JP2009133842A - Optical-waveguide sensor chip, method of manufacturing same, method of measuring substance, substance-measuring kit and optical-waveguide sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical-waveguide sensor for quantitatively measuring a substance to be measured of an analyte to be measured at a small amount in a short time. <P>SOLUTION: This optical-waveguide sensor includes an optical waveguide having a first substance immobilized on the surface thereof, the first substance being specifically reactive with the substance to be measured, and fine particles dispersed on the optical waveguide and having a second substance immobilized on the surface thereof, the second substance being specifically reactive with the substance to be measured. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an optical waveguide sensor chip, an optical waveguide sensor chip manufacturing method, a substance measuring method, a substance measuring kit, and an optical waveguide sensor.

  In conventional immunoassay using antigen-antibody reaction, a primary antibody corresponding to a protein or the like in a sample to be measured is usually immobilized on the surface of a well-shaped substrate. A predetermined amount of the sample solution to be measured, the secondary antibody solution, and the coloring reagent are sequentially dropped into each well. A predetermined cleaning solution is performed for each dropping of each solution. Such an immunoassay is performed by a complicated procedure in which a measurer adds and discharges while weighing. In this immunoassay, the volume of the sample to be measured needs to be at least about 5 μL to 25 μL.

On the other hand, in Patent Document 1 filed by the present applicant, the minimum amount of the sample to be measured is 1 μL, and the concentration of the measurement target substance in the sample to be measured can be measured even if the volume of the sample to be measured is inaccurate. A measurement method and a sensor chip are disclosed.
WO2005 / 022155

  However, in the conventional immunoassay system, the reaction between the primary antibody and the sample to be measured and the reaction between the sample to be measured and the secondary antibody each take about one hour, and it is necessary to follow the complicated procedure as described above. Therefore, there is a problem that it takes several hours to obtain a measurement result from collection of a sample to be measured. In addition, since the amount of sample to be measured is large, for example, in a blood test using a small animal such as a rat, one sample is sacrificed for performing several types of test items once. As a result, it is difficult to examine changes with time in the same specimen.

  The present invention further improves the above-described invention of Patent Document 1, and is an optical waveguide sensor chip capable of quantitatively measuring a measurement target substance of a sample to be measured in a shorter time with a smaller amount of sample to be measured. An optical waveguide sensor chip manufacturing method, a substance measuring method, a substance measuring kit, and an optical waveguide sensor are provided.

According to the first aspect of the present invention, the first substance that specifically reacts with the measurement target substance is immobilized on the surface; and is dispersed on the optical waveguide and specifically reacts with the measurement target substance. Fine particles having a second substance immobilized thereon;
An optical waveguide sensor chip is provided.

According to the second aspect of the present invention, an optical waveguide in which a first substance that specifically reacts with a substance to be measured is immobilized on the surface;
A support plate disposed opposite to the optical waveguide; and fine particles on which a second substance that is dispersed on the surface of the support plate facing the optical waveguide and specifically reacts with the measurement target substance is immobilized;
An optical waveguide sensor chip is provided.

According to the third aspect of the present invention, the first substance that specifically reacts with the measurement target substance is immobilized on the surface of the optical waveguide;
Applying a slurry containing fine particles on which a second substance that specifically reacts with the substance to be measured is immobilized on the optical waveguide; and drying after the application to disperse the fine particles on the optical waveguide. ;
A method for manufacturing an optical waveguide sensor chip is provided.

According to the fourth aspect of the present invention, the first substance that specifically reacts with the measurement target substance is immobilized on the surface of the optical waveguide;
Applying a slurry containing fine particles on which a second substance that specifically reacts with the measurement target substance is immobilized on the surface of the support plate;
Drying after the coating to disperse the fine particles on the support plate; and disposing the support plate on the optical waveguide at a certain distance so that the fine particle dispersion surface faces each other;
A method for manufacturing an optical waveguide sensor chip is provided.

According to the fifth aspect of the present invention, the first substance that specifically reacts with the measurement target substance is immobilized on the surface, and is dispersed on the optical waveguide and specifically reacts with the measurement target substance. Providing an optical waveguide sensor chip comprising fine particles having a second substance immobilized thereon;
The analyte solution to be measured is dropped on the surface of the optical waveguide of the sensor chip to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution, and the substance to be measured and the Reacting specifically with the second substance of the fine particles dispersed on the optical waveguide; and detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the measurement target substance. To do;
There is provided a method for measuring a substance characterized by comprising:

According to a sixth aspect of the present invention, providing an optical waveguide sensor chip comprising an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface;
Dropping the analyte solution to be measured on the surface of the optical waveguide of the sensor chip to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution;
Cleaning the surface of the optical waveguide;
A dispersion liquid of fine particles in which a second substance that specifically reacts with the measurement target substance of the sample to be measured is dropped onto the surface of the optical waveguide to drop between the measurement target substance of the measurement target solution and the second substance of the fine particles. And detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
There is provided a method for measuring a substance characterized by comprising:

According to a seventh aspect of the present invention, an optical waveguide sensor chip including an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface;
A sample solution and a microparticle to which a second substance that specifically reacts with the measurement target substance of the sample to be measured is mixed in advance, and the second substance of the microparticle and the measurement target substance of the sample solution to be measured are specified. Reacting automatically;
Dropping the mixed liquid onto the surface of the optical waveguide of the sensor chip and causing the first substance on the surface of the optical waveguide to react specifically with the measurement target substance of the analyte solution to be reacted with the second substance of the fine particles; Detecting an optical change caused by fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
There is provided a method for measuring a substance characterized by comprising:

According to an eighth aspect of the present invention, an optical waveguide sensor chip including an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface thereof is prepared;
Dropping the analyte solution to be measured on the surface of the optical waveguide of the sensor chip to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution;
A dispersion liquid of fine particles in which a second substance that specifically reacts with the measurement target substance of the specimen to be measured is dropped on the surface of the optical waveguide, and specific between the measurement target substance and the second fine substance substance. And detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
There is provided a method for measuring a substance characterized by comprising:

According to a ninth aspect of the present invention, an optical waveguide sensor chip including an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface;
Dropping a dispersion liquid of fine particles on which a second substance that specifically reacts with a measurement target substance of a sample to be measured is immobilized on the optical waveguide surface of the sensor chip;
A sample solution to be measured is dropped on the surface of the optical waveguide to which the dispersion liquid has been dropped to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the sample solution to be measured. Reacting specifically between the substance and the second substance of the fine particles in the dispersion; and detecting optical changes caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured To do;
There is provided a method for measuring a substance characterized by comprising:

According to the tenth aspect of the present invention, an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface, a support plate disposed to face the optical waveguide, and the support plate Providing an optical waveguide sensor chip comprising fine particles dispersed on a surface facing the optical waveguide and fixed with a second substance that specifically reacts with the substance to be measured;
The analyte solution to be measured is injected between the optical waveguide of the sensor chip and the support plate to cause a specific reaction between the first substance on the surface of the optical waveguide and the analyte to be measured in the analyte solution. Reacting specifically between the substance and the second substance of the fine particles dispersed on the support plate; and optical change by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured Detecting
There is provided a method for measuring a substance characterized by comprising:

According to the eleventh aspect of the present invention, an optical waveguide in which a first substance that specifically reacts with a measurement target substance of a specimen to be measured is immobilized on the surface, and disposed on the surface of the optical waveguide, An optical waveguide sensor chip having a recess for forming a measurement area between them and having a cap with an introduction hole and a discharge hole communicating with the measurement area; and a substance to be measured and a specific substance A package containing a dispersion of fine particles on which a second substance that reacts automatically is immobilized;
A substance measurement kit characterized by combining the above is provided.

According to a twelfth aspect of the present invention, in the use of the substance measurement kit,
A sample substance to be measured is dropped onto the surface of the optical waveguide in the measurement region through the introduction hole of the cap of the optical waveguide sensor chip, and the first substance immobilized on the surface of the optical waveguide and the substance to be measured of the sample solution to be measured are specifically determined. Reacting with;
While introducing the dispersion liquid of fine particles to the surface of the optical waveguide in the measurement region through the introduction hole of the cap, the measurement target substance and the fine particles of the measurement target solution specifically reacted while the measurement target solution is discharged through the discharge hole. Reacting specifically with the second substance; and detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
There is provided a method for measuring a substance characterized by comprising:

According to the thirteenth aspect of the present invention, the first substance that specifically reacts with the measurement target substance is immobilized on the surface, and is dispersed on the optical waveguide and specifically reacts with the measurement target substance. An optical waveguide sensor chip having fine particles on which a second substance is immobilized;
A light source that makes light incident on the optical waveguide; and a light receiving element that receives light emitted from the optical waveguide;
An optical waveguide sensor is provided.

