JP2012173220A - Optical waveguide type biochemical sensor chip, optical waveguide type biosensor, and substance measuring kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical waveguide type biochemical sensor chip which can improve detection sensitivity of a substance to be measured.SOLUTION: An optical waveguide type biochemical sensor chip includes an optical waveguide in which a first substance that specifically reacts with a substance to be measured is fixed to the surface thereof, and fine particles in which a second substance that specifically reacts with the substance to be measured and enzymes for catalyzing coloring reaction are fixed.

Description

Embodiments described herein relate generally to an optical waveguide biochemical sensor chip, an optical waveguide biosensor, and a substance measurement kit.

In conventional immunoassay using antigen-antibody reaction, a primary antibody corresponding to a substance to be measured (for example, protein) in a sample to be measured is usually immobilized on the surface of a well-shaped substrate. A predetermined amount of a sample solution, a secondary antibody solution, and a coloring reagent are sequentially dropped into each well. Cleaning is performed with a predetermined cleaning solution every time each solution is dropped. Such an immunoassay is performed by a complicated procedure in which a measurer adds and discharges each solution and washing solution while weighing them. Furthermore, since a solution having a scale of 100 μL is used, each reaction usually takes several tens of minutes to several hours.

Therefore, by using a coloring reagent that produces an enzyme reaction product that precipitates when color develops by an enzyme reaction, the change in the physical quantity of light caused by the precipitated enzyme reaction product is measured with an evanescent wave in the optical waveguide, thereby providing a sample solution. There has been proposed a method capable of measuring even if the capacity of the battery is inaccurate.

In these methods, an antibody or the like labeled with an enzyme that catalyzes a color reaction is used, and usually one enzyme (color-catalyzing enzyme) is labeled with one antibody. That is, color development for one color-forming catalytic enzyme is generated by a pair of antigen-antibody reactions. Therefore, the color development amount (that is, the sensitivity for detecting the substance to be measured) is defined by the number of antibodies, and it is difficult to achieve higher sensitivity.

On the other hand, by dispersing the microparticles on the optical waveguide of the optical waveguide biochemical sensor chip, the microparticles absorb and scatter the light, so that the measurement target substance can be removed without including the procedure of washing excess sample solution and secondary antibody. Techniques that can be quantified have been proposed. In this technique, it is considered that the sensitivity for detecting the measurement target substance is maximized when the fine particles are arranged in a close-packed state on the surface of the optical waveguide.

In any method, assuming an inspection item that requires detection with higher sensitivity, development of a technique for improving the detection sensitivity of the measurement target substance has been desired.

Japanese Patent No.4231051 JP 2009-133842 A

The problem to be solved by the present invention is to provide an optical waveguide biochemical sensor chip, an optical waveguide biosensor, and a substance measurement kit capable of improving the detection sensitivity of a substance to be measured.

  The optical waveguide biochemical sensor chip of the present embodiment includes an optical waveguide having a first substance that specifically reacts with a measurement target substance immobilized on the surface, a second substance that specifically reacts with the measurement target substance, and Fine particles on which an enzyme that catalyzes a color development reaction is immobilized.

Sectional drawing of the optical waveguide type biochemical sensor chip which concerns on 1st Embodiment Schematic diagram showing the form of fine particles according to the same embodiment Schematic diagram showing another form of fine particles according to the embodiment Schematic diagram of an optical waveguide biosensor according to the same embodiment The figure which shows the process of measuring the measuring object substance in the to-be-measured sample which concerns on the same embodiment The figure which shows the example of a time-dependent change of the detection signal strength which concerns on the same embodiment Sectional drawing of the substance measurement kit which concerns on 2nd Embodiment Sectional drawing of the substance measurement kit which concerns on 3rd Embodiment The figure explaining the procedure of the measuring method which concerns on 4th Embodiment

  Embodiments of the present invention will be described below with reference to the drawings.

