JP2015025820A - Optical waveguide type measurement system and measurement method for glycosylated hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily measure HbA1c with high accuracy.SOLUTION: An optical waveguide type measurement system has: a first optical waveguide having a first sensing area immobilized with a first substance capable of being specifically bonded to glycosylated hemoglobin; a plurality of superparamagnetic first magnetic fine particles immobilized with a second substance capable of being specifically bonded to the glycosylated hemoglobin in a part different from a part in which the first substance can be specifically bonded to the glycosylated hemoglobin; a first magnetic field application part provided above the first optical waveguide, and being able to move at least one of the plurality of first magnetic fine particles from the first sensing area by magnetic force; a first light source making light incident on the first optical waveguide, and being able to generate near-field light in the first sensing area; and a first light receiving element capable of receiving light emitted from the first optical waveguide.

Description

  Embodiments described herein relate generally to an optical waveguide measurement system and a method for measuring glycated hemoglobin.

  HbA1c (hemoglobin A1C) is produced by irreversibly binding to hemoglobin after sugar in the blood enters the erythrocytes, and reflects the average blood sugar level in the blood for the past 1-2 months To do. For this reason, HbA1c is widely used as an indicator for diabetes diagnosis, such as a screening test for diabetes and grasping the blood glucose control state of a diabetic patient.

  As a method for measuring HbA1c, there are a latex agglutination method and a method using a protease. Although the latex agglomeration method is simple in operation, it measures bulk turbidity, so that the apparatus becomes large and the sensitivity may be inferior to other methods. In the method using protease, only the glycated part of HbA1c is cut out with protease and measured. For this reason, complicated preprocessing is involved. Recently, the high performance liquid chromatography (HPLC) method is becoming the standard, but this method is expensive, has a large apparatus, and is complicated.

JP 2010-164579 A

  The problem to be solved by the present invention is to provide an optical waveguide measurement system and a method for measuring glycated hemoglobin capable of measuring HbA1c simply and accurately.

  The optical waveguide measurement system of the embodiment includes a first optical waveguide having a first sensing area on which a first substance capable of specifically binding to glycated hemoglobin is immobilized, and the first substance is the glycated hemoglobin. A plurality of first magnetic fine particles having superparamagnetism, in which a second substance capable of specifically binding to the glycated hemoglobin is immobilized at a location different from a location capable of specifically binding to glycated hemoglobin, A first magnetic field application unit provided above the first optical waveguide and capable of moving at least one of the plurality of first magnetic fine particles from the first sensing area by a magnetic force; A first light source capable of receiving light and generating near-field light in the first sensing area; and a first light receiving element capable of receiving light emitted from the first optical waveguide; Having.

It is a cross-sectional schematic diagram of the optical waveguide type measurement system according to the first embodiment, FIG. 1A is a schematic cross-sectional view showing the entire optical waveguide type measurement system, and FIG. It is the cross-sectional schematic diagram which expanded the 1st substance vicinity fixed on the waveguide. It is an image figure of HbA1c. FIG. 3A is a schematic diagram for illustrating the appearance of a magnetic fine particle, and FIGS. 3B and 3C are schematic cross-sectional views for illustrating a cross section of the magnetic fine particle. . It is process drawing which shows the method of measuring the glycated hemoglobin in the sample solution which concerns on 1st Embodiment. It is a cross-sectional schematic diagram of the optical waveguide type measurement system which concerns on 2nd Embodiment. It is process drawing which shows the method of measuring the glycated hemoglobin in the sample solution which concerns on 2nd Embodiment. It is a cross-sectional schematic diagram of the optical waveguide type measurement system which concerns on 3rd Embodiment. It is process drawing which shows the method of measuring the glycated hemoglobin in the sample solution which concerns on 3rd Embodiment. FIG. 9A is a schematic diagram of an optical waveguide type measurement system according to a fourth embodiment, FIG. 9A is a schematic plan view, and FIG. 9B is a cross-section at a position along line AB in FIG. It is a schematic diagram.

  Hereinafter, embodiments will be described with reference to the drawings. In the following description, the same members are denoted by the same reference numerals, and the description of the members once described is omitted as appropriate.

(First embodiment)
FIG. 1 is a schematic cross-sectional view of the optical waveguide type measurement system according to the first embodiment. FIG. 1A is a schematic cross-sectional view showing the entire optical waveguide type measurement system, and FIG. It is the cross-sectional schematic diagram which expanded the 1st substance vicinity fixed on the optical waveguide of a measurement system.

  The optical waveguide type measurement system 30 according to the first embodiment is fixed to an optical waveguide 3 (first optical waveguide) and a predetermined region (a sensing area 101 described later) on the optical waveguide 3, and is specific to glycated hemoglobin. A first medium 6 capable of binding to the second substance 13, a second substance 13, a plurality of magnetic fine particles 9 (first magnetic fine particles), and a dispersion medium 25 (first dispersion medium) such as a liquid (water, organic solvent). And comprising. A second substance 13 is immobilized on each of the plurality of magnetic fine particles 9. The second substance 13 can specifically bind to glycated hemoglobin at a place different from the place where the first substance 6 can specifically bind to glycated hemoglobin. A plurality of magnetic fine particles 9 are dispersed in the dispersion medium 25, and the dispersion medium 25 is in contact with the first substance 6.

  Further, the optical waveguide measurement system 30 is provided above the optical waveguide 3 and is capable of moving at least one of the plurality of magnetic fine particles 9 in the dispersion medium 25 by magnetic force (first magnetic field). An application unit), a light source 7 (first light source) capable of causing light to enter the optical waveguide 3 from outside the predetermined region, and light emitted from the optical waveguide 3 outside the predetermined region. A light-receiving element 8 (first light-receiving element) that can The light emitted from the light source 7 acts on the predetermined area. The magnetic field application unit 10 can apply a magnetic field that moves at least one of the plurality of magnetic fine particles 9 in a direction away from the optical waveguide 3.

  In addition, the optical waveguide measurement system 30 includes a substrate 1 that supports the optical waveguide 3, gratings 2 a and 2 b (incident side grating 2 a and outgoing side grating 2 b) provided in the optical waveguide 3, and the optical waveguide 3. A protective film 4 for protecting the surface and a frame 5 provided on the optical waveguide 3 are provided. The gratings 2a and 2b are formed of a material having a higher refractive index than that of the substrate 1. The optical waveguide 3 having a flat surface is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The protective film 4 is coated on the optical waveguide 3. The protective film 4 is a resin film having a low refractive index, for example. The protective film 4 is provided with an opening through which a part of the surface of the optical waveguide 3 located between the gratings 2a and 2b is exposed. The opening can be, for example, rectangular, and the surface of the optical waveguide 3 exposed in the opening serves as the sensing area 101. A space surrounded by the optical waveguide 3 and the frame 5 is filled with a dispersion medium 25. A portion filled with the dispersion medium 25 may be referred to as a reaction space 102.

  In the sensing area 101, the first substance 6 is immobilized on the optical waveguide 3. The sensing area 101 is exposed from the protective film 4. The frame 5 is formed on the protective film 4 so as to surround the sensing area 101. Further, in the optical waveguide measurement system 30, a configuration including the optical waveguide 3, the first substance 6, and the plurality of magnetic fine particles 9 on which the second substance 13 is immobilized is referred to as an optical waveguide sensor chip 100. . The optical waveguide sensor chip 100 is removed from the optical waveguide measurement system 30 and can be freely carried. Furthermore, the optical waveguide measurement system 30 includes a control unit 20 that controls each of the light source 7, the light receiving element 8, and the magnetic field application unit 10.

  As the optical waveguide 3, for example, a planar optical waveguide can be used. The material of the optical waveguide 3 is, for example, any one of thermosetting resin, photocurable resin, and alkali-free glass. The thermosetting resin or the photocurable resin is, for example, any one of a phenol resin, an epoxy resin, and an acrylic resin. Specifically, the material of the optical waveguide 3 is preferably a material having transparency to predetermined light, for example, a material having a refractive index higher than that of the substrate 1.

  Further, in the sensing area 101 that is the detection surface, the first substance 6 is immobilized by, for example, hydrophobic interaction or covalent bonding with the surface of the optical waveguide 3 in the sensing area 101. For example, the first substance 6 is fixed to the sensing area 101 by a hydrophobic treatment with a silane coupling agent. Alternatively, a functional group may be formed in the sensing area 101, and an appropriate linker molecule may act to immobilize the sensing area 101 by chemical bonding. As the first substance 6, for example, when glycated hemoglobin in the sample solution is an antigen, its antibody (primary antibody) can be used.