According to the fourteenth aspect of the present invention, an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface, a support plate disposed opposite to the optical waveguide, and the support plate An optical waveguide sensor chip having fine particles dispersed on a surface facing the optical waveguide and fixed with a second substance that specifically reacts with the substance to be measured;
A light source that makes light incident on the optical waveguide; and a light receiving element that receives light emitted from the optical waveguide;
An optical waveguide sensor is provided.

  According to the present invention, an optical waveguide sensor chip capable of quantitatively measuring a measurement target substance of a measured sample in a shorter time with a smaller amount of measured sample, a method for manufacturing an optical waveguide sensor chip, and An optical waveguide sensor can be provided.

  Further, according to the present invention, it is possible to provide a method for measuring a substance capable of quantifying the measurement target substance of the sample to be measured in a shorter time with a smaller amount of sample to be measured.

  Furthermore, according to the present invention, it is possible to provide a substance measurement kit capable of quantitatively measuring a measurement target substance of a sample to be measured in a shorter time with a smaller amount of sample to be measured.

  Hereinafter, an optical waveguide sensor, an optical waveguide sensor chip manufacturing method, a substance measuring method, a substance measuring kit, and an optical waveguide sensor will be described in detail as embodiments according to the present invention.

(First embodiment)
The optical waveguide sensor chip according to the first embodiment includes an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface, and is dispersed on the optical waveguide, and is specific to the measurement target substance. And a fine particle on which a second substance that reacts automatically is immobilized.

  Here, examples of the measurement target substance include proteins, peptides, genes and the like contained in blood, serum, plasma, biological samples, foods and the like. Specifically, insulin, casein, β-lactoglobulin, ovalbumin, calcitonin, C-peptide, leptin, β-2-microglobulin, retinol binding protein, α-1-microglobulin, α-fetoprotein, carcinoembryonicity Antigen, troponin-I, curcagon-like peptide, insulin-like peptide, tumor growth factor, fibroblast growth factor, platelet growth factor, epidermal growth factor, cortisol, triiodothyronine, hapten hormone such as thyroxine, digoxin, theophylline, etc. Examples include, but are not limited to, infectious substances such as drugs, bacteria, viruses, hepatitis antibodies, IgE, major protein complexes of buckwheat, and soluble proteins including peanut Arah2. In addition, the same thing is used for the measurement target substance in the following second to fifth embodiments.

  For example, a planar optical waveguide can be used as the optical waveguide. The planar optical waveguide can be formed from, for example, a thermosetting resin such as phenol resin or epoxy resin, or alkali-free glass. Specifically, the material used here is a material having a predetermined light transmission property, and is particularly preferably an epoxy resin having a main structure of polystyrene. The first substance that specifically reacts with the measurement target substance of the sample to be measured is fixed to the planar optical waveguide by, for example, the hydrophobic interaction of the substance on the surface hydrophobized with a silane coupling agent or the like. To do. As the first substance, for example, when the substance to be measured of the specimen to be measured is an antigen, an antibody can be used.

  Here, “fine particles are dispersed on the optical waveguide” means that the fine particles are directly or indirectly dispersed on the surface of the optical waveguide. Examples of the form in which “the fine particles are indirectly dispersed on the surface of the optical waveguide” include a form in which the fine particles are dispersed on the surface of the optical waveguide through a blocking layer. The blocking layer contains a water-soluble substance such as polyvinyl alcohol, bovine serum albumin (BSA), polyethylene glycol, phospholipid polymer, gelatin, saccharide (eg sucrose, trehalose). The blocking layer may further include a protein inhibitor.

  As fine particles, for example, resin beads such as polystyrene latex beads (trade name), metal colloids such as gold colloid, or inorganic oxide particles such as titanium oxide particles can be used. As the fine particles, proteins such as albumin, polysaccharides such as agarose, non-metallic particles such as silica particles and carbon particles can also be used. In particular, latex beads and metal colloids are preferable. Of the latex beads, when the light propagating through the optical waveguide described later is a red laser, blue latex beads are preferable.

  The fine particles preferably have a diameter of 50 nm to 10 μm.

  As the second substance, for example, when the substance to be measured of the sample to be measured is an antigen, an antibody can be used.

  Next, a method for manufacturing the optical waveguide sensor chip according to the first embodiment will be described.

  First, a first substance that specifically reacts with a measurement target substance is immobilized on the surface of the optical waveguide. Subsequently, the second substance that specifically reacts with the measurement target substance of the sample to be measured is immobilized on the fine particles by, for example, physical adsorption or chemical bonding via a carboxyl group, an amino group, or the like. Subsequently, a slurry is prepared by dispersing fine particles on which the second substance is immobilized in a physiological saline containing a water-soluble substance. After applying this slurry on the optical waveguide, it is dried to disperse the fine particles on the optical waveguide to produce an optical waveguide sensor chip.

  In such a production method, for example, polyvinyl alcohol, bovine serum albumin (BSA), polyethylene glycol, phospholipid polymer, gelatin, saccharides (for example, sucrose, trehalose) can be used as the water-soluble substance. Also, the drying is preferably lyophilized in order to improve the dispersibility of the fine particles.

  The optical waveguide sensor chip according to the first embodiment will be specifically described with reference to FIG. FIG. 1 is a cross-sectional view showing an optical waveguide sensor chip according to the first embodiment.

At both ends of the main surface of the glass substrate 1, an incident side grating 2a and an emission side grating 2b are provided. These gratings 2a and 2b are formed of, for example, titanium oxide (TiO ₂ ), tin oxide (SnO ₂ ), zinc oxide, lithium niobate, gallium arsenide (GaAs), indium tin oxide (ITO), polyimide, or the like. . For example, the planar optical waveguide 3 made of a thermosetting resin is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The low refractive index resin film 4 is coated on the planar optical waveguide 3. As the low refractive index resin, for example, commercially available Cytop (registered trademark) poly (perfluorobutenyl vinyl ether) manufactured by Asahi Glass Co., Ltd. can be used. For example, a rectangular reaction hole 5 is formed in the low refractive index resin film 4 so as to open a part of the planar optical waveguide 3 positioned between the gratings 2a and 2b. The frame-shaped cell wall 6 is formed on the low refractive index resin film 4 so as to surround the reaction hole 5 exposing the planar optical waveguide 3.

  The first substance 11 that specifically reacts with the measurement target substance of the sample to be measured is immobilized on the surface of the planar optical waveguide 3 exposed from the reaction hole (measurement area) 5 by, for example, a hydrophobic treatment with a silane coupling agent. . The fine particles 13 on which the second substance 12 that specifically reacts with the measurement target substance of the sample to be measured is immobilized are dispersed on the surface of the planar optical waveguide 3 on which the substance 11 is immobilized. The fine particles 13 are dispersed by, for example, applying a slurry containing fine particles and a water-soluble substance to the planar optical waveguide 3 and freeze-drying.

  The optical waveguide sensor according to the first embodiment includes a light source (for example, a red laser diode) 21 for causing light to enter the planar optical waveguide 3 from the incident side grating 2a of the optical waveguide sensor chip described above, and an output side grating 2b. The light receiving element (for example, photodiode) 22 which receives the light radiate | emitted from is provided.

  Next, a method for measuring a substance using the above-described optical waveguide sensor will be described with reference to FIGS.

  First, an optical waveguide sensor chip shown in FIG. This sensor chip includes a substrate 1 having gratings 2a and 2b. The planar optical waveguide 3 is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The low-refractive-index resin film 4 is coated on the planar optical waveguide 3 and is opened so that a part of the planar optical waveguide 3 located between the gratings 2a and 2b is exposed, for example, a rectangular reaction hole 5 is formed. Yes. A first substance (for example, a first antibody) 11 that specifically reacts with a measurement target substance (for example, an antigen) of a sample to be measured is immobilized on the surface of the planar optical waveguide 3 exposed to the reaction hole 5. A plurality of fine particles 13 on which a second substance (for example, a second antibody) 12 that specifically reacts with a measurement target substance of a sample to be measured is immobilized are dispersed on the planar optical waveguide 3.

  Next, the analyte solution to be measured is dropped on the surface of the planar optical waveguide 3 including the dispersed region of the fine particles 13 in the reaction hole 5. At this time, if there is no antigen that specifically reacts with the first antibody 11 on the surface of the planar optical waveguide 3 and the second antibody 12 of the fine particles 13 in the dropped sample solution to be measured, as shown in FIG. In addition, the second antibody 12 of the fine particles 13 is dispersed in the analyte solution 14 without being bonded to the first antibody 11 on the surface of the planar optical waveguide 3. In this state, red laser light from the red laser diode 21 is incident on the planar optical waveguide 3 from the incident side grating 2a and propagates through the planar optical waveguide 3 to emit evanescent light near the surface (exposed surface in the reaction hole 5). Even if it is generated, since the fine particles 13 in the sample solution 14 to be measured in the reaction hole 5 are dispersed, the fine particles 13 hardly exist in the evanescent light region. That is, since the fine particles 13 are hardly involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is hardly attenuated. As a result, when the red laser light emitted from the emission side grating 2b is received by the photodiode 22, the intensity of the laser light hardly changes.

