are shown in Figure 22. In Figure 22, the vertical axis shows the aggregate area of ​​fluorescent beads, the aggregate area of ​​multilayer portions, or the percentage of multilayer portions, and the horizontal axis shows the concentration of CEACAM-5 and the sodium chloride concentration in the measurement sample. Basic composition of the diluent used 10 mM phosphate buffer (pH 6.7, containing 0.00001% BSA) NaCl concentration of the diluent used 0.9 × 10 -3 %(w / v) or 0.9 × 10 -4 %(w / v) Light irradiation conditions Microchannel: 100 μm × 100 μm Syringe: Inner diameter 1.03 mm / Volume 50 μL Flow rate: 0.1 μL / min with 50 μL syringe setting. Laser intensity: 400mW for 5 minutes Beam waist position of x40 lens: 45 μm below the bottom of the channel

[0146] <Experiment 2> The NaCl concentration in the diluted solution is 0.9 × 10 -4 %(w / v) or 0.9 × 10 -5Except for using %(w / v), using a different lot of the fluorescent bead suspension modified with antibody B than in Experiment 1, and changing the "flow rate" under light irradiation conditions to 0.1 μL / min, 0.15 μL / min, or 0.2 μL / min depending on the situation, the experiment was conducted in the same manner as in Experiment 1, and the aggregate area of ​​fluorescent beads, the aggregate area of ​​the multilayer portion, and the percentage of the multilayer portion in the measurement field were determined by the prescribed method. The results are shown in Figure 22.

[0147] From the results in Figure 22, the NaCl concentration in the dilution was 0.9 × 10⁻⁶. -5 When the value was %(w / v), nonspecific adsorption of fluorescent beads was low when the CEACAM-5 concentration was 0 pg / mL, and the area and proportion of the multilayer portion increased in correlation with the CEACAM-5 concentration (results of Experiment 2 in Figure 22). More specifically, in Figure 19, the proportion of the multilayer portion saturated at 0.4 when the CEA concentration was 7.8 pg / mL or higher, whereas in Figure 22, when the NaCl concentration was 0.9 × 10⁻⁶ -5 At %(w / v), the proportion of the multilayer portion increased to 0.6 even at 15.6 pg / mL. This indicates that adding NaCl to an extent that does not cause salting out reduces the absolute value of the zeta potential (and thus the thickness of the electrical double layer in Figure 17), allowing CEA and the antibody to come into closer proximity and enabling a more efficient antigen-antibody reaction.

[0148] ≪8. Blocking in Microchannels≫ We investigated whether blocking (pre-coating) the microchannel with BSA or similar material would improve measurement accuracy (Figures 23 and 24). The specific procedure is as follows.

[0149] First, the polytetrafluoroethylene (PTFE) tube used to aspirate the sample was filled with a pre-coating buffer (10 mM phosphate buffer, pH 6.7, 0.0001% BSA), and the inside of the PTFE tube was blocked with BSA by discharging the buffer.

[0150] The NaCl concentration in the diluted solution is 0.9 × 10 -5Except for using %(w / v) and employing standard samples (5 types, 20 μL) with CEACAM-5 concentrations ranging from 0 to 15.6 pg / mL, the measurement samples were prepared in the same manner as in Experiment 2 in the section above (7. Effect of Salt Concentration (Ionic Strength) on Specificity of Liquid Samples). Next, the measurement sample (20 μL) was aspirated into the PTFE tube, and then the pre-coating buffer (20 μL) was aspirated consecutively (see Figure 23). The PTFE tube was set in a syringe pump and injected into a microfluidic channel, and the measurement sample was irradiated with light under the following conditions. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the percentage of multilayer portions in the measurement field were determined using the method described above. The results are shown in Figure 24. In Figure 24, the vertical axis represents the aggregate area of ​​fluorescent beads, the aggregate area of ​​multilayer portions, or the percentage of multilayer portions, and the horizontal axis represents the concentration of CEACAM-5 in the measurement sample. Light irradiation conditions Microchannel: 100 μm × 100 μm Syringe: Inner diameter 1.03 mm / Volume 50 μL Flow rate: 0.15 μL / min with a 50 μL syringe (however, when injecting pre-coating buffer, it was set to 4 μL / min). Laser intensity: 400mW for 5 minutes Beam waist position of x40 lens: 45 μm below the bottom of the channel

[0151] The results in Figure 24 show that blocking the microchannel beforehand stabilizes the flow velocity when injecting the sample, allowing for the creation of a calibration curve with smaller errors.