(First embodiment)
FIG. 1 is a sectional view of an optical waveguide biochemical sensor chip according to the first embodiment.

The optical waveguide biochemical sensor chip according to the first embodiment specifically reacts with the optical waveguide 3 on which the first substance 11 that specifically reacts with the measurement target substance is immobilized, and the measurement target substance. And a fine particle 13 on which an enzyme 14 that catalyzes a color development reaction 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 hormones 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.

As the optical waveguide 3, for example, a planar optical waveguide can be used. The optical waveguide 3 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 11 that specifically reacts with the measurement target substance in the sample to be measured is immobilized on the optical waveguide 3 by, for example, hydrophobic interaction or chemical bonding with the surface of the optical waveguide 3.

As the first substance 11, for example, when the measurement target substance of the sample to be measured is an antigen, an antibody (primary antibody) can be used.

The fine particles 13 are dispersed on the optical waveguide 3. Here, “fine particles are dispersed on the optical waveguide” means that the fine particles 13 are directly or indirectly dispersed above the optical waveguide 3 (surface opposite to the surface in contact with the substrate 1). The form in which “fine particles are indirectly dispersed above the optical waveguide” includes, for example, a form in which the fine particles 13 are dispersed on the surface of the optical waveguide 3 via a blocking layer. The blocking layer contains a water-soluble substance such as polyvinyl alcohol, bovine serum albumin (BSA), polyethylene glycol, phospholipid polymer, gelatin, casein, and sugars (eg, sucrose and trehalose). The blocking layer may further contain a protease inhibitor. Alternatively, FIG. 1 shows an example in which the fine particles 13 are arranged close to the optical waveguide. As another example, there is a form in which the fine particles 13 are arranged above the optical waveguide 3 with a space therebetween. . For example, a support plate (not shown) may be disposed facing the optical waveguide 3, and the fine particles 13 may be dispersed on the surface of the support plate facing the optical waveguide 3.

  The fine particles 13 are a colloid of any one of a metal, a metal oxide, a composite of a metal and a metal oxide, or a spherical resin. For example, as the fine particles 13, 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 13, 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 13 preferably have a diameter of 50 nm to 10 μm.

FIG. 2 is a schematic diagram showing the form of the fine particles 13. The second substance 12 and the enzyme 14 are immobilized on the surface of the fine particle 13. As the second substance 12, for example, when the measurement target substance of the sample to be measured is an antigen, an antibody (secondary antibody) can be used. The enzyme 14 is a color development catalytic enzyme that catalyzes a color development reaction. For example, peroxidase is used as the enzyme 14. In the present embodiment, a plurality of enzymes 14 can be immobilized on one fine particle 13.

FIG. 3 is a schematic diagram showing another form of the fine particles 13. As shown in FIG. 3, the second substance labeled with the enzyme 14 may be immobilized on the surface of the fine particle 13. The second substance enzyme-labeled with a chromogenic enzyme is, for example, a peroxidase-labeled secondary antibody.

Next, the optical waveguide biochemical sensor chip according to the first embodiment will be specifically described with reference to FIG.

At both ends of the main surface of the substrate 1, an incident side grating 2a and an emission side grating 2b are provided. The substrate 1 is, for example, alkali-free glass. The gratings 2a and 2b are formed of a material having a higher refractive index than the substrate. For example, the gratings 2a and 2b are made of titanium oxide (TiO2), tin oxide (SnO2), 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 sensing area 5 is formed in the low-refractive index resin film 4 so as to open a part of the planar optical waveguide 3 located 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 sensing area 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 sensing area 5 by, for example, a hydrophobic treatment using a silane coupling agent. The second substance 12 that specifically reacts with the measurement target substance of the sample to be measured is immobilized on the fine particles 13 by, for example, physical adsorption or chemical bonding via a carboxyl group, an amino group, or the like. Furthermore, an enzyme 14 that catalyzes a color development reaction is immobilized on the fine particles 13. The fine particles 13 on which the second substance 12 and the enzyme 14 are immobilized are dispersed on the surface of the planar optical waveguide 3 on which the substance 11 is immobilized. The dispersion of the fine particles 13 is formed, for example, by applying a slurry containing the fine particles 13 and a water-soluble substance to the planar optical waveguide 3 and freeze-drying. Alternatively, the fine particles 13 may be dissolved in a liquid and applied or dropped on the optical waveguide 3, or may be dispersed as a solid on the optical waveguide 3.