  The second substance 13 that specifically reacts with glycated hemoglobin in the sample solution is immobilized on the surface of the magnetic fine particle 9 by, for example, physical adsorption or chemical bonding via a carboxyl group, an amino group, or the like. . The magnetic fine particles 9 on which the second substance 13 is immobilized are dispersed and held in the sensing area 101 on which the first substance 6 is immobilized. The dispersion and holding of the magnetic fine particles 9 are formed by, for example, applying and drying a slurry containing the magnetic fine particles 9 and a water-soluble substance on the sensing area 101 or a surface (not shown) facing the sensing area 101. . Alternatively, the magnetic fine particles 9 may be dispersed in a liquid and held in a space different from the reaction space 102 or a container (not shown).

  The light source 7 irradiates the above-described optical waveguide sensor chip 100 with light. The light source 7 is, for example, a red laser diode. The light incident from the light source 7 is diffracted by the incident side grating 2 a and propagates through the optical waveguide 3. Thereafter, the light is diffracted and emitted by the emission side grating 2b. The light emitted from the emission side grating 2b is received by the light receiving element 8, and the light intensity is measured. The light receiving element 8 is, for example, a photodiode. The amount of the magnetic fine particles 9 is measured by comparing the intensity of the incident light and the emitted light and measuring the light absorption rate. Then, the antigen concentration in the sample solution is obtained based on the measured amount of the magnetic fine particles 9. Details regarding the determination of the antigen concentration in the specimen solution based on the measured amount of the magnetic fine particles 9 will be described later.

  The magnetic field application unit 10 applies a magnetic field to the optical waveguide sensor chip 100. The magnetic field application unit 10 generates a magnetic field and applies the generated magnetic field to the optical waveguide sensor chip 100 to move the magnetic fine particles 9 according to the magnetic field. The magnetic field application unit 10 is disposed in a direction opposite to the direction in which the optical waveguide 3 exists when viewed from the magnetic fine particles 9. In the first embodiment, the magnetic field application unit 10 is installed in the upward direction in FIG. The magnetic field application unit 10 includes, for example, a magnet or an electromagnet. In order to dynamically adjust the magnetic field strength, a method of adjusting with an electric current using an electromagnet is desirable, but using a ferrite magnet or the like, the magnetic field strength is adjusted according to the strength of the magnet itself or the distance from the optical waveguide sensor chip 100. May be.

  For example, the magnetic field strength can be adjusted by disposing a ferrite magnet above the optical waveguide sensor chip 100 and changing the thickness of the ferrite magnet between the magnet and the optical waveguide sensor chip 100 via a spacer. In addition, the magnetic field strength can be adjusted by changing the relative position of the ferrite magnet and the optical waveguide sensor chip 100 using an actuator such as a linear motor.

  In the case of using an electromagnet, the coil is disposed on the side opposite to the settling direction (the direction of the optical waveguide 3) when viewed from the magnetic fine particles 9, and a current is applied to the coil. Can be adjusted.

  In the first embodiment, by applying a magnetic field to the magnetic fine particles 9 by the magnetic field application unit 10, the magnetic fine particles 9 adsorbed on the sensing area 101 without depending on the antigen-antibody reaction are peeled off from the sensing area 101. be able to. Thereby, it is possible to measure absorbance due to only the magnetic fine particles 9 bound to the sensing area 101 via glycated hemoglobin by an antigen-antibody reaction, and reduce measurement errors.

  At this time, it is preferable to use the magnetic fine particles 9 having superparamagnetism that quickly loses magnetization when the application of the magnetic field is stopped. Thereby, even if the magnetic fine particles 9 aggregate due to magnetization when a magnetic field is applied, the magnetic fine particles 9 can be redispersed by stopping the application of the magnetic field. For example, even when a magnetic field is applied when glycated hemoglobin is not present in the sample solution, an aggregate of the magnetic fine particles 9 may be generated and hardly peel from the sensing area 101 in some cases. Such agglomerates of magnetic fine particles 9 cause measurement errors. In this case, if the magnetic fine particles 9 have superparamagnetism, aggregation of the magnetic fine particles 9 can be suppressed, so that generation of measurement errors can be suppressed.

  Further, in order to further improve the redispersibility when the application of the magnetic field is stopped, the surface of the magnetic fine particles 9 may have a positive or negative charge. Alternatively, a dispersant such as a surfactant may be added to the dispersion medium of the magnetic fine particles 9.

  Furthermore, in the first embodiment, the naturally settled magnetic fine particles 9 can be pulled back upward by the magnetic field application unit 10. By repeating the natural sedimentation of the magnetic fine particles 9 and the upward pulling back by the magnetic field applying unit 10, the sample solution and the magnetic fine particles 9 can be stirred. Thereby, the coupling | bonding by the antigen antibody reaction of the magnetic microparticle 9 and the sensing area 101 through the antigen (glycated hemoglobin) contained in the sample solution is promoted, and high detection sensitivity can be obtained in a shorter time. Therefore, when the glycated hemoglobin has a low concentration, the detection sensitivity can be increased.

  If the surface of the magnetic fine particles 9 is positively or negatively charged, or a dispersant such as a surfactant is added, the magnetic fine particles 9 are easily redispersed when the application of the magnetic field is stopped, and stirring is performed. Further promote. Thereby, detection sensitivity further improves.

  Further, hemoglobin (Hb) in glycated hemoglobin is present in erythrocytes and is known to bind to oxygen in the lung, for example. Among these, HbA (hemoglobin A) known as hemoglobin for adults has a tetrameric structure composed of two α subunits and two β subunits. The β subunit is unique to HbA. The α subunit is composed of 141 amino acids, and the β subunit is composed of 146 amino acids. The molecular weight of HbA (α2β2) is about 64500.

  Glycated hemoglobin is obtained by binding sugars such as Glc (glucose), Fru (fructose), Suc (sucrose), Mal (maltose) to hemoglobin.

  Moreover, HbA1 (hemoglobin A1) is obtained by binding Glc (glucose), phosphorylated sugar, etc. to the β chain of HbA. HbA1c (hemoglobin A1C) is obtained by binding Glc (glucose) to the β-chain N-terminus (Val (valine)) of HbA1.

FIG. 2 is an image diagram of HbA1c.
As shown in FIG. 2, HbA1c has two α subunits and two β subunits. In HbA1c, Glc (glucose) is bound to the β chain N-terminus of HbA1.

  HbA1c is glycated according to blood glucose and accumulated until the life of red blood cells. Therefore, the value of HbA1c is proportional to the blood glucose level from the time when red blood cells are made to the present, and reflects the average blood glucose level in the past 1 to 2 months.

  As the first substance 6, a monoclonal antibody whose antigen is a glycated peptide at the N-terminus of the β chain of HbA1c is selected. As the second substance 13, a monoclonal antibody in which HbA1c other than the glycated peptide at the β-chain N-terminal of HbA1c is an antigen, or a monoclonal antibody in which the β subunit of HbA1c other than the glycated peptide at the β-chain N-terminal of HbA1c is the antigen Is selected. The role of the first substance 6 and the second substance 13 will be described later.

  The glycated peptide is a fructosyl peptide. More specifically, Fru (fructose) -Val (valine) -His (histidine) -Leu (leucine) -Thr (threonine) -Pro (proline) -Glu (glutamic acid) It is.

  3A and 3B are schematic views of the magnetic fine particles. FIG. 3A is a schematic view for illustrating the appearance of the magnetic fine particles, and FIGS. 3B and 3C are cross sections for illustrating the cross section of the magnetic fine particles. It is a schematic diagram.

Before describing the specific structure of the magnetic fine particles 9, an outline thereof will be described.
The magnetic fine particles 9 are held in a dispersed state on the sensing area 101 or are held in another space or a container (not shown). Here, “the magnetic fine particles are held in a dispersed state on the sensing area” means that the magnetic fine particles 9 are held in a dispersed state directly or indirectly above the sensing area 101. Examples of the form “the magnetic fine particles are indirectly dispersed above the sensing area 101” include a form in which the magnetic fine particles 9 are dispersed on the surface of the sensing area 101 via a blocking layer.

  The blocking layer includes a water-soluble substance such as polyvinyl alcohol, bovine serum albumin (BSA), polyethylene glycol, phospholipid polymer, gelatin, casein, saccharide (eg, sucrose, trehalose), and synthetic polymer.