  On the other hand, when an antigen is present in the dropped sample solution 14 to be measured, the antigen 15 binds to the first antibody 11 on the surface of the planar optical waveguide 3 by causing an antigen-antibody reaction as shown in FIG. The second antibody 12 of the fine particle 13 binds to the antigen 15 by causing an antigen-antibody reaction. That is, in order to cause an antigen-antibody reaction via the antigen 15 between the first antibody 11 on the surface of the planar optical waveguide 3 and the second antibody 12 of the particulate 13, the particulate 13 is immobilized on the surface of the planar optical waveguide 3. The

  Immediately after the sample solution to be measured is dropped, red laser light from the red laser diode 21 is incident on the planar optical waveguide 3 from the incident side grating 2a, and propagates through the planar optical waveguide 3 to expose the surface (exposed surface in the reaction hole 5). When evanescent light is generated in the vicinity, the fine particles 13 are fixed to the surface of the planar optical waveguide 3, and therefore the fine particles 13 exist in the evanescent light region. That is, since the fine particles 13 are involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is attenuated. As a result, when the red laser light emitted from the emission side grating 2b is received by the photodiode 22, the intensity of the laser light decreases with time due to the influence of the fixed fine particles 13.

  The rate of decrease in the intensity of the laser beam received by the photodiode 22 is proportional to the amount of fine particles 13 immobilized on the surface of the planar optical waveguide 3, that is, the antigen concentration in the analyte solution 14 to be measured involved in the antigen-antibody reaction. . Therefore, a laser light intensity decrease curve with the passage of time is created in a sample solution whose antigen concentration is known, and the rate of decrease of the laser light intensity over a predetermined time of this curve is obtained. A calibration curve showing the relationship with the decrease rate is prepared in advance. By calculating the laser light intensity decrease rate at a predetermined time from the time measured by the above method and the laser light intensity decrease curve, and comparing the laser light intensity decrease rate with the calibration curve, Can be measured.

  In the concentration measurement, the dispersibility of the fine particles is improved by dispersing the fine particles together with the water-soluble substance on the planar optical waveguide. In addition, when the sample solution to be measured is dropped onto the planar optical waveguide, the water-soluble substance coexisting with the fine particles dissolves to enable the movement of the fine particles, so that the measurement target substance in the sample solution to be measured and the second substance of the fine particles are smooth. Can achieve a good reaction.

  As described above, according to the first embodiment, the concentration of the measurement target substance in the sample to be measured is reduced by a single operation of dropping the sample to be measured into the measurement area with a small amount of the minimum sample to be measured (for example, 10 μL or less). An optical waveguide sensor chip that can be quantified, a manufacturing method thereof, and an optical waveguide sensor can be provided.

  In addition, according to the first embodiment, the concentration of the measurement target substance in the sample to be measured is reduced by a single operation of dropping the sample to be measured into the measurement area with a small amount of the minimum sample to be measured (for example, 10 μL or less). A method for measuring a substance that can be quantified can be provided.

(Second Embodiment)
The optical waveguide sensor chip according to the second embodiment includes an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface, a support plate disposed to face the optical waveguide, Dispersed on the surface of the support plate facing the optical waveguide, and fine particles on which a second substance that specifically reacts with the substance to be measured is immobilized.

  For example, a planar optical waveguide can be used as the optical waveguide. This planar optical waveguide can be formed from a thermosetting resin or non-alkali glass, as described in the first embodiment.

  The first substance that specifically reacts with the measurement target substance of the analyte to be measured on the planar optical waveguide is immobilized by the same method as described in the first embodiment. As the first substance, for example, when the substance to be measured of the specimen to be measured is an antigen, an antibody can be used.

  As described in the first embodiment, for example, resin beads such as latex beads, metal colloids such as gold colloids, or inorganic oxide particles such as titanium oxide particles can be used as the fine particles. As the fine particles, proteins such as albumin, polysaccharides such as agarose, non-metallic particles such as silica particles and carbon particles can also be used. Latex beads and metal colloids are particularly preferable. The fine particles preferably have a diameter of 50 nm to 10 μm.

  As the second substance, for example, when the substance to be measured of the sample to be measured is an antigen, an antibody can be used.

  Next, a method for manufacturing an optical waveguide sensor chip according to the second embodiment will be described.

  First, a first substance that specifically reacts with a measurement target substance is immobilized on the surface of the optical waveguide. Subsequently, the second substance that specifically reacts with the measurement target substance of the sample to be measured is immobilized on the fine particles by, for example, physical adsorption or chemical bonding via a carboxyl group, an amino group, or the like. Subsequently, a slurry is prepared by dispersing fine particles on which the second substance is immobilized in a physiological saline containing a water-soluble substance. The slurry is applied to the surface of the support plate and then dried to disperse the fine particles on the support plate. Thereafter, an optical waveguide sensor chip is manufactured by disposing a support plate on the optical waveguide at a predetermined distance so that the fine particle dispersion surface faces the optical waveguide.

  In such a production method, for example, polyvinyl alcohol, bovine serum albumin (BSA), polyethylene glycol, phospholipid polymer, gelatin, saccharides (for example, sucrose, trehalose) can be used as the water-soluble substance. Also, the drying is preferably lyophilized in order to improve the dispersibility of the fine particles.

  An optical waveguide sensor according to the second embodiment will be specifically described with reference to FIG. FIG. 3 is a cross-sectional view showing an optical waveguide sensor chip according to the second embodiment.

  At both ends of the main surface of the glass substrate 1, an incident side grating 2a and an emission side grating 2b made of, for example, titanium oxide are provided. For example, the planar optical waveguide 3 made of a thermosetting resin is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The low refractive index resin film 4 is coated on the planar optical waveguide 3. For example, a rectangular reaction hole 5 is formed in the low refractive index resin film 4 so as to open a part of the planar optical waveguide 3 positioned between the gratings 2a and 2b. For example, the support plate 7 made of synthetic resin is formed on the low refractive index resin film 4 so as to cover the reaction hole 5. A hole (not shown) for dropping the sample solution to be measured is drilled from the surface of the support plate 7 toward the reaction hole 5.

  The first substance 11 that specifically reacts with the measurement target substance of the sample to be measured is immobilized on the surface of the planar optical waveguide 3 exposed to the reaction hole (measurement area) 5 by, for example, a hydrophobic treatment with a silane coupling agent. Yes. The fine particles 13 on which the second substance 12 that specifically reacts with the measurement target substance of the sample to be measured is immobilized are exposed to the reaction hole 5 and dispersed on the surface (lower surface) of the support plate 7 facing the planar optical waveguide 3. ing. The dispersion of the fine particles 13 is formed, for example, by applying a slurry containing fine particles and a water-soluble substance to the surface of the support plate and freeze-drying.

  An optical waveguide sensor according to the second embodiment includes a light source (for example, a red laser diode) 21 for causing light to enter the planar optical waveguide 3 from the incident side grating 2a of the optical waveguide sensor chip described above, and an output side grating 2b. The light receiving element (for example, photodiode) 22 which receives the light radiate | emitted from is provided.

  Next, a method for measuring a substance using the above-described optical waveguide sensor will be described with reference to FIGS.

  First, an optical waveguide sensor chip shown in FIG. This sensor chip includes a substrate 1 having gratings 2a and 2b. The planar optical waveguide 3 is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The low-refractive-index resin film 4 is coated on the planar optical waveguide 3 and is opened so that a part of the planar optical waveguide 3 located between the gratings 2a and 2b is exposed, for example, a rectangular reaction hole 5 is formed. Yes. A first substance (for example, a first antibody) 11 that specifically reacts with a measurement target substance (for example, an antigen) of a sample to be measured is immobilized on the surface of the planar optical waveguide 3 exposed to the reaction hole 5. The support plate 7 is formed on the low refractive index resin film 4 so as to cover the reaction hole 5. A plurality of fine particles 13 on which a second substance (for example, a second antibody) 12 that specifically reacts with the measurement target substance of the sample to be measured is immobilized are dispersed on the lower surface of the support plate 7 exposed to the reaction hole 5. .