[0152] 9. Stabilization of multilayer structures by mixing beads of different sizes. <Preparation of a suspension of antibody-modified fluorescent beads> The following experiment investigated whether mixing beads of different sizes stabilizes a multilayer structure (Figures 25 and 26). First, suspensions A1, A2, B1, and B2 of the following four types of antibody-modified fluorescent beads were prepared. Antibody modification of the fluorescent beads was carried out in accordance with the section "Investigation of the amount of antibody modification when using fluorescent beads" described above. (Suspension of antibody-modified fluorescent beads) (A1) Fluorescent beads modified with antibody A (size: 1 μm, antibody modification amount: half, density in suspension: 4.2 × 10⁻⁶) 9 pieces / mL) (A2) Fluorescent beads modified with antibody A (size: 2 μm, antibody modification amount: half, density in suspension: 5.3 × 10) 8 pieces / mL) (B1) Fluorescent beads modified with antibody B (size: 1 μm, antibody modification amount: normal amount, density in suspension: 4.2 × 10⁻⁶) 9 pieces / mL) (B2) Fluorescent beads modified with antibody B (size: 2 μm, antibody modification amount: 4 times, density in suspension: 5.3 × 10⁻¹⁴) 8 pieces / mL)

[0153] <Experiment A> As a diluent, 10 mM phosphate buffer (pH 6.7, containing 0.00001% BSA) was used to prepare five standard samples with CEACAM-5 concentrations ranging from 0 to 15.6 pg / mL. 20 μL of each of the five standard samples was transferred to a microtube, and the aforementioned suspensions A1 (5 μL after 20-fold dilution), A2 (5 μL), B1 (5 μL after 20-fold dilution), and B2 (5 μL) were added and thoroughly mixed. The measurement samples were prepared using the above procedure. In the measurement samples prepared in Experiment A, the ratio of 2 μm beads to 1 μm beads was calculated to be approximately 1:0.4, and the volume ratio was calculated to be approximately 1:0.05.

[0154] The prepared sample was injected into the microchannel of the microfluidic chip in the photoconcentration system, and the sample was irradiated with light under the following conditions. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the proportion of multilayer portions in the measurement field were determined using the method described above. The results are shown in Figure 25. In Figure 25, the vertical axis represents the aggregate area of ​​fluorescent beads, the aggregate area of ​​multilayer portions, or the proportion of multilayer portions, and the horizontal axis represents the concentration of CEACAM-5 in the sample. Light irradiation conditions Microchannel: 100 μm × 100 μm Syringe: Inner diameter 1.03 mm / Volume 50 μL Flow rate: 0.1 μL / min with 50 μL syringe setting. Laser intensity: 400mW for 5 minutes Beam waist position of x40 lens: 45 μm below the bottom of the channel

[0155] <Experiment B> As a diluent, 10 mM phosphate buffer (pH 6.7, containing 0.00001% BSA) was used to prepare five standard samples with CEACAM-5 concentrations ranging from 0 to 15.6 pg / mL. 20 μL of each of the five standard samples was transferred to a microtube, and the aforementioned suspensions A2 (10 μL) and B2 (10 μL) were added and thoroughly mixed. In Experiment B, suspensions A1 and B1 were not used. That is, the measurement sample prepared in Experiment B had a ratio of 2 μm beads to 1 μm beads of 1:0 (volume ratio was also 1:0). The measurement sample was prepared using the above procedure. The prepared measurement sample was injected into the microchannel of the microfluidic chip in the photoconcentration system, and the measurement sample was irradiated with light under the same conditions as in Experiment A. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the proportion of multilayer portions in the measurement field of view were determined.

[0156] <Experiment C> As a diluent, 10 mM phosphate buffer (pH 6.7, containing 0.00001% BSA) was used to prepare five standard samples with CEACAM-5 concentrations ranging from 0 to 15.6 pg / mL. 20 μL of each of the five standard samples was transferred to a microtube, and the above-mentioned suspensions A1 (5 μL after 10-fold dilution), A2 (5 μL), B1 (5 μL after 10-fold dilution), and B2 (5 μL) were added and thoroughly mixed. The measurement samples were prepared using the above procedure. In the measurement samples prepared in Experiment C, the ratio of 2 μm beads to 1 μm beads was calculated to be approximately 1:0.8, and the volume ratio was calculated to be approximately 1:0.1. The prepared measurement samples were injected into the microchannels of the microfluidic chip in the photoconcentration system, and the measurement samples were irradiated with light under the same conditions as in Experiment A. Subsequently, the aggregate area of ​​beads, the aggregate area of ​​multilayer portions, and the proportion of multilayer portions in the measurement field of view were determined.