  As another example, there is a form in which the magnetic fine particles 9 are arranged above the sensing area 101 with a space therebetween. For example, a support plate (not shown) facing the sensing area 101 may be disposed, and the magnetic fine particles 9 may be held in a dispersed state on the surface where the support plate faces the sensing area 101.

  In this case, it is desirable that the magnetic fine particles 9 be held in a dry or semi-dry state. Although it is desirable to easily re-disperse when it comes into contact with a dispersion medium such as a sample solution, the form held in a dry or semi-dry state does not necessarily need to be completely dispersed. When held in another space or a container, it may be in a state of being dispersed in a dispersion medium, a state of being settled in a dispersion medium, in addition to a dry or semi-dry state.

A specific structure of the magnetic fine particles 9 will be described.
As shown in FIG. 3A, the second substance 13 is immobilized on the surface of the magnetic fine particle 9. For example, when the glycated hemoglobin in the sample solution is an antigen, an antibody (secondary antibody) can be used as the second substance 13.

  In this case, as shown in FIG. 3B, the magnetic fine particles 9 in which the magnetic nano-particles 9a are covered with the polymer material 9b, and as shown in FIG. 3C, the core 9c and the shell 9d that covers the core 9c. The magnetic fine particles 9 can be obtained.

  The core 9c can be formed from a polymer material. The shell 9d is formed of a polymer material and can include the magnetic nanoparticle 9a.

Alternatively, it may be a fine particle itself made of a magnetic material, and in this case, it is desirable to have a functional group that binds the substance to be measured to the surface of the fine particle. Examples of the magnetic material used for the magnetic fine particles 9 include various ferrites such as γ-Fe ₂ O ₃ . In this case, it is preferable to use a material having superparamagnetism that quickly loses magnetism when the application of the magnetic field is stopped.

  In general, superparamagnetism is a phenomenon that occurs in nanoparticles of several tens of nanometers (nanometers) or less. On the other hand, the size of the fine particles that cause light scattering needs to be several hundred nm or more. Therefore, as the magnetic fine particles 9 in the first embodiment, those in which the magnetic nanoparticles 9a are wrapped with the polymer material 9b as shown in FIG. 3B or FIG. 3C are suitable.

  In general, the refractive index is mostly 1.5 to 1.6 for polymer materials, and about 3.0 for ferrites. Further, when the magnetic fine particles 9 are in the vicinity of the surface of the optical waveguide 3, the higher the refractive index, the easier it is to scatter light. Therefore, it is considered that detection can be made with higher sensitivity when the magnetic nanoparticle 9 a having a high refractive index is distributed in the vicinity of the surface of the magnetic particle 9.

  As shown in FIG. 3B, in the magnetic fine particles 9 in which the magnetic nanoparticles 9a are simply covered with the polymer material 9b, the magnetic nanoparticles 9a are distributed throughout the fine particles. Therefore, from the viewpoint of detection sensitivity, a structure in which the core-shell type magnetic fine particles 9 are used and the magnetic nanoparticles 9a are contained in the shell 9d at a high density as shown in FIG. 3C is suitable.

  The particle size of the magnetic fine particles 9 is desirably 0.05 μm (micrometer) or more and 200 μm or less. When the particle size is smaller than 0.05 μm, the light scattering efficiency is lowered. In addition, when the particle size is larger than 200 μm, the reaction efficiency between the second substance 13 and glycated hemoglobin decreases, or the magnetic fine particles 9 do not move sufficiently due to the own weight of the magnetic fine particles 9 even when a magnetic force is applied. There is. In order to further improve the light scattering efficiency and reactivity, the particle size of the magnetic fine particles 9 is desirably set to 0.2 μm or more and 20 μm or less. By using a particle size in this range, the light scattering efficiency and reactivity are enhanced, so that the detection sensitivity is improved in the optical waveguide measurement system 30 that detects glycated hemoglobin using light.

FIG. 4 is a process diagram showing a method for measuring glycated hemoglobin in a sample solution.
Here, a method of measuring the amount of glycated hemoglobin using the optical waveguide type measurement system 30 described above will be described with reference to FIGS. 4 (a) to 4 (d). HbA1c is selected as the glycated hemoglobin. In FIG. 4A to FIG. 4D, the state in the reaction space 102 is illustrated.

  As a procedure for measuring glycated hemoglobin in the sample solution, first, the dispersion medium 25 is prepared. In the dispersion medium 25, a plurality of magnetic fine particles 9 on which the second substance 13 is immobilized are dispersed, and glycated hemoglobin is mixed. Next, the dispersion medium 25 is brought into contact with the first substance 9 on the optical waveguide 3. Next, the light intensity of the light emitted from the optical waveguide 3 is measured as the first light intensity. Next, a magnetic field is applied to the dispersion medium 25. Next, after applying the magnetic field, the light intensity of the light emitted from the optical waveguide 3 is measured as the second light intensity. Then, glycated hemoglobin is quantified based on the difference between the first light intensity and the second light intensity.

  Specifically, first, as shown in FIG. 4A, the sample solution is introduced onto the optical waveguide 3 in which the magnetic fine particles 9 are dispersed and held, and the magnetic fine particles 9 are redispersed. When the magnetic fine particles 9 are held in a space other than the optical waveguide 3 or in a separate container, a mixed dispersion of the sample solution and the magnetic fine particles 9 is introduced. Alternatively, first, the introduction of the dispersion of the magnetic fine particles 9 and the introduction of the sample solution may be performed separately, and the dispersion of the magnetic fine particles 9 and the sample solution may be introduced separately. As an introduction method, for example, dripping or inflow can be considered.

  That is, the first substance provided in the sensing area 101 with the dispersion medium 25 including HbA1c (14) and the magnetic fine particles 9 in which the second substance 13 that specifically binds to HbA1c (14) is immobilized and having magnetism. 6 is contacted.

  Next, as shown in FIG. 4B, the magnetic fine particles 9 settle toward the sensing area 101 due to their own weight. At this time, the first substance 6 immobilized on the sensing area 101 binds to the N-terminal of the β chain of HbA1c (14), and the second substance 13 immobilized on the surface of the magnetic fine particle 9 is, for example, HbA1c. It binds to the β subunit of HbA1c other than the β-chain N-terminal glycated peptide. This state is shown in FIG. Thereby, the magnetic fine particle 9 is couple | bonded with the sensing area 101 via HbA1c (14). At this stage, there are also magnetic fine particles 9 adsorbed on the sensing area 101 without passing through the HbA1c (14).

  Next, as shown in FIG. 4D, by applying a magnetic field from a direction different from the sedimentation direction (for example, upward) as seen from the magnetic fine particles 9, the magnetic particles 9 are attracted to the sensing area 101 without passing through the HbA1c (14). The magnetic fine particles 9 being moved are moved in a direction different from the settling direction (for example, upward) and removed from the sensing area 101. That is, the magnetic fine particles 9 bonded to the sensing area 101 via HbA1c (14) are left in the sensing area 101.

  For example, by adjusting the magnetic field strength to an appropriate value, the magnetic fine particles 9 bound to the sensing area 101 via the HbA1c (14) by the antigen-antibody reaction are not peeled off from the first substance 6, and the HbA1c (14) is removed. The magnetic fine particles 9 adsorbed on the sensing area 101 without being interposed can be removed from the sensing area 101.

  In the first embodiment, it is conceivable to obtain an appropriate magnetic field strength as follows. The states of the magnetic fine particles 9 that can be detected by near-field light such as evanescent light can be classified into the following states A to C depending on the strength of interaction with the sensing area 101.

Stated in the order of strong interaction, state A is a state of magnetic fine particles 9 bound to sensing area 101 by binding of HbA1c (14) and a molecule that specifically binds to it, such as antigen-antibody binding, and state B is a molecule. The state C of the magnetic fine particles 9 adsorbed to the sensing area 101 non-specifically due to an interatomic force or hydrophobic interaction, and the state C are states of the magnetic fine particles 9 floating in the vicinity of the sensing area 101. The magnetic fine particles 9 in the state A are the magnetic fine particles 9 that should contribute to the detection of the concentration of HbA1c (14), and the magnetic fine particles 9 in the state B or the state C are magnetic fine particles 9 that can cause measurement errors (noise). . The magnetic fine particles 9 in the state A will be appropriately referred to as magnetic fine particles 9 coupled to the sensing area 101.
Further, the magnetic fine particles 9 in the state B will be appropriately referred to as magnetic fine particles 9 adsorbed on the sensing area 101.