FIG. 4 is a schematic diagram of the optical waveguide biosensor according to the first embodiment. The optical waveguide biosensor according to the first embodiment includes a light source (for example, a red laser diode) 21 for causing light to enter the optical waveguide 3 from the incident-side grating 2a of the optical waveguide biochemical sensor chip described above, and an emission. A light receiving element (for example, a photodiode) 22 that receives light emitted from the side grating 2b is further provided. Light is irradiated from the light source 21 to the optical waveguide type biochemical sensor chip, and the light is diffracted by the incident side grating 2a. The light incident from the incident side grating 2a propagates through the optical waveguide 3, and is diffracted and emitted by the output side grating 2b. The emitted light is received by the light receiving element 22 and the intensity of the light is measured. The concentration of the object to be measured is measured by comparing the intensity of the incident light and the emitted light and measuring the light absorption rate.

Next, a measurement method for measuring a substance to be measured using the above-described optical waveguide biosensor will be described with reference to (a) to (c) of FIG.

First, an optical waveguide biosensor shown in FIG. 4 is prepared. Next, as shown in FIG. 5 (a), the analyte solution, the second substance (secondary antibody) 12, and the enzyme 14 are immobilized on the surface of the optical waveguide 3 including the dispersed region of the fine particles 13 in the sensing area 5. A mixed solution with the dispersion of the fine particles 13 is introduced. As an introduction method, for example, dripping or inflow can be considered. When a measurement target substance (antigen) that specifically reacts with the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the microparticles 13 exists in the introduced analyte solution, As shown in FIG. 5A, the antigen 15 is bound to the first substance (primary antibody) 11 on the surface of the optical waveguide 3 by causing an antigen-antibody reaction, and the second substance (secondary antibody) 12 of the fine particles 13 is the antigen. It produces an antigen-antibody reaction and binds. That is, since the antigen-antibody reaction is caused between the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the microparticle 13 via the antigen, the microparticle 13 is on the surface of the optical waveguide 3. Is fixed against.

Further, as shown in FIG. 5B, after the excess specimen liquid and the fine particles 13 are washed and removed, a coloring reagent is introduced into the surface of the optical waveguide 3 including the dispersed region of the fine particles 13 in the sensing area 5. As an introduction method, for example, dripping or inflow can be considered. When a measurement target substance (antigen) that specifically reacts with the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the fine particles 13 is present in the sample solution to be measured, color development By introducing the reagent, the enzyme 14 catalyzes the color development, and the dye develops color. By the way, as described above, one substance to be measured (antigen) binds to one first substance (primary antibody) 11 by causing an antigen-antibody reaction, and further, one second substance (secondary antibody) of the fine particles 13. ) 12 binds to one substance to be measured (antigen) by causing an antigen-antibody reaction. In the present embodiment, since a plurality of enzymes are immobilized on the fine particles 13, a plurality of enzymes catalyze a coloring reaction for a pair of antigen-antibody reactions, and a coloring reaction corresponding to the plurality of enzymes occurs.

In this state, red laser light is incident on the planar optical waveguide 3 from the incident side grating 2a from the red laser diode 21, and propagates through the optical waveguide 3 to generate evanescent light near the surface (exposed surface in the sensing area 5). Let Then, since the fine particles 13 are fixed to the surface of the optical waveguide 3, 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. Furthermore, since the dye is colored by the enzyme and the coloring reagent, the evanescent light is absorbed by the dye. 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 emitted laser light decreases with time due to the influence of the fixed fine particles 13 and the coloring dye. .