[0157] Figure 26 shows a comparison of the proportions of the multilayer portion obtained in Experiments A, B, and C. In Figure 26, the "bead mixing ratio" is shown as a volume ratio. From the results in Figure 26, it was found that when the ratio of 2 μm beads to 1 μm beads was approximately 1:0.4 (1:0.05 by volume) (Experiment A), there was little nonspecific adsorption of beads and a stable multilayer structure was formed.

[0158] The embodiments and examples disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the claims rather than the embodiments and examples described above, and all modifications within the scope of the claims are intended to be included in the meaning of equivalents and within the scope. [Explanation of symbols]

[0159] B1, B2, B3 Beads, X, Y Detected substance, B0 Bead body, B11 First antibody, B21 Second antibody, B12, B22 Avidin, B13, B23 Biotin, B31 Antibody, 90 Microfluidic chip, 91 Substrate, 92 Microfluidic channel, 921 Inlet, 922 Outlet.

Claims

1. A kit for detecting glycoproteins contained in a sample using a photoconcentration system, Microparticles with modified host molecules, A diluent for diluting the aforementioned sample, The host molecule specifically binds to the glycoprotein, The aforementioned diluent comprises a blocking agent and a buffering agent. The pH of the diluent is higher than the isoelectric point of the glycoprotein. The concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. The salt concentration of the diluent is such that the fine particles modified with the host molecule do not precipitate by salting out, according to the kit.

2. The kit according to claim 1, wherein the diluent is neutral.

3. The kit according to claim 1 or claim 2, wherein the salt concentration of the diluent is such that the fine particles modified with the host molecule do not settle by salting out, and the thickness of the electrical double layer in the fine particles decreases.

4. The kit according to claim 1 or claim 2, wherein the fine particles further include an additive that enhances electrostatic repulsion.

5. The kit according to claim 1 or claim 2, wherein the fine particles include two or more types of fine particles of different sizes.

6. The host molecule is an antibody, a Fab fragment, and F(ab'). ２ The kit according to claim 1 or claim 2, comprising at least one selected from the group consisting of a fragment, an Fv fragment, and scFv.

7. The kit according to claim 1 or claim 2, wherein the blocking agent comprises at least one selected from the group consisting of albumin, gelatin, casein, and goat serum.

8. The kit according to claim 1 or claim 2, wherein the concentration of the blocking agent is 0.000001% by mass or more and less than 0.001% by mass relative to the diluent.

9. The kit according to claim 1 or claim 2, wherein the buffering agent comprises at least one selected from the group consisting of a phosphate compound, trishydroxymethylaminomethane, HEPES, and MES.

10. The dispersion further comprises a dispersion for dispersing the fine particles modified with the aforementioned host molecule, The dispersion comprises the blocking agent and the buffering agent. The kit according to claim 1 or claim 2, wherein the concentration of the blocking agent is 0.00000001% by mass or more and 0.001% by mass or less, relative to the dispersion.

11. The kit according to claim 1 or claim 2, wherein the glycoprotein is a cancer marker protein.

12. The kit according to claim 11, wherein the cancer marker protein comprises at least one selected from the group consisting of carcinoembryonic antigens and carbohydrate antigens.

13. The aforementioned sample is a sample that has been frozen after collection, or frozen after being refrigerated for a predetermined period of time. The kit according to claim 1 or claim 2, wherein the glycoprotein contained in the sample aggregates to form a polymer or nanoparticles.

14. The kit according to claim 1 or claim 2, further comprising a microfluidic chip.

15. A kit for detecting glycoproteins contained in a sample using a photoconcentration system, Microparticles with modified host molecules, A diluent for diluting the aforementioned sample, A dispersion for dispersing the fine particles modified with the aforementioned host molecule, The host molecule specifically binds to the glycoprotein, The aforementioned diluent contains a buffering agent, The dispersion comprises a blocking agent and a buffering agent. The pH of the diluent is higher than the isoelectric point of the glycoprotein. The concentration of the blocking agent is lower than the concentration that suppresses the nonspecific adsorption of the host molecules to each other in an environment where photo-induced force is not acting on the host molecules. The salt concentration of the diluent is such that the fine particles modified with the host molecule do not precipitate by salting out, according to the kit.

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