Here, “the vicinity of the surface” of the optical waveguide 3 that can be detected by near-field light is, for example, evanescent light that permeates the surface of the propagation body when light propagates by total reflection, and the permeation distance d is expressed by the following equation: It is calculated | required by (1). From formula (1), it can be seen that the seepage distance d is approximately a fraction of the wavelength of light used for measurement.
d = λ / {2π (n ₁ × sin ² θ−n ₂ ² ) ^1/2 } (1)
Here, d is the evanescent light penetration distance, λ is the wavelength of light used for measurement, n ₁ is the refractive index of the optical waveguide 3, n ₂ is the refractive index of the dispersion medium 25 in which the magnetic fine particles 9 are dispersed, and θ is The total reflection angle.

Therefore, the magnetic field application unit 10 applies a magnetic field having such a magnetic field strength that the magnetic fine particles 9 are separated from the sensing area 101 by a distance L that satisfies the following expression (2).
L> λ / {2π (n ₁ × sin ² θ−n ₂ ² ) ^1/2 } (2)
Here, L is the distance that the magnetic fine particles 9 are separated from the sensing area 101, λ is the wavelength of light used for measurement, n ₁ is the refractive index of the optical waveguide 3, and n ₂ is the refraction of the dispersion medium 25 in which the magnetic fine particles 9 are dispersed. The rate, θ, is the total reflection angle.

For example, when λ = 635 nm, n ₁ = 1.58, n ₂ = 1.33 (when the dispersion medium 25 is water), and θ = 78 °, L> 130 nm. Therefore, the measurement error can be sufficiently reduced by applying the magnetic field and moving the magnetic fine particles 9 in the state B and the state C slightly from the sensing area 101 by about several hundred nm. Therefore, the time required to move the magnetic fine particles 9 in the state B and the state C away from the sensing area 101 to a distance that does not cause an error in detection sensitivity is very short.

  Further, if the time is within an allowable range, the magnetic fine particles 9 in the state B and the state C can be moved to a distance that does not cause a measurement error with a weak magnetic field intensity even if a certain amount of time is required. Become. Thereby, possibility that the magnetic fine particle 9 required for the measurement of the state A will be excessively peeled off can be reduced. That is, the magnetic fine particles 9 in the state A that should contribute to the measurement are not peeled off from the sensing area 101, and the magnetic fine particles 9 in the state B and the state C that may cause measurement noise are separated from the sensing area 101 without affecting the measurement. The S / N ratio can be improved.

  As described above, the appropriate magnetic field strength means that the magnetic fine particles 9 in the state B and the state C that may become measurement noise are removed from the sensing area 101 without peeling off the magnetic fine particles 9 in the state A that should contribute to the measurement from the sensing area 101. The magnetic field strength is suitable for peeling to a distance that does not affect the measurement.

  As described above, a method of optimally adjusting the magnetic field intensity with an electric current using an electromagnet is desirable. However, using a ferrite magnet or the like, the strength of the magnet itself, or the relative position between the optical waveguide sensor chip 100 and the magnet. May be changed to adjust the magnetic field strength. In the case of using an electromagnet, the coil is disposed on the side opposite to the settling direction (the direction of the optical waveguide 3) when viewed from the magnetic fine particles 9, and a current is applied to the coil. Can be adjusted.

  Further, in order to optimally adjust the magnetic field strength, the optical waveguide type measurement system 30 of the first embodiment may further include a control unit 20 that controls the magnetic field strength of the magnetic field applied by the magnetic field application unit 10. Good. By performing the control as described above by the control unit 20, the magnetic field strength can be adjusted to an appropriate strength. For example, without separating the magnetic fine particles 9 in the state A that should contribute to the measurement from the sensing area 101, the magnetic fine particles 9 in the state B and the state C that may cause measurement noise are away from the sensing area 101 without affecting the measurement. It can be adjusted so that the magnetic field intensity can be removed.

  Further, when the magnetic field intensity is adjusted as needed, it can be dynamically controlled by adjusting the control unit 20. For example, the control unit 20 that controls at least one of the timing and the length of time at which the magnetic field applying unit 10 applies the magnetic field may be used.

  Then, by measuring the difference in the detection signal intensity ratio in the light receiving element 8, the amount of HbA1c (14) in the sample solution (that is, the antigen concentration) can be measured. Specifically, in FIG. 1, laser light is incident from the light source 7 to the optical waveguide 3 from the incident side grating 2a, propagates through the optical waveguide 3, and evanescent light or the like near the surface (exposed surface in the sensing area 101). Of near-field light. In this state, when a mixed dispersion of the sample solution and the magnetic fine particles 9 is introduced onto the sensing area 101, the magnetic fine particles 9 settle immediately after that (FIG. 4A), in the vicinity of the sensing area 101, for example, evanescent light. The region is reached (FIG. 4B). Since the magnetic fine particles 9 are involved in the absorption and scattering of the evanescent light, the intensity of the reflected light is attenuated.

  As a result, when the laser beam emitted from the emission side grating 2 b is received by the light receiving element 8, the intensity of the emitted laser beam decreases with the passage of time due to the influence of the combined magnetic fine particles 9. Thereafter, when the upper magnetic field is applied by the magnetic field application unit 10, the magnetic fine particles 9 in the state B and the state C move out of the evanescent light region (FIG. 4D), so that the light reception intensity is restored to a predetermined value. To do. The received light intensity at this time is compared with the received light intensity in the state of FIG. 4A, that is, immediately after the introduction of the mixed dispersion, and can be quantified as, for example, a reduction rate.

  In addition, the light intensity of light emitted from the optical waveguide sensor chip 100 (corresponding to an example of the first light intensity) after the sample solution or the like is introduced into the optical waveguide sensor chip 100 and before the magnetic field is applied. ). Further, after application of the magnetic field, the light intensity of light emitted from the optical waveguide sensor chip 100 (corresponding to an example of the second light intensity) is measured. And HbA1c (14) can be quantified based on the difference of these light intensity.

  The rate of decrease in the intensity of the laser beam received by the light receiving element 8 depends on the amount of the magnetic fine particles 9 bound to the sensing area 101 mainly by an antigen-antibody reaction or the like. That is, the rate of decrease is proportional to the antigen concentration in the sample solution involved in the antigen-antibody reaction. Therefore, a variation curve of the intensity of the laser beam with the passage of time in a specimen solution with a known antigen concentration is obtained, and a decrease rate of the intensity of the laser beam at a predetermined time after application of the upper magnetic field of this variation curve is obtained, A calibration curve showing the relationship between the antigen concentration and the rate of decrease in the intensity of the laser beam is created in advance. Next, the decrease rate of the laser light intensity at a predetermined time is obtained from the time measured by the above method in the specimen solution with the unknown antigen concentration and the fluctuation curve of the laser light intensity, and the decrease rate of the laser light intensity is calculated. By collating with the calibration curve, the antigen concentration in the sample solution can be measured.

  Next, a more specific example in which the measurement of the first embodiment is performed by experiment will be described. The following specific numerical values and materials are examples, and are not limited to these numerical values and materials.

  In the experiment, a titanium oxide film having a refractive index of 2.2 to 2.4 is formed on a light-transmitting substrate 1 such as glass to a thickness of 50 nm by a sputtering method, and a lithography method and a dry etching method are performed. Thus, gratings 2a and 2b were formed. An ultraviolet curable acrylic resin film having a film thickness of about 10 μm was formed on the substrate 1 on which the gratings 2 a and 2 b were formed by a spin coating method and ultraviolet irradiation to obtain an optical waveguide 3. The refractive index after curing is 1.58.

  The protective film 4, which is a low refractive index resin film, includes a region corresponding to the upper side of the gratings 2 a and 2 b on the surface of the optical waveguide 3, and screen printing is performed so as to surround the antibody immobilization region that is the sensing area 101. Formed using. The refractive index after drying of the protective film 4 is 1.34. In order to form a liquid reservoir for holding the sample solution and the like, the resin frame 5 was fixed with double-sided tape. A first substance 6 for HbA1c (14) was immobilized on the surface of a region where a protective film between the gratings was not formed by a covalent bond method.

  In the first embodiment, the magnetic fine particles 9 are of a core-shell type in which the magnetic nanoparticles 9a are densely contained in the shell 9d. The average particle size of the magnetic fine particles 9 was 1.1 μm. A dispersion containing such magnetic fine particles 9 was separately prepared.