  Next, the sample solution to be measured is dropped into the reaction hole 5 through a hole (not shown) for dropping the sample solution to be measured. At this time, if there is no antigen that specifically reacts with the first antibody 11 on the surface of the planar optical waveguide 3 and the second antibody 12 of the fine particles 13 in the dropped sample solution to be measured, as shown in FIG. In addition, the second antibody 12 of the fine particles 13 is dispersed in the analyte solution 14 without being bonded to the first antibody 11 on the surface of the planar optical waveguide 3. In this state, red laser light from the red laser diode 21 is incident on the planar optical waveguide 3 from the incident side grating 2a and propagates through the planar optical waveguide 3 to emit evanescent light near the surface (exposed surface in the reaction hole 5). Even if it is generated, the fine particles 13 are dispersed in the sample solution 14 to be measured, so that the fine particles 13 hardly exist in the evanescent light region. That is, since the fine particles 13 are hardly involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is hardly attenuated. As a result, when the red laser light emitted from the emission side grating 2b is received by the photodiode 22, the intensity of the laser light hardly changes.

  On the other hand, when an antigen is present in the dropped sample solution 14 to be measured, the antigen 15 binds to the first antibody 11 on the surface of the planar optical waveguide 3 by causing an antigen-antibody reaction as shown in FIG. The second antibody 12 of the fine particle 13 binds to the antigen 15 by causing an antigen-antibody reaction. That is, in order to cause an antigen-antibody reaction via the antigen 15 between the first antibody 11 on the surface of the planar optical waveguide 3 and the second antibody 12 of the particulate 13, the particulate 13 is immobilized on the surface of the planar optical waveguide 3. The

  Immediately after the sample solution to be measured is dropped, red laser light from the red laser diode 21 is incident on the planar optical waveguide 3 from the incident side grating 2a, and propagates through the planar optical waveguide 3 to expose the surface (exposed surface in the reaction hole 5). When evanescent light is generated in the vicinity, the fine particles 13 are fixed to the surface of the planar optical waveguide 3, and therefore the fine particles 13 exist in the evanescent light region. That is, since the fine particles 13 are involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is attenuated. As a result, when the red laser light emitted from the emission side grating 2b is received by the photodiode 22, the intensity of the laser light decreases with time due to the influence of the fixed fine particles 13.

  The rate of decrease in the intensity of the laser beam received by the photodiode 22 is proportional to the amount of fine particles 13 immobilized on the surface of the planar optical waveguide 3, that is, the antigen concentration in the analyte solution 14 to be measured involved in the antigen-antibody reaction. . Therefore, a laser light intensity decrease curve with the passage of time is created in a sample solution whose antigen concentration is known, and the rate of decrease of the laser light intensity over a predetermined time of this curve is obtained. A calibration curve showing the relationship with the decrease rate is prepared in advance. By calculating the laser light intensity decrease rate at a predetermined time from the time measured by the above method and the laser light intensity decrease curve, and comparing the laser light intensity decrease rate with the calibration curve, Can be measured.

  In the concentration measurement, the dispersibility of the fine particles is improved by dispersing the fine particles together with the water-soluble substance on the planar optical waveguide. In addition, when the sample solution to be measured is dropped onto the planar optical waveguide, the water-soluble substance coexisting with the fine particles dissolves to enable the movement of the fine particles, so that the measurement target substance in the sample solution to be measured and the second substance of the fine particles are smooth. Can achieve a good reaction.

  As described above, according to the second embodiment, the concentration of the measurement target substance in the sample to be measured is reduced by a single operation of dropping the sample to be measured into the measurement area with a small amount of the minimum sample to be measured (for example, 10 μL or less). An optical waveguide sensor chip that can be quantified, a manufacturing method thereof, and an optical waveguide sensor can be provided.

  Moreover, according to the second embodiment, the concentration of the measurement target substance in the sample to be measured is reduced by a single operation of dropping the sample to be measured in a measurement area with a small amount of the minimum sample to be measured (for example, 10 μL or less). A method for measuring a substance that can be quantified can be provided.

(Third embodiment)
A method for measuring a substance according to the third embodiment will be described below.

  First, an optical waveguide sensor chip including an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface is prepared. Subsequently, the analyte solution to be measured is dropped on the surface of the optical waveguide to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution to be measured. Subsequently, the surface of the optical waveguide is cleaned. Next, a dispersion liquid of fine particles on which a second substance that specifically reacts with the measurement target substance is immobilized is dropped on the surface of the optical waveguide, so that the measurement target substance in the sample solution is measured between the measurement target substance and the second fine substance substance. React specifically. Thereafter, the concentration of the measurement target substance in the sample solution to be measured is measured by detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the measurement target substance.

  For example, a planar optical waveguide can be used as the optical waveguide. This planar optical waveguide can be formed from a thermosetting resin or non-alkali glass, as described in the first embodiment. The first substance that specifically reacts with the measurement target substance of the analyte to be measured on the planar optical waveguide is immobilized by the same method as described in the first embodiment. As the first substance, for example, when the substance to be measured of the specimen to be measured is an antigen, an antibody can be used.

  For washing, for example, a solution in which a buffer solution and a surfactant are combined, a phosphate buffered saline solution (PBS) containing a surfactant, a Tris-HCl buffered saline solution, a Good buffer buffered saline solution, a phosphate buffer solution. It can be carried out using a cleaning liquid comprising a liquid or the like.

  As described in the first embodiment, for example, resin beads such as latex beads, metal colloids such as gold colloids, or inorganic oxide particles such as titanium oxide particles can be used as the fine particles. As the fine particles, proteins such as albumin, polysaccharides such as agarose, non-metallic particles such as silica particles and carbon particles can also be used. In particular, latex beads and metal colloids are preferable. The fine particles preferably have a diameter of 50 nm to 10 μm.

  The second substance is immobilized on the fine particles by the same method as described in the first embodiment. As the second substance, for example, when the substance to be measured of the sample to be measured is an antigen, an antibody can be used.

  The dispersion of fine particles is stabilized with bovine serum albumin (BSA), casein, polyethylene glycol, etc. in a buffer or good buffer containing, for example, phosphoric acid, trishydroxymethylaminomethane, boric acid, acetic acid, citric acid, carbonic acid, etc. Agents, non-ionic surfactants such as Tween and Triton-X, or phosphate buffered saline (PBS).

  A method for measuring a substance according to the third embodiment will be specifically described with reference to the optical waveguide sensor chip shown in FIG. 5 and (A) to (C) of FIG.

  First, an optical waveguide sensor chip shown in FIGS. 5 and 6A is prepared. This sensor chip has a similar structure except that it does not have the fine particle dispersion layer of FIG. 1 described in the first embodiment. That is, a substrate 1 having gratings 2a and 2b is provided. The planar optical waveguide 3 is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The low-refractive-index resin film 4 is coated on the planar optical waveguide 3 and is opened so that a part of the planar optical waveguide 3 located between the gratings 2a and 2b is exposed, for example, a rectangular reaction hole 5 is formed. Yes. A first substance (for example, a first antibody) 11 that specifically reacts with a measurement target substance (for example, an antigen) of a sample to be measured is immobilized on the surface of the planar optical waveguide 3 exposed to the reaction hole 5. In FIG. 5, in order to measure the change of the evanescent light in the planar optical waveguide 3 located in the reaction hole 5, a laser oscillator (for example, a red laser diode) 21 for making light incident on the incident side grating 2a is installed, and the emission side grating is provided. A photoelectric conversion element (photodiode) 22 that receives light emitted from 2b is installed.

  Next, the sample solution to be measured is dropped into the reaction hole 5. At this time, as shown in FIG. 6B, the antigen 15 in the sample solution to be dropped is bonded to the first antibody 11 on the surface of the planar optical waveguide 3 by causing an antigen-antibody reaction.

  Next, a washing process is performed to wash away the antigen 15 that does not react with the first antibody 11 on the surface of the planar optical waveguide 3. Subsequently, a dispersion liquid of fine particles on which a second substance (for example, a second antibody) that specifically reacts with an antigen as a measurement target substance is immobilized is dropped into the reaction hole 5. At this time, as shown in FIG. 6C, the antigen 15 as the measurement target substance that has undergone an antigen-antibody reaction with the first antibody 11 on the surface of the planar optical waveguide 3 in the dispersion liquid 16 and the second antibody 12 of the fine particles 13 are present. It is bound by antigen-antibody reaction. That is, in order to cause an antigen-antibody reaction via the antigen 15 between the first antibody 11 on the surface of the planar optical waveguide 3 and the second antibody 12 of the particulate 13, the particulate 13 is immobilized on the surface of the planar optical waveguide 3. The

  Immediately after the dispersion of the fine particle is dropped, red laser light is incident from the incident side grating 2a to the planar optical waveguide 3 and propagates through the planar optical waveguide 3 to be near the surface (exposed surface in the reaction hole 5). When evanescent light is generated, the fine particles 13 are fixed to the surface of the planar optical waveguide 3, and therefore the fine particles 13 exist in the evanescent light region. That is, since the fine particles 13 are involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is attenuated. As a result, when the red laser light emitted from the emission side grating 2b is received by the photodiode 22, the intensity of the laser light decreases with time due to the influence of the fixed fine particles 13.