The rate of decrease in the intensity of the laser beam received by the photodiode 22 depends on the amount of the fine particles 13 immobilized on the surface of the optical waveguide 3 and the concentration of the coloring dye. That is, it is proportional to the antigen concentration in the analyte solution 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.

On the other hand, when there is no antigen specifically reacting with the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the microparticles 13 in the introduced analyte solution, the microparticles 13 The second substance (secondary antibody) 12 is dispersed in the analyte solution without binding to the first substance (primary antibody) 11 on the surface of the optical waveguide 3. Furthermore, even if a coloring reagent is introduced into the surface of the optical waveguide 3 including the dispersed region of the fine particles 13 in the sensing area 5 after washing away and removing the excess specimen liquid and the fine particles 13, the light is introduced into the introduced analyte solution. When there is no antigen that specifically reacts with the first substance (primary antibody) 11 on the surface of the waveguide 3 and the second substance (secondary antibody) 12 of the fine particles 13, no antigen-antibody reaction occurs, and thus no enzyme exists. No color reaction occurs.

In this state, red laser light is incident on the planar optical waveguide 3 from the incident side grating 2a from the red laser diode 21, and propagates through the optical waveguide 3 to generate evanescent light near the surface (exposed surface in the sensing area 5). Even if it makes it, the microparticles | fine-particles 13 in the to-be-measured sample solution in the sensing area 5 will hardly exist in an evanescent light area | 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. Further, since the red laser light is not absorbed by the coloring dye, 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. As a result, when the decrease rate of the received laser beam intensity is collated with the calibration curve, it can be seen that the measurement target substance (antigen) does not exist in the sample solution to be measured.

In the present embodiment, the sample solution, the second substance (secondary antibody) 12 and the dispersion liquid of the fine particles 13 on which the enzyme 14 is immobilized are mixed and then introduced into the sensing area 5. At this time, for example, after introducing the fine particle dispersion, the fine particle dispersion and the sample solution may be separately introduced into the sensing area 5 such as introducing and mixing the sample solution.

  FIG. 6 shows an example of a change over time in the detection signal intensity ratio according to the present embodiment. In FIG. 6, as the signal intensity ratio [%] decreases, the incident laser light is absorbed and attenuated, and the sensitivity for detecting the measurement target substance is high. The solid line in FIG. 6 indicates the method of the present invention, and the long two-dot chain line indicates the comparison method. Here, the comparative method is a case of only an antigen-antibody reaction using fine particles not labeled with an enzyme. In the experiment, horseradish peroxidase was used as an enzyme.

In the method of the present invention, when the bead solution and the sample solution are first mixed and introduced into the detection surface, the detection signal intensity decreases according to the amount of the beads bound to the surface of the optical waveguide due to the antigen-antibody reaction. % Signal reduction rate was obtained. Thereafter, after the excess beads and the sample mixed solution are washed, when a coloring reagent solution is dropped, a coloring reaction by the enzyme bound to the fine particles captured on the chip surface by the antigen-antibody reaction starts. A large signal decrease was confirmed by this color reaction, and a signal decrease rate of about 72% was obtained 1500 seconds after the start of measurement.

On the other hand, it can be seen that the signal decrease rate after 1500 seconds is about 47% in the comparative method using only antigen-antibody reaction using fine particles not labeled with an enzyme.

That is, from the result example of FIG. 6, the present system (solid line) is combined with the color amplification of the enzyme as compared with the comparison system (long two-dot chain line) only of the antigen-antibody reaction using fine particles not labeled with the enzyme. As a result, the measurement sensitivity can be improved by about 25%.