  Next, light having a central wavelength of 635 nm from the light emitting diode 7 is incident from the grating 2a on the incident side, and the light intensity of the light emitted from the grating 2b on the emitting side is measured by the photodiode 8, while the sample solution and the magnetic fine particles are measured. After mixing with 9 dispersion liquid, it was introduced into the sensing area 101 (inside the frame 5). Thereafter, measurement was performed according to the measurement procedure described above.

  In the first embodiment, a ferrite magnet is disposed above the optical waveguide sensor chip 100, a spacer is provided between the ferrite magnet and the optical waveguide sensor chip 100, and the magnetic field strength is increased by changing the thickness of the spacer. Changed.

  The photodiode detection signal intensity at this stage is compared with the intensity immediately after the introduction of the mixed solution (initial intensity), and the difference is measured. The attenuation amount of the signal intensity indicated by this difference corresponds to the number of magnetic fine particles 9 bonded to the surface of the optical waveguide 3 through HbA1c (14). Therefore, the concentration of HbA1c (14) that is the measurement target can be calculated.

  In this way, using the optical waveguide 3 in which the first substance 6 that specifically reacts with HbA1c is immobilized and the magnetic fine particles 9 in which the second substance 13 is immobilized, the noise particles of the non-specifically adsorbed magnetic fine particles 9 are subjected to a magnetic field. With the method of removing by application, binding-free separation (B / F separation) is not required, but extremely low concentration of HbA1c can be measured with high sensitivity.

  There are various immunoassay methods for measuring the concentration of a substance to be measured by using an antibody against the substance to be measured, or when the substance to be measured is an antibody, using an antigen or an antibody against the antibody.

  For example, in enzyme immunoassay (EIA or ELISA), a primary antibody corresponding to a substance to be measured in a sample to be measured is immobilized on the surface of a well-shaped substrate, and a predetermined amount of sample solution is placed in the well. In addition, a primary reaction is performed. After that, add the secondary antibody solution labeled with the enzyme that catalyzes the color development reaction of the dye, perform the secondary reaction, remove the excess secondary antibody by washing, add the color reagent and measure the absorbance. doing. However, these procedures are complicated.

  As a more sensitive immunoassay method, there is a method of detecting the amount of luminescence by chemiluminescence using a secondary antibody labeled with an enzyme that catalyzes a chemiluminescent reaction in the secondary reaction, that is, the CLEIA method. Furthermore, there is an immunochromatography method that can determine the presence or absence of a substance to be measured simply by dropping a sample liquid.

  However, in these immunoassay methods, a secondary antibody that is not bound to a test substance becomes a background or noise component. For this reason, the immunoassay method commonly requires a step (Binding-Free separation (B / F separation) step) of sufficiently removing excess secondary antibodies by washing. As a result, the work time becomes long. In addition, automating these processes will increase the cost and size of the apparatus. On the other hand, the simple immunochromatography method generally has low quantification, and there are problems that the determination in the low concentration region varies depending on the measurer and the measurement result cannot be managed as a digital numerical value.

  In order to solve this problem, a method for detecting fine particles bonded to the surface by an evanescent wave of an optical waveguide has been started. This method does not require a B / F separation step. However, in the conventional method, in the process in which the fine particles are bound to the surface of the optical waveguide by an antigen-antibody reaction or the like, the actual state is that the binding is advanced by the so-called “deposition” or diffusion due to Brownian motion.

  For this reason, the antigen that contributes to the binding of the microparticles to the surface within a predetermined measurement time is estimated to be a very small part of the total antigen in the system. In addition, when the liquid property of the specimen fluctuates, the diffusion rate, dispersion state, and the like fluctuate accordingly, and measurement errors are caused thereby. Furthermore, it is difficult to completely suppress the phenomenon in which fine particles are physically adsorbed nonspecifically on the surface of the optical waveguide, and this can be a noise component.

  In another method using an evanescent wave, a measurement target substance, a first reactant having both a reaction part and a localization induction part that specifically bind to the measurement target substance, and a measurement target substance are specifically specified. A second reactant having both a reaction part to be bonded and a photoactive component is reacted. Then, the first reactant-measurement target substance-second reactant combination obtained by this reaction is localized in the near-field light region by the localization means, and the measurement target substance is determined by the difference in signal obtained before and after that. Quantify.

  However, when the first reactant is a magnetic fine particle, the free first reactant not bound to the antigen or the second reactant is also localized in the near-field light region at the same time. In addition, the free second reactant is present in the near-field light region throughout the detection process. When the distribution of these free reactants in the near-field light region varies depending on the liquidity of the biological sample, this variation can be a noise component. In addition, no positive countermeasure is taken against noise components caused by non-specific physical adsorption of each reactant and bound substance on the surface of the detection means that generates near-field light.

  There is also an immunosensor method using a Lamb wave mode acoustic device. In this sensor method, when latex or the like, which is a labeling substance for detection, is introduced into the reaction system, the viscosity changes thereby causing a phase difference change, or the latex floating without adsorbing to the sensor section is noisy. It can be a cause.

  To solve this problem, use magnetic microparticles as a labeling substance, apply a magnetic field after the reaction to keep the labeling substance floating near the sensor unit away, and make the environment around the acoustic device the same as before the labeling substance was charged. To.

However, in the acoustic device in contact with the liquid, the depth in the liquid that is interlocked with the vibration of the sensor surface is considered to be several μm to several tens of μm as determined from the following equation (3). That is, the thickness d of the layer hydrodynamically interlocking with the sensor surface is
d = (2η / ωρ) ^1/2 (3)
(Η: viscosity of liquid, ω: angular frequency, ρ: density of liquid)
It is represented by Here, when the frequency is F, ω = 2πF. If F is 5 MHz, the viscosity of water is about 1 cp and the density is 1 g / cm ³ (= 103 kg / m ³ ), so d is about 8 μm.

  Therefore, in order to apply a magnetic field after the reaction so that the environment around the acoustic device becomes the same as before the labeling substance is charged, it is necessary to keep the labeling substance floating in the vicinity of the sensor unit about 10 μm or more away. In order to shorten the measurement time, it is necessary to apply a stronger magnetic force. However, if it is too strong, the antigen-antibody binding is also separated, and the sensitivity is lowered.

  On the other hand, in the first embodiment, by applying a magnetic field to the magnetic fine particles 9 in a direction different from the settling direction, the magnetic fine particles can be adsorbed to the sensing area 101 without causing an antigen-antibody reaction and become noise. 9 is peeled off from the sensing area 101. Thereby, the absorbance due to the magnetic fine particles 9 bound to the sensing area 101 via, for example, glycated hemoglobin such as HbA1c can be measured by an antigen-antibody reaction or the like, so that measurement errors can be reduced.

  In addition, since the magnetic fine particles 9 that can become noise by applying a magnetic field can be removed, the work of removing such magnetic fine particles 9 by washing becomes unnecessary.

  According to the first embodiment, since the optical waveguide sensor chip 100 is used and measurement is performed with near-field light such as evanescent light, the distance by which the magnetic fine particles 9 are peeled from the sensing area 101 to a range that does not affect the measurement is small. It is short. Thereby, the time required for peeling off the magnetic fine particles 9 from the sensing area 101 by applying the upper magnetic field can be shortened. Alternatively, the magnetic fine particles 9 can be peeled from the sensing area 101 to a range that does not affect the measurement by a weaker magnetic field.

  In addition, according to the first embodiment, since the magnetic field strength can be controlled, the magnetic fine particles 9 that can be measurement noise can be removed without peeling the magnetic fine particles 9 that should contribute to the measurement from the sensing area 101. To a distance that does not affect the measurement. As a result, the S / N ratio can be improved.

  In addition, according to the first embodiment, the control accuracy can be kept high by dynamically controlling the magnetic field intensity by the control unit 20.

  In addition, if the magnetic fine particles 9 have superparamagnetism that quickly loses magnetization when the application of the magnetic field is stopped, the magnetic fine particles 9 are easily redispersed when the application of the magnetic field is stopped. For this reason, even when glycated hemoglobin is not present in the sample solution, the generation of aggregates of the magnetic fine particles 9 is suppressed, so that occurrence of measurement errors can be suppressed.

  Furthermore, the scattering intensity of the evanescent light can be increased by using a core-shell type in which the magnetic nanoparticle 9a includes the magnetic nanoparticle 9a in the shell 9d. As a result, highly sensitive detection can be performed.