  The rate of decrease in the intensity of the laser beam received by the photodiode 22 is proportional to the amount of fine particles 13 immobilized on the surface of the planar optical waveguide 3, that is, the antigen concentration in the analyte solution 14 to be measured involved in the antigen-antibody reaction. . Therefore, a laser light intensity decrease curve with the passage of time is created in a sample solution whose antigen concentration is known, and the rate of decrease of the laser light intensity over a predetermined time of this curve is obtained. A calibration curve showing the relationship with the decrease rate is prepared in advance. By calculating the laser light intensity decrease rate at a predetermined time from the time measured by the above method and the laser light intensity decrease curve, and comparing the laser light intensity decrease rate with the calibration curve, Can be measured.

  As described above, according to the third embodiment, the minimum required amount of sample to be measured is small (for example, 10 μL or less), and the dropping of the sample solution to be measured into the measurement region, washing, and dropping of the fine particle dispersion into the measurement region are performed three times. Thus, it is possible to provide a method for measuring a measurement object of a sample to be measured, which can quantify the concentration of a substance to be measured in the sample to be measured.

  In particular, the third embodiment is effective when the concentration of the substance to be measured (for example, antigen) in the sample solution to be measured is high because the washing is performed after dropping the sample solution to be measured to the measurement area.

(Fourth embodiment)
A method for measuring a substance according to the fourth embodiment will be described below.

  First, an optical waveguide sensor chip including an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on a surface is prepared. In advance, fine particles on which a second substance that specifically reacts with a measurement target solution and a measurement target substance of the measurement target is mixed in a container such as a microtube, and the second substance and the measurement target are mixed. Specifically react with the substance to be measured in the sample solution. Subsequently, the mixed solution is dropped on the surface of the optical waveguide of the sensor chip to specifically react the first substance on the surface of the optical waveguide and the measurement target substance of the sample solution to be measured that has reacted with the second substance of the fine particles. Next, the measurement target in the sample solution to be measured is detected by detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the measurement target substance, that is, the microparticles immobilized on the surface of the optical waveguide. Measure the concentration of the substance.

  For example, a planar optical waveguide can be used as the optical waveguide. This planar optical waveguide can be formed from a thermosetting resin or non-alkali glass, as described in the first embodiment. The first substance that specifically reacts with the measurement target substance of the analyte to be measured on the planar optical waveguide is immobilized by the same method as described in the first embodiment. As the first substance, for example, when the substance to be measured of the specimen to be measured is an antigen, an antibody can be used.

  As described in the first embodiment, for example, resin beads such as latex beads, metal colloids such as gold colloid, or inorganic oxide particles such as titanium oxide particles can be used as the fine particles. Latex beads and metal colloids are preferred.

  The fine particles preferably have a diameter of 50 nm to 10 μm.

  The second substance is immobilized on the fine particles by the same method as described in the first embodiment. As the second substance, for example, when the substance to be measured of the sample to be measured is an antigen, an antibody can be used.

  For example, the microparticles are prepared by adding a buffer such as phosphoric acid, trishydroxymethylaminomethane, boric acid, acetic acid, citric acid, carbonic acid or the like, a good buffer, a stabilizer such as bovine serum alpmin (BSA), casein, polyethylene glycol, A dispersion of fine particles can be prepared by dispersing in a non-ionic surfactant such as Triton-X or in phosphate buffered saline (PBS).

  In the mixing of microparticles in which the sample solution to be measured and the second substance that specifically reacts with the measurement target substance of the sample to be measured are immobilized, the microparticles are in a solid state (for example, dried solids, frozen A product or a powder). Specifically, a dispersion of fine particles may be prepared in advance, and this dispersion and the sample solution to be measured may be mixed in a container such as a microtube. In addition, when mixing the sample solution to be measured and the fine particles on which the second substance is immobilized, for example, in a container such as a microtube, a dispersion of fine particles containing a water-soluble substance is first put in the container and dried, for example, It is possible to freeze-dry and disperse the fine particles on the inner surface of the container with a water-soluble substance interposed therebetween, and then put the sample solution to be measured in the container and mix it. As the water-soluble substance, for example, polyvinyl alcohol, bovine serum albumin (BSA), polyethylene glycol, phospholipid polymer, gelatin, and saccharides (for example, sucrose and trehalose) can be used.

  A measurement method according to the fourth embodiment will be specifically described with reference to the optical waveguide sensor chip shown in FIG. 5 and FIGS. 7A and 7B.

  First, as shown in FIG. 5 and FIG. 7A, a first substance (for example, a first substance) that specifically reacts with a measurement target substance (for example, an antigen) of a sample to be measured on the surface of the planar optical waveguide 3 exposed in the reaction hole 5. An optical waveguide sensor having 1 antibody 11 immobilized thereon is prepared.

  In advance, for example, in a microtube, a dispersion liquid of fine particles on which a sample substance to be measured and a second substance (for example, a second antibody) that specifically reacts with an antigen that is a measurement target substance is immobilized is mixed, and the sample solution to be measured is obtained. An antigen-antibody reaction is caused between the light source and the second antibody of fine particles.

  Next, the mixed solution is dropped into the reaction hole 5. At this time, as shown in FIG. 7B, the antigen 15 in the sample solution that has already undergone an antigen-antibody reaction with the second antibody in the mixed solution causes an antigen-antibody reaction with the first antibody 11 on the surface of the planar optical waveguide 3. And combine. That is, the antigen 15 to which the second antibody 12 of the fine particle 13 is previously bound is bonded to the first antibody 11 on the surface of the planar optical waveguide 3 to cause an antigen-antibody reaction. As a result, the particulate 13 is fixed to the surface of the planar optical waveguide 3. It becomes.

  Immediately after dropping of the analyte solution to be measured and the dispersion liquid of the fine particles, red laser light is incident from the incident side grating 2a to the planar optical waveguide 3 through the incident side grating 2a, and propagates through the planar optical waveguide 3 to cause the surface (reaction hole 5). When evanescent light is generated in the vicinity of the exposed surface), the fine particles 13 are fixed to the surface of the planar optical waveguide 3, so that the fine particles 13 exist in the evanescent light region. That is, since the fine particles 13 are involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is attenuated. As a result, when the red laser light emitted from the emission side grating 2b is received by the photodiode 22, the intensity of the laser light decreases with time due to the influence of the fixed fine particles 13.

  The rate of decrease in the intensity of the laser beam received by the photodiode 22 is proportional to the amount of fine particles 13 immobilized on the surface of the planar optical waveguide 3, that is, the antigen concentration in the analyte solution 14 to be measured involved in the antigen-antibody reaction. . Therefore, a laser light intensity decrease curve with the passage of time is created in a sample solution whose antigen concentration is known, and the rate of decrease of the laser light intensity over a predetermined time of this curve is obtained. A calibration curve showing the relationship with the decrease rate is prepared in advance. By calculating the laser light intensity decrease rate at a predetermined time from the time measured by the above method and the laser light intensity decrease curve, and comparing the laser light intensity decrease rate with the calibration curve, Can be measured.

  As described above, according to the fourth embodiment, the measurement target sample is measured by a single operation of dropping the sample solution to be measured and the fine particle dispersion liquid into the measurement region with a small amount of the minimum sample to be measured (for example, 10 μL or less). It is possible to provide a method for measuring an object to be measured of a sample to be measured capable of quantifying the concentration of a substance.

  In the above-described fourth embodiment, the sample solution to be measured and the dispersion liquid of the fine particles dropped on the surface of the optical waveguide are simultaneously dropped on the surface of the optical waveguide instead of mixing in advance. Also good.

  In the fourth embodiment described above, the sample solution to be measured and the fine particle dispersion are dropped on the optical waveguide surface after mixing, but the order in which the fine particle dispersion is dropped after dropping the sample solution to be measured, and the fine particle dispersion are dropped. The order of dropping the sample solution to be measured later may be used. Even in the method for measuring substances in such an order, the concentration of the substance to be measured in the sample to be measured can be quantified even if the minimum amount of sample to be measured is small (for example, 10 μL or less) as in the fourth embodiment.

(Fifth embodiment)
A substance measurement kit according to the fifth embodiment will be described below.

  The substance measurement kit is configured by combining an optical waveguide sensor chip and a package containing a dispersion of fine particles on which a second substance that specifically reacts with a measurement target substance of a sample to be measured is immobilized. . The optical waveguide sensor chip includes an optical waveguide in which a first substance that specifically reacts with a measurement target substance of a sample to be measured is immobilized on the surface, and is disposed on the optical waveguide and faces the optical waveguide in a measurement region. And a cap having an introduction hole and a discharge hole communicating with the measurement area.