As described above, according to the first embodiment, in addition to absorption and scattering of light by the fine particles, absorption due to coloration catalyzed by the enzyme occurs, so that the conventional measurement method using only the fine particles or the measurement method using only the color development of the enzyme. Also, the sensitivity for detecting the target substance can be improved. Furthermore, by introducing fine particles in which a second substance (secondary antibody) and a plurality of enzymes are immobilized before the dye reaction, a plurality of chromogenic catalytic enzymes can act on a pair of antigen-antibody reactions. As a result, the amount of color produced by a pair of antigen-antibody reactions increases, so that even a very low concentration of a substance to be measured is likely to develop color and can be quantified with high sensitivity.

(Second Embodiment)
The substance measurement kit according to the second embodiment will be described below. FIG. 7 shows a cross-sectional view of the substance measuring kit according to the present embodiment.

  As shown in FIG. 7, the substance measurement kit of the present embodiment includes the optical waveguide biochemical sensor chip and the cap 31 of the first embodiment. Therefore, the substrate 1, gratings 2a and 2b, optical waveguide 3, low refractive index resin film 4, and sensing area 5 in FIG. 7 are the same as those of the optical waveguide biochemical sensor chip of the first embodiment.

The cap 31 is disposed so as to cover the main surface and the side surface of the optical waveguide 3 in the optical waveguide biochemical sensor chip, and has a recess for forming a sensing area 5 between the surface of the optical waveguide 3.

Similarly to FIG. 2 or FIG. 3 in the first embodiment, the fine particles 13 are configured such that the second substance (secondary antibody) 12 and the enzyme 14 are immobilized. However, in the present embodiment, the fine particles 13 are arranged above the optical waveguide 3 with a space therebetween. That is, the fine particles 13 are attached or contained on the surface of the cap 31 facing the optical waveguide 3. The fine particles 13 may be attached to the surface of the cap 31 in a dry solid state, or a package (not shown) may contain a dispersion containing the fine particles 13.

The dispersion containing the fine particles 13 may be, for example, a buffer solution containing phosphoric acid, trishydroxymethylaminomethane, boric acid, acetic acid, citric acid, carbonic acid, or a good buffer, bovine serum alpmin (BSA), casein, polyethylene glycol, etc. A stabilizer, a nonionic surfactant such as Tween or Triton-X, or an amphoteric surfactant such as CHAPS, or phosphate buffered saline (PBS) is included.

The package can be made of, for example, a polyethylene film or a laminated film of polyethylene and polyethylene terephthalate. The package is a micro tube, plastic bottle,
Glass bottles can be used.

The cap 31 has a rectangular recess for forming, for example, a rectangular sensing area 5 between the surface of the optical waveguide 3. The cap 31 has an introduction hole 30 communicating with the sensing area 5. From this introduction hole, for example, a sample solution or a coloring reagent is introduced into the sensing area 5 using a pump or the like. The cap 31 may have a discharge hole for discharging various solutions separately from the introduction hole 30. The cap 31 is made of a resin such as an acrylic resin. As the material of the cap 31, other resins having a predetermined low refractive index can be substituted.

Next, a measurement method using the substance measurement kit according to the present embodiment will be described.

First, a substance measurement kit shown in FIG. 7 is prepared. Next, the analyte solution to be measured is introduced into the sensing area 5 through the introduction hole 30. As an introduction method, for example, dripping or inflow can be considered. At this time, the substance to be measured (antigen) in the introduced sample solution to be measured is the optical waveguide 3.
An antigen-antibody reaction is caused to bind to the surface first substance (primary antibody) 11.

  Next, the analyte solution to be measured is mixed with the analyte solution to be measured by sucking and extruding the analyte solution to be measured through the introduction hole 30 with a pump, for example. Alternatively, the dispersion liquid containing the fine particles 3 held in the package and the sample solution to be measured are mixed. Thereby, the fine particles 13 are introduced into the sensing area 5, and the second substance (secondary antibody) 12 of the fine particles 13 reacts with the first substance (primary antibody) 11 on the surface of the optical waveguide 3 by antigen-antibody reaction (antigen). And is bound by antigen-antibody reaction. That is, in order to cause an antigen-antibody reaction between the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the fine particle 13 via the measurement target substance (antigen), the fine particle 13 Is immobilized on the surface of the optical waveguide 3.