  Furthermore, by adding a positive or negative charge to the surface of the magnetic fine particles 9 or adding a dispersant such as a surfactant, the magnetic fine particles 9 are easily redispersed when the application of the magnetic field is stopped. It is also possible to reduce the measurement error.

  Further, according to the first embodiment, the naturally settled magnetic fine particles 9 can be pulled back by applying a magnetic field in a direction different from the settling direction. By repeating the natural sedimentation of the magnetic fine particles 9 and the upward pulling back by the magnetic field applying unit 10, the sample solution and the magnetic fine particles 9 are agitated, so that glycated hemoglobin (for example, antigen) and magnetic fine particles contained in the sample solution are stirred. The antigen-antibody reaction with 9 is promoted, and high detection sensitivity can be obtained in a shorter time. Therefore, when the glycated hemoglobin has a low concentration, the detection sensitivity can be increased.

  At this time, by adding a positive or negative charge to the surface of the magnetic fine particles 9 or adding a dispersing agent such as a surfactant, the magnetic fine particles 9 are redispersed when the application of the magnetic field is stopped. It can be facilitated, stirring can be promoted, and detection sensitivity can be improved.

  In addition, according to the first embodiment, the optical waveguide sensor chip 100 is used to measure the amount and concentration of glycated hemoglobin using near-field light such as evanescent light. In this case, if the magnetic fine particles 9 having a particle size of 0.05 μm or more and 200 μm or less, preferably 0.2 μm or more and 20 μm or less are used, the light scattering efficiency can be increased, and thus the detection sensitivity of glycated hemoglobin Can be improved.

(Second Embodiment)
FIG. 5 is a schematic cross-sectional view of an optical waveguide measurement system according to the second embodiment.

  An optical waveguide measurement system 31 according to the second embodiment is obtained by further adding a magnetic field application unit 11 (second magnetic field application unit) to the optical waveguide measurement system 30 of the first embodiment. The magnetic field application unit 11 is provided below the optical waveguide 3. Other configurations are the same as those in the first embodiment.

  The magnetic field applying unit 11 applies a magnetic field to the optical waveguide sensor chip 100 in the direction of the optical waveguide 3 as viewed from the magnetic fine particles 9. The magnetic field application unit 11 can apply a magnetic field that moves at least one of the plurality of magnetic fine particles 9 in a direction approaching the optical waveguide 3.

  The magnetic field application unit 11 is provided in the direction in which the optical waveguide 3 exists as viewed from the magnetic fine particles 9. In the second embodiment, the magnetic field application unit 11 is provided below the sensor chip 100.

  Similar to the magnetic field application unit 10, the magnetic field application unit 11 includes a magnet or an electromagnet. In order to dynamically adjust the magnetic field strength, a method of adjusting the current by using an electromagnet is desirable, but using a ferrite magnet or the like, the strength of the magnet itself or the relative position of the optical waveguide sensor chip 100 and the magnet is changed. To adjust the magnetic field strength. For example, the magnetic field strength can be adjusted by disposing a ferrite magnet below the optical waveguide sensor chip 100 and changing the thickness of the ferrite magnet through a spacer between the magnet and the optical waveguide sensor chip 100. In the case of using an electromagnet, the coil is disposed in the direction of the optical waveguide 3 when viewed from the magnetic fine particles, and a current is applied to the coil, and the magnetic field intensity can be adjusted by changing the current value.

  Here, the optical waveguide measurement system 31 of the second embodiment includes the control unit 20. The control unit 20 controls the strength of the magnetic field applied to the sensing area 101 from at least one of the magnetic field application unit 10 and the magnetic field application unit 11. In this case, for example, as shown in FIG. 5, a common control unit 20 and a changeover switch 20 s can be provided for the magnetic field application unit 10 and the magnetic field application unit 11. Independent control units may be provided for the magnetic field application unit 10 and the magnetic field application unit 11. In addition, a control unit that controls the magnetic field strength at the same time for the magnetic field application unit 10 and the magnetic field application unit 11 may be provided. Further, the control unit 20 may dynamically adjust the magnetic field strength as needed to dynamically adjust the magnetic field strength.

  Further, the control unit 20 may control the timing of applying the magnetic field in each of the magnetic field applying unit 10 and the magnetic field applying unit 11. Thereby, the magnetic field application unit 10 and the magnetic field application unit 11 can alternately apply a magnetic field according to a predetermined condition (for example, a predetermined time or a time during which a predetermined magnetic field is continuously applied).

FIG. 6 is a process diagram showing a method for measuring glycated hemoglobin in a sample solution according to the second embodiment.
6A to 6C illustrate states in the sensing area 101. FIG. 6 (a) and 6 (c) are the same as the states shown in FIGS. 4 (a) and 4 (d), and a description thereof will be omitted.

  Further, the measurement of the antigen concentration in the sample solution by measuring the difference in the detection signal intensity ratio in the light receiving element 8 is the same as in the first embodiment, and thus the description thereof is omitted.

The state in FIG. 6B will be described.
6B, the magnetic field application unit 11 applies a lower magnetic field in the settling direction (in the direction of the optical waveguide 3, for example, the lower side in FIG. 6) as viewed from the magnetic fine particles 9. Thereby, the magnetic fine particles 9 are attracted to the sensing area 101. At this time, the first substance 6 (for example, a primary antibody) immobilized on the sensing area 101 and the second substance 13 (for example, a secondary antibody) immobilized on the magnetic fine particles 9 are passed through HbA1c (14). It binds by antigen-antibody reaction. Thereby, the magnetic fine particles 9 are coupled to the sensing area 101.

  In the second embodiment, the downward magnetic field application shown in FIG. 6B and the upward magnetic field application shown in FIG. 6C may be alternately repeated.

  When the magnetic fine particles 9 are attracted to the optical waveguide 3 by applying a magnetic field in the downward direction shown in FIG. 6B, HbA1c (14) is contained in both the first substance 6 and the second substance 13 in the sample solution. It remains in a state where it is not bonded, or in a state where it is bonded to the second substance 13 immobilized on the surface of the magnetic fine particle 9 but not bonded to the first substance 6 immobilized in the sensing area 101. In addition, the magnetic fine particles 9 adsorbed nonspecifically exist in the sensing area 101.

  Therefore, in FIG. 6C, a magnetic field having a strength that does not peel off the magnetic fine particles 9 bound by the antigen-antibody reaction or the like is applied, and the magnetic fine particles 9 not bound by the antigen-antibody reaction or the like are directed in a direction different from the optical waveguide 3. Move.

  Thereafter, again, as shown in FIG. 6B, a magnetic field is applied in the direction of the optical waveguide 3 to attract the magnetic fine particles 9 that are not bound by an antigen-antibody reaction or the like. Then, HbA1c (14) and HbA1c (14) bound to the second substance 13 immobilized on the surface of the magnetic fine particle 9 are newly bound to the first substance 6 immobilized in the sensing area 101.

  By repeating this, the number of magnetic fine particles 9 not bound to the sensing area 101 due to antigen-antibody reaction or the like is reduced, and the number of magnetic fine particles 9 bound to the sensing area 101 due to antigen-antibody reaction or the like is increased. As a result, the S / N ratio is further improved.

  Also in the second embodiment, the same effect as in the first embodiment is obtained. Furthermore, in the second embodiment, by repeating the lower magnetic field application and the upper magnetic field application during the initial sedimentation, the binding speed to the magnetic fine particles 9, the reaction efficiency, and the reproducibility are further improved.

  For example, according to the second embodiment, the magnetic fine particles 9 can be attracted to the sensing area 101 by applying a magnetic field to the magnetic fine particles 9 by the magnetic field applying unit 11. As a result, the magnetic fine particles 9 are more easily coupled to the sensing area 101, and the detection sensitivity of HbA1c (14) is further improved.

  In addition, since the magnetic fine particles 9 and the sample solution are introduced into the reaction space 102 and the magnetic fine particles 9 are quickly drawn in the direction of the sensing area 101, the time for waiting for the natural precipitation of the magnetic fine particles 9 can be shortened. Measurement can be performed in a short time. In addition, the coupling between the magnetic fine particles 9 and the sensing area 101 can be promoted before the reaction or aggregation of the magnetic fine particles 9 proceeds. Thereby, since the utilization factor of HbA1c (14) with respect to the coupling | bonding of the magnetic fine particle 9 and the sensing area 101 can be raised more, higher detection sensitivity is obtained.