  The first substance that specifically reacts with the measurement target substance of the analyte to be measured on the planar optical waveguide is immobilized by the same method as described in the first embodiment. As the first substance, for example, when the substance to be measured of the specimen to be measured is an antigen, an antibody can be used.

  As described in the first embodiment, the fine particles contained in the package are, for example, resin beads such as latex beads made of styrene, metal colloids such as gold colloid, or inorganic oxides such as titanium oxide particles. Particles or the like can be used. As the fine particles, proteins such as albumin, polysaccharides such as agarose, non-metallic particles such as silica particles and carbon particles can also be used. In particular, latex beads and metal colloids are preferable. The fine particles preferably have a diameter of 50 nm to 10 μm.

  The second substance is immobilized on the fine particles by the same method as described in the first embodiment. As the second substance, for example, when the substance to be measured of the sample to be measured is an antigen, an antibody can be used.

  The fine particle dispersion is stabilized with bovine serum alpmin (BSA), casein, polyethylene glycol, etc. in a buffer or good buffer containing, for example, phosphoric acid, trishydroxymethylaminomethane, boric acid, acetic acid, citric acid, carbonic acid, etc. Agents, non-ionic surfactants such as Tween and Triton-X, or phosphate buffered saline (PBS).

  The package can be made of, for example, a polyethylene film or a laminated film of polyethylene and polyethylene terephthalate. Moreover, a microtube, a plastic bottle, and a glass bottle can be used for a package.

  The substance measurement kit according to the fifth embodiment will be specifically described with reference to FIGS. 8 (a) and 8 (b). FIG. 8A is a plan view showing an optical waveguide sensor chip, and FIG. 8B is a cross-sectional view of FIG.

  At both ends of the main surface of the glass substrate 31, an incident side grating 32a and an emission side grating 32b made of, for example, titanium oxide are provided. For example, the planar optical waveguide 33 made of a thermosetting resin is formed on the main surface of the substrate 31 including the gratings 32a and 32b. For example, the cap 34 made of a resin such as an acrylic resin is disposed so as to cover the main surface and the side surface of the planar optical waveguide 33. Here, it is also possible to substitute another resin having a predetermined low refractive index for the material of the cap 34. The cap 34 has a rectangular recess 36 for forming, for example, a rectangular measurement area 35 between the surface of the planar optical waveguide 33. The cap 34 has an introduction hole 37 and a discharge hole 38 extending from the surface thereof to the measurement area 35. The first substance 11 that specifically reacts with the measurement target substance of the sample to be measured is immobilized on the surface of the planar optical waveguide 33 exposed to the measurement region 35 by, for example, a hydrophobic treatment with a silane coupling agent. The planar optical waveguide 33, the cap 34, and the like constitute an optical waveguide sensor chip.

  A dispersion liquid of fine particles in which a second substance that specifically reacts with a measurement target substance of a sample to be measured is immobilized is accommodated in, for example, a polyethylene package (not shown), and the above-described optical waveguide sensor chip. The measurement kit is combined with the above.

  Next, a method for measuring a substance using the kit described above will be described with reference to FIGS. In order to measure the change of the evanescent light in the planar optical waveguide exposed in the measurement area, a laser oscillator (for example, a red laser diode) 21 that makes light incident on the incident side grating 32a is installed, and is emitted from the output side grating 32b. A photoelectric conversion element (photodiode) 22 that receives light is installed.

  First, as shown in FIG. 8 and FIG. 9A, a first substance (for example, a substance that specifically reacts with a measurement target substance (for example, an antigen) of the sample to be measured on the surface of the planar optical waveguide 33 exposed in the measurement area 35). An optical waveguide sensor chip having a first antibody 11 immobilized thereon is prepared.

  Next, the analyte solution to be measured is dropped into the measurement area 35 through the introduction hole 37. At this time, as shown in FIG. 9B, the antigen 15 in the sample solution 14 dropped is bonded to the first antibody 11 on the surface of the planar optical waveguide 33 by causing an antigen-antibody reaction.

  Next, the dispersion liquid of fine particles in the package is introduced to the surface of the planar optical waveguide 33 in the measurement area 35 through the introduction hole 37 of the cap 34, and the sample solution to be measured is discharged to the outside through the discharge hole 38. During this time, the unreacted antigen in the sample liquid to be measured is washed away together with the dispersion. At the same time, as shown in FIG. 9C, the second antibody 12 of the fine particles 13 in the dispersion 16 reacts with the first antibody 11 on the surface of the planar optical waveguide 33 and undergoes an antigen-antibody reaction. Combined in reaction. That is, in order to cause an antigen-antibody reaction via the antigen 15 between the first antibody 11 on the surface of the planar optical waveguide 33 and the second antibody 12 of the particulate 13, the particulate 13 is immobilized on the surface of the planar optical waveguide 33. The

  Immediately after the dispersion of fine particles is introduced, red laser light is incident on the planar optical waveguide 33 from the incident side grating 32a and propagated through the planar optical waveguide 33 to be exposed (surface exposed in the measurement area 35). When evanescent light is generated in the vicinity, the fine particles 13 are fixed to the surface of the planar optical waveguide 33 in the dispersion liquid 16, so that the fine particles 13 exist in the evanescent light region. That is, since the fine particles 13 are involved in the absorption and scattering of the evanescent light, the intensity of the evanescent light is attenuated. As a result, when the red laser light emitted from the emission side grating 32b is received by the photodiode 22, the intensity of the laser light decreases with time due to the influence of the fixed fine particles 13.

  The rate of decrease in the intensity of the laser beam received by the photodiode 22 is proportional to the amount of fine particles 13 immobilized on the surface of the planar optical waveguide 33, that is, the antigen concentration in the analyte solution 14 to be measured involved in the antigen-antibody reaction. . Therefore, a laser light intensity decrease curve with the passage of time is created in a sample solution whose antigen concentration is known, and the rate of decrease of the laser light intensity over a predetermined time of this curve is obtained. A calibration curve showing the relationship with the decrease rate is prepared in advance. By calculating the laser light intensity decrease rate at a predetermined time from the time measured by the above method and the laser light intensity decrease curve, and comparing the laser light intensity decrease rate with the calibration curve, Can be measured.

  As described above, according to the substance measurement kit of the fifth embodiment, the optical waveguide sensor chip having a structure capable of dropping the analyte solution to be measured into the optical waveguide and the measurement region and introducing / discharging the fine particle dispersion, and the fine particle Since the package containing the dispersion liquid is combined, the required minimum amount of sample to be measured is a small amount (for example, 10 μL or less), the sample solution to be measured is dropped into the measurement area, and the dispersion of fine particles into the measurement area. The concentration of the substance to be measured in the sample to be measured can be quantified by two operations of introduction and discharge.

  In particular, in the measurement method of the measurement object of the sample to be measured by using the substance measurement kit, since the dispersion liquid of fine particles is introduced into and discharged from the measurement region after dropping the sample solution to be measured, This is also effective when the concentration of the substance to be measured (for example, antigen) in the measurement sample solution is high.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings described above.

Example 1
A titanium oxide film having a thickness of 50 nm is formed by sputtering titanium oxide having a refractive index of 2.2 to 2.4 on an alkali-free glass substrate 1 having a refractive index of 1.52. Then, the glass substrate 1 is formed by lithography and dry etching. The gratings 2a and 2b were formed on the top. Subsequently, an epoxy resin solution was spin-coated on the glass substrate 1 including the gratings 2a and 2b, and then fired to form the planar optical waveguide 3 having a thickness of about 30 μm. The refractive index of the planar optical waveguide 3 after firing was 1.56. Subsequently, a rectangular reaction hole (by a low refractive index resin on the planar optical waveguide 3 and screen-printed with a commercially available Cytop (registered trademark) poly (perfluorobutenyl vinyl ether) manufactured by Asahi Glass Co., Ltd. ( A low refractive index resin 4 having an opening 5 in the measurement area) was formed.

  Next, the surface of the planar optical waveguide 3 exposed from the reaction hole 5 was hydrophobized with a silane coupling agent, and the anti-insulin antibody 11 was immobilized thereon by hydrophobic interaction. Thereafter, a frame-like cell wall 6 was formed on the low refractive index resin 4 so as to surround the reaction hole 5.

  Further, blocking one (manufactured by Nacalai Tesque Co., Ltd.) was added to phosphate buffered saline (PBS) so as to be diluted 2.5 times to prepare a solution. Subsequently, blue latex beads having an average particle diameter of 760 nm on which the anti-insulin antibody was immobilized were dispersed in this solution to prepare a bead dispersion having a bead dispersion concentration of 4% by weight. Blocking One is a blocking agent for suppressing non-specific adsorption, 4 to 8% by weight of tris (hydroxymethyl) aminomethane, 1 to 2% by weight of albumin, 2 to 6% by weight of casein, 10% by weight. % Aqueous polymer compound, 1% by weight or less of an antiseptic and about 3% by weight of a 4M sodium hydroxide solution.