  Thereafter, the coloring reagent is introduced into the sensing area 5 through the introduction hole 30 by, for example, a pump. The coloring reaction is catalyzed by the enzyme 14 immobilized on the fine particles 13, and the dye develops color.

  Before introducing the coloring reagent, an excessive mixture of the microparticles 3 and the sample solution to be measured may be sucked and discharged by a pump or the like. Or you may discharge outside from a discharge hole.

  In this state, similarly to the first embodiment, laser light is incident on the optical waveguide 3 from the light source 21 via the incident-side grating 2a. The laser light propagates through the optical waveguide 3 and is emitted from the emission side grating 2 b to the light receiving element 22. The concentration of the substance to be measured (antigen) in the sample solution to be measured can be measured from the rate of decrease in the intensity of the emitted laser light.

As described above, according to the substance measurement kit according to the second embodiment, the same effects as those of the first embodiment can be obtained. That is, in addition to absorption and scattering of light by the fine particles, absorption due to coloring catalyzed by the enzyme occurs, so that the sensitivity of detecting the target substance can be improved. Furthermore, since a plurality of color-catalyzing catalytic enzymes are allowed to act on a pair of antigen-antibody reactions, even a very low concentration of a substance to be measured is likely to develop color and can be quantified with high sensitivity.

(Third embodiment)
A substance measurement kit according to the third embodiment will be described below. FIG. 8 shows a cross-sectional view of the substance measuring kit according to the present embodiment.

  As shown in FIG. 8, the substance measurement kit of the present embodiment has a configuration in which the optical waveguide biochemical sensor chip of the first embodiment is installed on the cap 44 in the reverse manner.

Similarly to FIG. 2 or FIG. 3 in the first embodiment, the fine particles 13 are configured such that the second substance (secondary antibody) 12 and the enzyme 14 are immobilized. However, in the present embodiment, the fine particles 13 are not disposed in the sensing area 5 but are accommodated in the gap 48 provided in the cap 44. The fine particles 13 may be attached to the inner surface of the void 48 in a dry solid state, or a dispersion containing the fine particles 13 may be accommodated in the void 48.

The dispersion containing the fine particles 13 may be, for example, a buffer solution containing phosphoric acid, trishydroxymethylaminomethane, boric acid, acetic acid, citric acid, carbonic acid, or a good buffer, bovine serum alpmin (BSA), casein, polyethylene glycol, etc. A stabilizer, a nonionic surfactant such as Tween or Triton-X, or an amphoteric surfactant such as CHAPS, or phosphate buffered saline (PBS) is included.

The cap 44 has a gap 43 for forming, for example, a rectangular sensing area 5 between the surface of the optical waveguide 3. The cap 44 has an introduction hole 45 communicating with the sensing area 5. From this introduction hole, for example, a sample solution to be measured, a coloring reagent and the like are introduced into the sensing area 5 using a pump 40 or the like. The cap 44 may have a discharge hole 41 for discharging various solutions. The discharge hole 41 includes a check valve 42 and controls the discharge of the solution.

The cap 44 is made of a resin such as an acrylic resin. As the material of the cap 44, other resins having a predetermined low refractive index can be substituted.

The cap 44 has a gap 46 for introducing the sample solution to be measured.

Next, a measurement method using the substance measurement kit according to the present embodiment will be described.

  First, a substance measurement kit shown in FIG. 8 is prepared. Next, the analyte solution to be measured is introduced through the gap 46. The analyte solution to be measured is introduced from the gap 46 into the gap 48 by sucking air using the pump 40. In the gap 48, the sample solution to be measured and the fine particles 13 are mixed. At this time, mixing may be promoted by sucking and pushing out air using the pump 40.