  Furthermore, the specimen solution and the magnetic fine particles 9 can be stirred by moving the magnetic fine particles 9 by both or one of the magnetic field applying unit 10 and the magnetic field applying unit 11. As a result, the antigen-antibody reaction between HbA1c (14) and the magnetic fine particles 9 contained in the sample solution is promoted, and high detection sensitivity can be measured in a shorter time. Further, the magnetic fine particles 9 can be further stirred by repeatedly applying the upper magnetic field by the magnetic field applying unit 10 and applying the lower magnetic field by the magnetic field applying unit 11.

  This increases the chance that the magnetic fine particles 9 are coupled to the sensing area 101 via HbA1c (14), so that HbA1c (14) can be detected in a shorter time. Further, it is possible to improve the probability that the magnetic fine particles 9 are bonded to the sensing area 101, and to improve the detection sensitivity and measurement accuracy of HbA1c (14). For example, it is effective when HbA1c (14) has a low concentration.

  In the second embodiment, since the magnetic fine particles 9 are agitated using a magnetic field, a manual agitation operation or an agitation mechanism having a pump or the like is not required, and a simple operation and a small measurement system can be realized. For example, if the application of the magnetic field by the control unit 20 is automated, measurement can be performed with only one operation in which the measurer introduces the sample solution into the sensor chip 100.

  Furthermore, if the magnetic fine particles 9 have superparamagnetism that quickly loses magnetization when the application of the magnetic field is stopped, even if the magnetic fine particles 9 aggregate due to magnetization when the magnetic field is applied, The magnetic fine particles 9 can be redispersed by stopping the application. Even if the magnetic fine particles 9 aggregate when the magnetic field is applied, by stopping the application of the magnetic field before the aggregate of the magnetic fine particles 9 reaches the vicinity of the sensing area 101, the aggregate of the magnetic fine particles 9 is regenerated. Can be dispersed. Therefore, the magnetic fine particles 9 can reach the sensing area 101 in a dispersed state. Accordingly, it is possible to prevent an increase in measurement noise due to aggregation of the magnetic fine particles 9.

  Further, in order to further improve the redispersibility when the application of the magnetic field is stopped, the surface of the magnetic fine particles 9 may have a positive or negative charge. Alternatively, a dispersant such as a surfactant may be added as a dispersion medium to the surface of the magnetic fine particles 9.

  Moreover, according to 2nd Embodiment, the detection sensitivity and measurement precision of HbA1c (14) improve by controlling the magnetic field intensity of the magnetic field application part 10 and the magnetic field application part 11 appropriately by the control part 20. FIG.

(Third embodiment)
In the first and second embodiments, the case where the optical waveguide is disposed in the natural sedimentation direction as viewed from the magnetic fine particles has been described, but in the third embodiment, the optical waveguide is disposed in a direction opposite to the natural sedimentation direction as viewed from the magnetic fine particles. The case of an existing configuration will be described.

FIG. 7 is a schematic cross-sectional view of an optical waveguide measurement system according to the third embodiment.
The optical waveguide type measurement system 32 according to the third embodiment uses a cap 15 having an enclosure shape in which liquid does not fall, instead of the frame 5 in the optical waveguide type measurement system 31 of the second embodiment. The entire optical waveguide type measurement system 31 is inverted up and down. That is, in the third embodiment, the magnetic field application unit 10 is disposed below the optical waveguide sensor chip 100 and the magnetic field application unit 11 is disposed above the optical waveguide sensor chip 100. Therefore, in 3rd Embodiment, the magnetic field application part 10 applies a lower magnetic field, and the magnetic field application part 11 applies an upper magnetic field. The magnetic field application unit 10 is not always necessary. Other configurations are the same as those of the second embodiment.

  The optical waveguide measurement system 32 includes a cap 15 having a concave cross section instead of the frame 5 in order to hold a mixed dispersion of the sample solution and the magnetic fine particles 9. The cap 15 and the sensing area 101 form a reaction space 102 that is a semi-enclosed space except for an opening for introducing liquid and an air vent hole (both not shown).

  Here, the optical waveguide measurement system 32 of the third embodiment includes the control unit 20. The control unit 20 controls the intensity of the magnetic field applied to the sensing area 101 by at least one of the magnetic field application unit 10 and the magnetic field application unit 11. In this case, independent control units may be provided for the magnetic field application unit 10 and the magnetic field application unit 11. Further, a common control unit and a changeover switch (not shown) may be provided for the magnetic field application unit 10 and the magnetic field application unit 11. In addition, a control unit that controls the magnetic field strength at the same time for the magnetic field application unit 10 and the magnetic field application unit 11 may be provided. Further, by controlling the magnetic field strength as needed, it may be controlled so as to dynamically become an appropriate magnetic field strength.

  Further, the control unit 20 may control the timing of applying the magnetic field in each of the magnetic field application unit 10 and the magnetic field application unit 11. Thereby, the magnetic field application unit 10 and the magnetic field application unit 11 can alternately apply a magnetic field to the dispersion medium 25 according to a predetermined condition (for example, a predetermined time or a time during which a predetermined magnetic field is continuously applied).

FIG. 8 is a process diagram showing a method for measuring glycated hemoglobin in a sample solution according to the third embodiment.
8A to 8C illustrate the state in the reaction space 102. FIG.

  The amount and concentration (for example, antigen concentration) of HbA1c (14) in the sample solution by measuring the difference in the detection signal intensity ratio in the light receiving element 8 is the same as in the first embodiment. The description is omitted.

  First, as shown in FIG. 8A, the reaction space 102 formed by the frame 5 and the sensing area 101 is filled with a mixed dispersion of the sample solution and the magnetic fine particles 9. The method for this is the same as the method described in the first embodiment. In addition, it is desirable that the sample solution or the like is introduced by inflow through an opening (not shown) for introducing the liquid. Here, the sample solution may contain a contaminant 17 that settles under its own weight. Examples of the contaminant 17 include blood cell components in blood. If such a contaminant 17 exists in the vicinity of the sensing area 101, it becomes a scatterer itself and causes measurement noise, or the reaction of the magnetic fine particles 9 binding to the sensing area 101 is hindered. Measurement accuracy may be reduced.

  Next, as shown in FIG. 8B, the magnetic field application unit 11 applies a magnetic field in the direction of the sensing area 101 as viewed from the magnetic fine particles 9. Thereby, the magnetic fine particles 9 are attracted to the sensing area 101. At this time, the first substance 6 (for example, primary antibody) immobilized on the sensing area 101 and the second substance 13 (for example, secondary antibody) immobilized on the surface of the magnetic microparticle 9 are HbA1c (14). It binds by antigen-antibody reaction via Thereby, the magnetic fine particles 9 are coupled to the sensing area 101. At the same time, the sedimentary contaminant 17 moves downward (in the direction opposite to the sensing area 101) in FIG.

  Next, as shown in FIG. 8C, the magnetic field applying unit 10 applies a downward magnetic field shown in FIG. Then, regardless of the antigen-antibody reaction, the magnetic fine particles 9 adsorbed on the sensing area 101 without passing through the HbA1c (14) move in the settling direction and are removed from the sensing area 101. Here, even if the application of the magnetic field in the upward direction shown in FIG. 8 (b) is simply stopped using a measurement system that does not have the magnetic field application unit 10, it does not go through HbA1c (14) regardless of the antigen-antibody reaction or the like. The magnetic fine particles 9 adsorbed on the sensing area 101 can be moved downward by their own weight.

  However, with this method, it is difficult to remove the magnetic fine particles 9 adsorbed on the sensing area 101 when the attractive force of the magnetic fine particles 9 on the sensing area 101 is superior to the downward force corresponding to its own weight. It becomes. In addition, also in the process shown in FIG. 8C, the sedimentary contaminant 17 continues to move downward (in the direction opposite to the optical waveguide 3) in FIG. 8C due to its own weight.

  Also in the third embodiment, the magnetic field application in the upward direction shown in FIG. 8B and the magnetic field application in the downward direction shown in FIG. 8C or sedimentation by the weight of the magnetic fine particles 9 may be alternately repeated. .

  Also in the third embodiment, the same effect as in the first and second embodiments can be obtained. Furthermore, according to the third embodiment, the sensing area 101 is located above the magnetic fine particles 9 and the magnetic field applying unit 11 applies a magnetic field to the magnetic fine particles 9. Therefore, at the same time as the magnetic fine particles 9 are attracted to the sensing area 101, the sedimentary contaminant 17 can be allowed to settle downward. Thereby, the contaminant 17 can be naturally moved outside the evanescent light region in the vicinity of the sensing area 101. As a result, the measurement accuracy can be further improved without removing the contaminant 17 by filtration or the like in advance.