  1 μL of this anti-insulin antibody-immobilized bead dispersion is dropped into the reaction hole 5 and pre-frozen at −80 ° C., and then freeze-dried for about 1 day to place the beads in advance. A waveguide sensor chip was manufactured. At the time of this lyophilization, 3% by weight of trehalose as a disaccharide and 0.1% by weight of Tween as a surfactant were added to the bead dispersion having the above composition. These components were added for the purpose of improving the redispersibility of the bead dispersion.

  10 μL each of insulin solutions having concentrations of 1.6 ng / mL and 6.4 ng / mL, which are analyte solutions, were dropped into the reaction hole of the obtained sensor chip, and antigen-antibody reaction was performed. Immediately after the sample solution to be measured is dropped, red light having a wavelength of 655 nm is incident on the planar optical waveguide 3 through the incident side grating 2a and propagates through the planar optical waveguide 3 to be near the surface (exposed surface in the reaction hole 5). Then, evanescent light was generated, red light emitted from the emission side grating 2b was received by the photodiode 22, and the light intensity was measured. That is, the change in light intensity over time was measured.

  About the 6.4 ng / mL insulin solution, the same operation was performed 3 times (a total of 4 times), and the change of the light intensity with time passage was measured. These results are shown in FIG.

  In FIG. 10, the light intensity immediately after dropping of the sample solution to be measured is assumed to be 100%, and the change in light intensity over time is shown. FIG. 10 shows that the result of the insulin solution having a concentration of 1.6 ng / mL is S1, and the results of the four insulin solutions having a concentration of 6.4 ng / mL are S2-1, S2-2, S2-3, and S2-4. Show. Moreover, the same operation was performed using only the insulin dilution solvent (blank) as the sample solution to be measured, and the result of measuring the change in the laser light intensity over time is shown as B in FIG.

  As is apparent from FIG. 10, it can be seen that the rate of decrease of the laser light intensity at a predetermined time correlates with the insulin concentration in the sample solution to be measured. In addition, it can be seen that, in the four analyte solutions having the same insulin concentration (6.4 ng / mL), the rate of decrease of the laser light intensity at a predetermined time is approximate, and concentration measurement with good reproducibility is possible.

(Example 2)
A titanium oxide film having a thickness of 50 nm is formed by sputtering titanium oxide having a refractive index of 2.2 to 2.4 on an alkali-free glass substrate 1 having a refractive index of 1.52. Then, the glass substrate 1 is formed by lithography and dry etching. The gratings 2a and 2b were formed on the top. Subsequently, an epoxy resin solution was spin-coated on the glass substrate 1 including the gratings 2a and 2b, and then fired to form the planar optical waveguide 3 having a thickness of about 30 μm. The refractive index of the planar optical waveguide 3 after firing was 1.56. Subsequently, a rectangular reaction hole (by a low refractive index resin on the planar optical waveguide 3 and screen-printed with a commercially available Cytop (registered trademark) poly (perfluorobutenyl vinyl ether) manufactured by Asahi Glass Co., Ltd. ( An optical waveguide sensor chip shown in FIG. 5 was manufactured by forming a low refractive index resin 4 having a measurement area 5 opened.

  10 μL each of insulin solutions having concentrations of 1.6 and 6.4 ng / mL, which are analyte solutions, were dropped into the reaction hole of the obtained optical waveguide sensor chip, and an antigen-antibody reaction was performed at 37 ° C. for 10 minutes. Subsequently, excess insulin in the reaction hole was washed with a washing buffer solution composed of Tris-buffered saline (TBS). 20 μL of the same bead dispersion as in Example 1 was dropped into the reaction hole after washing. The change in light intensity was measured using the red LED 21 and the photodiode 22 in the same manner as in Example 1 immediately after the bead-dispersed droplet.

  About the 6.4 ng / mL insulin solution, the same operation was performed 3 times (a total of 4 times), and the change of the light intensity with time passage was measured. The light intensity immediately after dropping of the sample solution to be measured is assumed to be 100%, and the change in light intensity over time is shown in FIG. In FIG. 11, the result of the insulin solution with a concentration of 1.6 ng / mL is S1, and the results of the four insulin solutions with a concentration of 6.4 ng / mL are S2-1, S2-2, S2-3, and S2-4. Show. Moreover, the same operation was performed using only the insulin dilution solvent (blank) as the sample solution to be measured, and the result of measuring the change in the intensity of the laser beam with the lapse of time is shown as B in FIG.

  As is clear from FIG. 11, it can be seen that the rate of decrease in laser light intensity at a predetermined time correlates with the insulin concentration in the sample solution to be measured. In addition, it can be seen that, in the four analyte solutions having the same insulin concentration (6.4 ng / mL), the rate of decrease of the laser light intensity at a predetermined time is approximate, and concentration measurement with good reproducibility is possible.

(Example 3)
10 μL of the same bead dispersion as in Example 1 is dropped into the reaction hole of the optical waveguide sensor chip similar to that in Example 2, and immediately after that, concentrations of the analyte solution to be measured are 1.6 ng / mL and 6.4 ng / mL. 10 μL of each insulin solution was added dropwise and stirred by pipetting. Immediately after stirring, the change in light intensity was measured using the red LED 21 and the photodiode 22 in the same manner as in Example 1.

  About the 6.4 ng / mL insulin solution, the same operation was performed 3 times (a total of 4 times), and the change of the light intensity with time passage was measured. The light intensity immediately after dropping of the sample solution to be measured is assumed to be 100%, and the change in light intensity over time is shown in FIG. FIG. 12 shows that the result of the insulin solution with a concentration of 1.6 ng / mL is S1, and the results of the four insulin solutions with a concentration of 6.4 ng / mL are S2-1, S2-2, S2-3, and S2-4. Show. Moreover, the same operation was performed using only the diluted solvent of insulin (blank) as the sample solution to be measured, and the result of measuring the change in the laser light intensity over time is shown as B in FIG.

  As is clear from FIG. 12, it can be seen that the rate of decrease of the laser light intensity at a predetermined time correlates with the insulin concentration in the sample solution to be measured. In addition, it can be seen that, in the four analyte solutions having the same insulin concentration (6.4 ng / mL), the rate of decrease of the laser light intensity at a predetermined time is approximate, and concentration measurement with good reproducibility is possible.

  In Example 3, whether the bead dispersion liquid and the insulin solution were dropped in the reverse order or simultaneously, the rate of decrease in laser light intensity was similar to the insulin concentration in the sample solution to be measured, as in Example 3. It was confirmed that there was a correlation.

Example 4
In advance, 50 μL of the same bead dispersion as in Example 1 was placed in a microtube and freeze-dried. At the time of freeze-drying, 3% by weight of trehalose as a disaccharide and 0.1% by weight of Tween as a surfactant were added to the bead dispersion as in Example 1. At the same time, 50 μL each of insulin solutions having a concentration of 1.6 ng / mL and 6.4 ng / mL, which are sample solution to be measured, were dropped into a microtube and mixed to perform an antigen-antibody reaction. Next, 20 μL of the mixed solution in the microtube is dropped into the reaction hole of the optical waveguide sensor chip shown in FIG. 5 similar to that of the second embodiment, and the red LED 21 and the photodiode 22 are used just as in the first embodiment immediately after the dropping. The change in light intensity was measured.

  About the 6.4 ng / mL insulin solution, the same operation was performed 3 times (a total of 4 times), and the change of the light intensity with time passage was measured. The light intensity immediately after dropping of the sample solution to be measured is assumed to be 100%, and the change in light intensity over time is shown in FIG. FIG. 13 shows that the result of the insulin solution with a concentration of 1.6 ng / mL is S1, and the results of the four insulin solutions with a concentration of 6.4 ng / mL are S2-1, S2-2, S2-3, and S2-4. Show. Moreover, the same operation was performed using only the diluted solvent of insulin (blank) as the sample solution to be measured, and the result of measuring the change in laser light intensity over time is shown as B in FIG.

  As is apparent from FIG. 13, it can be seen that the rate of decrease of the laser light intensity at a predetermined time correlates with the insulin concentration in the sample solution to be measured. In addition, it can be seen that, in the four analyte solutions having the same insulin concentration (6.4 ng / mL), the rate of decrease of the laser light intensity at a predetermined time is approximate, and concentration measurement with good reproducibility is possible.