  Next, air is pushed out from the pump 40 to introduce the mixed solution of the sample solution to be measured and the fine particles 13 into the gap 43. The substance to be measured (antigen) in the introduced mixed solution is bound to the first substance (primary antibody) 11 on the surface of the optical waveguide 3 by causing an antigen-antibody reaction. Further, since the fine particles 13 are introduced into the sensing area 5, the second substance (secondary antibody) 12 of the fine particles 13 undergoes an antigen-antibody reaction with the first substance (primary antibody) 11 on the surface of the optical waveguide 3 (antigen). ) And antigen-antibody reaction and binding. That is, in order to cause an antigen-antibody reaction between the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the fine particle 13 via the measurement target substance (antigen), the fine particle 13 Is immobilized on the surface of the optical waveguide 3.

  After that, by pushing out air with a pump, the excess mixed liquid of the fine particles 3 and the sample solution to be measured is discharged from the discharge hole 41 to the outside. Thereby, the surplus measurement target substance (antigen) that has not reacted with the antibody is washed away.

  Thereafter, similarly, the pump is sucked and pushed out to introduce the coloring reagent into the gap 43 through the gap 46, thereby providing the coloring reagent in the sensing area 5. Alternatively, the optical waveguide type biochemical sensor chip may be removed from the cap 44 and the coloring reagent may be dropped onto the sensing area 5. By providing the coloring reagent in the sensing area 5, the coloring reaction is catalyzed by the enzyme 14 immobilized on the fine particles 13, and the dye develops color.

  In this state, similarly to the first embodiment and the second embodiment, laser light is incident on the optical waveguide 3 from the light source 21 via the incident-side grating 2a. The laser light propagates through the optical waveguide 3 and is emitted from the emission side grating 2 b to the light receiving element 22. The concentration of the substance to be measured (antigen) in the sample solution to be measured can be measured from the rate of decrease in the intensity of the emitted laser light.

As described above, according to the substance measurement kit according to the third embodiment, the same effects as those of the first embodiment can be obtained. That is, in addition to absorption and scattering of light by the fine particles, absorption due to coloring catalyzed by the enzyme occurs, so that the sensitivity of detecting the target substance can be improved. Furthermore, since a plurality of color-catalyzing catalytic enzymes are allowed to act on a pair of antigen-antibody reactions, even a very low concentration of a substance to be measured is likely to develop color and can be quantified with high sensitivity.

(Fourth embodiment)
A method for measuring a substance to be measured according to the fourth embodiment will be described below. FIG. 9 shows a diagram for explaining the measurement method according to the present embodiment.

  As shown in FIG. 9, the optical waveguide type biochemical sensor chip used in this embodiment has the same configuration as that of the first embodiment. Similarly to FIG. 2 or FIG. 3 in the first embodiment, the fine particles 13 are configured such that the second substance (secondary antibody) 12 and the enzyme 14 are immobilized. However, in the present embodiment, the fine particles 13 are not disposed in the sensing area 5 but are accommodated in another container or package. The fine particles 13 may be in a dry solid state or a dispersion containing the fine particles 13. Here, the fine particles 13 contained in another container or package are referred to as fine particle reagents.

First, an optical waveguide type biochemical sensor chip and a fine particle reagent shown in FIG. 9 are prepared. Next, the analyte solution to be measured is dropped on the sensing area 5 of the optical waveguide type biochemical sensor chip. Subsequently, the particulate reagent is dropped onto the sensing area 5 of the optical waveguide type biochemical sensor chip. As a result, an antigen-antibody reaction occurs between the first substance (primary antibody) 11 on the surface of the optical waveguide 3 and the second substance (secondary antibody) 12 of the fine particles 13 via the measurement target substance (antigen). 13 is fixed to the surface of the optical waveguide 3.

Thereafter, as in the first embodiment, after the excess specimen liquid and the fine particles 13 are washed away, a coloring reagent is dropped on the sensing area 5, and the coloring reaction is catalyzed by the enzyme 14 immobilized on the fine particles 13. The pigment develops color. Laser light is incident from the light source 21 and the concentration of the measurement target substance (antigen) in the sample solution to be measured is measured from the rate of decrease in the intensity of the laser light emitted to the light receiving element 22.

The order in which the sample solution to be measured and the particulate reagent are dropped onto the sensing area 5 may be opposite to the order described above. That is, the particulate reagent may be dropped first on the sensing area 5 and then the sample solution to be measured may be dropped on the sensing area 5.

  Furthermore, instead of dropping the sample solution to be measured and the fine particle reagent separately on the sensing area 5, a mixed solution is prepared by mixing the sample solution to be measured and the fine particle reagent, and this mixed solution is dropped on the sensing area 5. Also good.

As described above, according to the measurement method according to the fourth embodiment, the same effect as in the first embodiment can be obtained. That is, in addition to absorption and scattering of light by the fine particles, absorption due to coloring catalyzed by the enzyme occurs, so that the sensitivity of detecting the target substance can be improved. Furthermore, since a plurality of color-catalyzing catalytic enzymes are allowed to act on a pair of antigen-antibody reactions, even a very low concentration of a substance to be measured is likely to develop color and can be quantified with high sensitivity.

Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are also included in the invention described in the claims and the equivalents thereof.

DESCRIPTION OF SYMBOLS 1 ... Substrate 2a, 2b ... Grating 3 ... Optical waveguide 4 ... Low refractive index resin film 5 ... Sensing area 6 ... Cell wall 10 ... Enzyme label (secondary) antibody 11 ... 1st substance (primary antibody)
12 ... Second substance (secondary antibody)
13 ... Fine particles 14 ... Enzyme 21 ... Light source 22 ... Light receiving element

Claims (9)

An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface;
An optical waveguide type biochemical sensor chip comprising: a second substance that specifically reacts with the measurement target substance; and fine particles on which an enzyme that catalyzes a color development reaction is immobilized. The optical waveguide biochemical sensor chip according to claim 1, wherein a plurality of the enzymes are immobilized on one fine particle. The optical waveguide biochemical sensor chip according to claim 1, wherein the enzyme is labeled on the second substance. 4. The optical waveguide biochemical sensor chip according to claim 1, wherein the fine particles are dispersed on the optical waveguide. Further comprising a support plate disposed facing the optical waveguide;
4. The optical waveguide biochemical sensor chip according to claim 1, wherein the fine particles are dispersed on a surface of the support plate facing the optical waveguide. 5. An optical waveguide biochemical sensor chip according to any one of claims 1 to 5,
A light source for making light incident on the optical waveguide;
An optical waveguide biosensor comprising: a light receiving element that receives light emitted from the optical waveguide. An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface;
A cap that is disposed so as to cover a main surface and a side surface of the optical waveguide, and that has a recess for forming a sensing area with the surface of the optical waveguide;
A substance measurement kit comprising: a second substance that is dispersed inside the recess of the cap and that specifically reacts with the measurement target substance; and fine particles on which an enzyme that catalyzes a color development reaction is immobilized. An optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized on the surface;
A second substance that specifically reacts with the measurement target substance, and fine particles on which an enzyme that catalyzes a color development reaction is immobilized
A first gap for forming a rectangular sensing area between the surface of the optical waveguide, a second gap for introducing a sample solution containing the substance to be measured, and the fine particles are accommodated therein. A substance measurement kit comprising: a cap having a third gap. An optical waveguide type biochemical sensor chip having an optical waveguide in which a first substance that specifically reacts with a measurement target substance is immobilized;
A second substance that specifically reacts with the measurement target substance, and fine particles on which an enzyme that catalyzes a color development reaction is immobilized,
A substance measurement kit for a substance to be measured, wherein the optical waveguide biochemical sensor chip and the fine particles are individually accommodated and combined.

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