Sectional drawing which shows the optical waveguide type sensor which has the optical waveguide type sensor chip concerning 1st Embodiment. Schematic which shows the measurement process of the measuring object substance of the to-be-measured sample in 1st Embodiment. Sectional drawing which shows the optical waveguide type sensor which has the optical waveguide type sensor chip concerning 2nd Embodiment. Schematic which shows the measurement process of the substance in 2nd Embodiment. Sectional drawing which shows the optical waveguide type sensor chip used for the measuring method of the substance which concerns on 3rd Embodiment. Schematic which shows the measurement process of the substance in 3rd Embodiment. Schematic which shows the measurement process of the substance in 4th Embodiment. Sectional drawing which shows the optical waveguide type sensor chip of the substance measurement kit which concerns on 5th Embodiment. Schematic which shows the measurement process of the substance in 5th Embodiment. The characteristic view which shows the change of the laser beam intensity with time progress in the density | concentration measurement of the insulin of Example 1. FIG. The characteristic view which shows the change of the laser beam intensity with progress of time in the density | concentration measurement of the insulin of Example 2. FIG. FIG. 5 is a characteristic diagram showing a change in laser light intensity with time in measuring the concentration of insulin in Example 3. The characteristic view which shows the change of the laser beam intensity with time progress in the density | concentration measurement of the insulin of Example 4. FIG.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1,31 ... Glass substrate, 2a, 2b, 32a, 32b ... Grating, 3, 33 ... Planar optical waveguide, 5 ... Reaction hole, 11 ... 1st substance, 12 ... 2nd substance, 13 ... Fine particle, 15 ... Measurement object Substance, 34 ... cap, 35 ... measurement area, 37 ... introduction hole, 38 ... discharge hole.

Claims (23)

An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface; and fine particles that are dispersed on the optical waveguide and in which a second substance that specifically reacts with the measurement target substance is immobilized ;
An optical waveguide sensor chip comprising: An optical waveguide in which a first substance that specifically reacts with a substance to be measured is immobilized on the surface;
A support plate disposed opposite to the optical waveguide; and fine particles on which a second substance that is dispersed on the surface of the support plate facing the optical waveguide and specifically reacts with the measurement target substance is immobilized;
An optical waveguide sensor chip comprising:   3. The optical waveguide sensor chip according to claim 1, wherein the optical waveguide is a plate-like glass.   The optical waveguide sensor chip according to claim 1, wherein the optical waveguide is an organic resin film having a thickness of 3 to 300 μm.   3. The optical waveguide sensor chip according to claim 1, wherein the fine particles are resin beads.   3. The optical waveguide sensor chip according to claim 1, wherein the fine particles are metal colloids.   The first and second substances that specifically react with the measurement target substance immobilized on the surface of the optical waveguide and the fine particles are antigens, respectively, and the measurement target substance is an antibody. 7. The optical waveguide sensor chip according to claim 6.   2. The optical waveguide sensor chip according to claim 1, wherein the fine particles are dispersed on the surface of the optical waveguide via a blocking layer. Immobilizing a first substance that specifically reacts with the substance to be measured on the surface of the optical waveguide;
Applying a slurry containing fine particles on which a second substance that specifically reacts with the substance to be measured is immobilized on the optical waveguide; and drying after the application to disperse the fine particles on the optical waveguide. ;
A method for manufacturing an optical waveguide sensor chip, comprising: Immobilizing a first substance that specifically reacts with the substance to be measured on the surface of the optical waveguide;
Applying a slurry containing fine particles on which a second substance that specifically reacts with the measurement target substance is immobilized on the surface of the support plate;
Drying after the coating to disperse the fine particles on the support plate; and disposing the support plate on the optical waveguide at a certain distance so that the fine particle dispersion surface faces each other;
A method for manufacturing an optical waveguide sensor chip, comprising:   11. The method for manufacturing an optical waveguide chip according to claim 9, wherein the slurry contains a water-soluble substance. An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface, and fine particles that are dispersed on the optical waveguide and in which a second substance that specifically reacts with the measurement target substance is immobilized An optical waveguide sensor chip comprising:
The analyte solution to be measured is dropped on the surface of the optical waveguide of the sensor chip to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution, and the substance to be measured and the Reacting specifically with the second substance of the fine particles dispersed on the optical waveguide; and detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the measurement target substance. To do;
A method for measuring a substance, comprising: Providing an optical waveguide sensor chip including an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface;
Dropping the analyte solution to be measured on the surface of the optical waveguide of the sensor chip to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution;
Cleaning the surface of the optical waveguide;
A dispersion liquid of fine particles in which a second substance that specifically reacts with the measurement target substance of the sample to be measured is dropped onto the surface of the optical waveguide to drop between the measurement target substance of the measurement target solution and the second substance of the fine particles. And detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
A method for measuring a substance, comprising: Providing an optical waveguide sensor chip including an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface;
A sample solution and a microparticle to which a second substance that specifically reacts with the measurement target substance of the sample to be measured is mixed in advance, and the second substance of the microparticle and the measurement target substance of the sample solution are specified Reacting automatically;
Dropping the mixed liquid onto the surface of the optical waveguide of the sensor chip and causing the first substance on the surface of the optical waveguide to react specifically with the measurement target substance of the analyte solution to be reacted with the second substance of the fine particles; Detecting an optical change caused by fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
A method for measuring a substance, comprising: Providing an optical waveguide sensor chip including an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface;
Dropping the analyte solution to be measured on the surface of the optical waveguide of the sensor chip to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the analyte solution;
A dispersion liquid of fine particles in which a second substance that specifically reacts with the measurement target substance of the specimen to be measured is dropped on the surface of the optical waveguide, and specific between the measurement target substance and the second fine substance substance. And detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
A method for measuring a substance, comprising: Providing an optical waveguide sensor chip including an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface;
Dropping a dispersion liquid of fine particles on which a second substance that specifically reacts with a measurement target substance of a sample to be measured is immobilized on the optical waveguide surface of the sensor chip;
A sample solution to be measured is dropped on the surface of the optical waveguide to which the dispersion liquid has been dropped to cause a specific reaction between the first substance on the surface of the optical waveguide and the substance to be measured in the sample solution to be measured. Reacting specifically between the substance and the second substance of the fine particles in the dispersion; and detecting optical changes caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured To do;
A method for measuring a substance, comprising: An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface, a support plate disposed to face the optical waveguide, and a surface of the support plate that is opposed to the optical waveguide are dispersed on the surface. And providing an optical waveguide sensor chip comprising fine particles on which a second substance that specifically reacts with the measurement target substance is immobilized;
The analyte solution to be measured is injected between the optical waveguide of the sensor chip and the support plate to cause a specific reaction between the first substance on the surface of the optical waveguide and the analyte to be measured in the analyte solution. Reacting specifically between the substance and the second substance of the fine particles dispersed on the support plate; and optical change by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured Detecting
A method for measuring a substance, comprising: An optical waveguide in which a first substance that specifically reacts with a measurement target substance of a sample to be measured is immobilized on the surface, and disposed on the surface of the optical waveguide to form a measurement area between the surface of the optical waveguide An optical waveguide sensor chip having a recess and a cap having an introduction hole and a discharge hole communicating with the measurement area; and a second substance that specifically reacts with the measurement target substance of the sample to be measured is fixed A package containing a dispersion of micronized fine particles;
A substance measurement kit characterized by combining the above. Use of the substance measurement kit according to claim 18,
A sample substance to be measured is dropped onto the surface of the optical waveguide in the measurement region through the introduction hole of the cap of the optical waveguide sensor chip, and the first substance immobilized on the surface of the optical waveguide and the substance to be measured of the sample solution to be measured are specifically determined. Reacting with;
While the dispersion of fine particles in the package is introduced to the surface of the optical waveguide in the measurement area through the introduction hole of the cap, the measurement target of the measurement target solution specifically reacted while the measurement target solution is discharged through the discharge hole Reacting specifically between the substance and the second substance of the fine particles; and detecting an optical change caused by the fine particles immobilized on the surface of the optical waveguide via the first substance and the substance to be measured;
A method for measuring a substance, comprising:   20. The measurement target substance of the sample solution to be measured is an antigen, and the first and second substances that specifically react with the measurement target substance are antibodies. The method for measuring a substance according to item 1.   19. The substance measurement kit according to claim 18, wherein the substance to be measured in the sample solution to be measured is an antigen, and the first and second substances that react specifically with the substance to be measured are antibodies. An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface, and fine particles that are dispersed on the optical waveguide and in which a second substance that specifically reacts with the measurement target substance is immobilized An optical waveguide sensor chip having:
A light source that makes light incident on the optical waveguide; and a light receiving element that receives light emitted from the optical waveguide;
An optical waveguide sensor comprising: The first substance that specifically reacts with the measurement target substance is dispersed on the surface of the optical waveguide fixed on the surface, the support plate disposed opposite to the optical waveguide, and the surface of the support plate facing the optical waveguide. An optical waveguide sensor chip having fine particles on which a second substance that specifically reacts with the substance to be measured is immobilized;
A light source that makes light incident on the optical waveguide; and a light receiving element that receives light emitted from the optical waveguide;
An optical waveguide sensor comprising:

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