(Fourth embodiment)
FIG. 9 is a schematic diagram of an optical waveguide measurement system according to the fourth embodiment. FIG. 9A is a schematic plan view, and FIG. 9B is along the line AB in FIG. 9A. It is a cross-sectional schematic diagram in a position.

  The cross section at the position along the line CD in FIG. 9A corresponds to the cross section of the optical waveguide measurement system 30 described above. Further, in FIG. 9A, the above-described control unit 20, magnetic field application unit 10, light source 7, and light receiving element 8 are not displayed. In FIG. 9A, the plane of the optical waveguide sensor chip is shown.

  In the optical waveguide type measurement system 300 according to the fourth embodiment, the optical waveguide type measurement system 30 described above and an optical waveguide type measurement system 35 different from the optical waveguide type measurement system 30 are provided flat. In the optical waveguide type measurement system 35, the concentration of total hemoglobin can be measured. That is, in the optical waveguide measurement system 300, the ratio of HbA1c to the total hemoglobin can be calculated in one measurement by arranging the hemoglobin antibody on one line and the HbA1c antibody on the other line. This is because it is necessary to express the concentration of HbA1c as a concentration (%) with respect to the total amount of hemoglobin, so one of the two chips is used for measuring the hemoglobin concentration and the other is used for measuring the HbA1c concentration.

  The optical waveguide type measurement system 35 according to the fourth embodiment is fixed on the optical waveguide 3 (the second optical waveguide in the fourth embodiment) and the optical waveguide 3 and can specifically bind to hemoglobin. The third substance 60, the fourth substance 130, the plurality of magnetic fine particles 9 (second magnetic fine particles in the fourth embodiment), and the dispersion medium 25 (in the fourth embodiment, such as liquid (water, organic solvent)). , A second dispersion medium). A fourth substance 130 is immobilized on each of the plurality of magnetic fine particles 9. The fourth substance 130 can specifically bind to hemoglobin at a place different from the place where the third substance 60 can specifically bind to hemoglobin. A plurality of magnetic fine particles 9 are dispersed in the dispersion medium 25, and the dispersion medium 25 is in contact with the third substance 60.

  Further, the optical waveguide type measurement system 30 is provided above the optical waveguide 3 and is capable of moving at least one of the plurality of magnetic fine particles 9 in the dispersion medium 25 by magnetic force (fourth embodiment). In the embodiment, the second magnetic field applying unit), the light source 7 (in the fourth embodiment, the second light source) capable of making light incident on the optical waveguide 3, and the light emitted from the optical waveguide 3 are received. Light receiving element 8 (in the fourth embodiment, a second light receiving element).

  In addition, the optical waveguide measurement system 35 includes a substrate 1 that supports the optical waveguide 3, gratings 2 a and 2 b (incident side grating 2 a and outgoing side grating 2 b) provided in the optical waveguide 3, and the optical waveguide 3. A protective film 4 for protecting the surface and a frame 5 provided on the optical waveguide 3 are provided. The gratings 2a and 2b are formed of a material having a higher refractive index than that of the substrate 1. The optical waveguide 3 having a flat surface is formed on the main surface of the substrate 1 including the gratings 2a and 2b. The protective film 4 is coated on the optical waveguide 3. The protective film 4 is a resin film having a low refractive index, for example. The protective film 4 is provided with an opening through which a part of the surface of the optical waveguide 3 located between the gratings 2a and 2b is exposed. The opening can be rectangular, for example, and the surface of the optical waveguide 3 exposed to the opening serves as the sensing area 201. A space surrounded by the optical waveguide 3 and the frame 5 is filled with a dispersion medium 25. A portion filled with the dispersion medium 25 may be referred to as a reaction space 202.

  In the sensing area 201, the third substance 60 is fixed on the optical waveguide 3. The sensing area 201 is exposed from the protective film 4. The frame 5 is formed on the protective film 4 so as to surround the sensing area 201. In the optical waveguide measurement system 35, a configuration including the optical waveguide 3, the third substance 60, and the plurality of magnetic fine particles 9 on which the fourth substance 130 is immobilized is referred to as an optical waveguide sensor chip 200. . The optical waveguide sensor chip that combines the optical waveguide sensor chip 100 and the optical waveguide sensor chip 200 is free to carry. Further, in the optical waveguide type measurement system 35, each of the light source 7, the light receiving element 8, and the magnetic field application unit 10 is controlled by the control unit 20.

  Further, in the sensing area 201 that is the detection surface, the third substance 60 is immobilized by, for example, hydrophobic interaction or covalent bond with the surface of the optical waveguide 3 in the sensing area 201. For example, the third substance 60 is fixed to the sensing area 201 by a hydrophobic treatment with a silane coupling agent. Alternatively, a functional group may be formed in the sensing area 201, and an appropriate linker molecule may be allowed to act to be immobilized by chemical bonding. As the third substance 60, for example, when hemoglobin in the sample solution is an antigen, its antibody (primary antibody) can be used.

  The fourth substance 130 that specifically reacts with hemoglobin in the sample solution is immobilized on the surface of the magnetic fine particle 9 by, for example, physical adsorption or chemical bonding via a carboxyl group, an amino group, or the like. The magnetic fine particles 9 on which the fourth substance 130 is immobilized are dispersed and held in the sensing area 201 on which the third substance 60 is immobilized. The dispersion and holding of the magnetic fine particles 9 are formed by, for example, applying and drying a slurry containing the magnetic fine particles 9 and a water-soluble substance on the sensing area 201 or a surface (not shown) facing the sensing area 201. The Alternatively, the magnetic fine particles 9 may be dispersed in a liquid and held in a space different from the reaction space 202 or in a container (not shown).

  As the third substance 60 and the fourth substance 130, since all hemoglobin has an α subunit in common, each of the third substance 60 and the fourth substance 130 is a monoclonal antibody in which the α subunit of hemoglobin is an antigen. It is desirable to be. For example, the third substance 60 is a monoclonal antibody in which the α subunit of hemoglobin is an antigen. For example, the fourth substance 130 is a monoclonal antibody in which an α subunit other than the α subunit of hemoglobin to which the third substance 60 specifically reacts becomes an antigen. That is, the third substance 60 and the fourth substance 130 have different hemoglobin α subunits as antigens.

  The optical waveguide measurement system 35 may include the magnetic field application unit 11 described above in addition to the magnetic field application unit 10. Furthermore, the optical waveguide measurement system 35 may be a system in which the top and bottom are inverted as shown in FIG. Further, instead of the optical waveguide type measurement system 30, any one of the optical waveguide type measurement systems 31 and 32 may be used.

  According to such an optical waveguide type measurement system 300, the same effect as in the first to third embodiments can be obtained, and the ratio of HbA1c to total hemoglobin can be quickly calculated by one measurement.

  The embodiment has been described above with reference to specific examples. However, the embodiments are not limited to these specific examples. In other words, those specific examples that have been appropriately modified by those skilled in the art are also included in the scope of the embodiments as long as they include the features of the embodiments. Each element included in each of the specific examples described above and their arrangement, material, condition, shape, size, and the like are not limited to those illustrated, and can be appropriately changed.

  In addition, each element included in each of the above-described embodiments can be combined as long as technically possible, and combinations thereof are also included in the scope of the embodiment as long as they include the features of the embodiment. In addition, in the category of the idea of the embodiment, those skilled in the art can conceive various changes and modifications, and it is understood that these changes and modifications also belong to the scope of the embodiment. .

  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 novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the scope of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are included in the invention described in the claims and the equivalents thereof.

  1 substrate, 2a, 2b grating, 3 optical waveguide, 4 protective film, 5 frame, 6 first material, 7 light source (light emitting diode), 8 light receiving element (photodiode), 9 magnetic fine particle, 9a magnetic nano fine particle, 9b high Molecular material, 9c core, 9d shell, 10 magnetic field application unit, 13 second material, 14 HbA1c, 15 cap, 17 contaminated material, 20 control unit, 25, 26 dispersion medium, 30, 31, 32, 35, 300 Optical waveguide Type measurement system, 60 third substance, 100, 200 optical waveguide sensor chip, 101, 201 sensing area, 102, 202 reaction space, 130 fourth substance

Claims (19)

A first optical waveguide having a first sensing area on which a first substance capable of specifically binding to glycated hemoglobin is immobilized;
Superparamagnetism in which the second substance capable of specifically binding to the glycated hemoglobin is immobilized at a location different from the location where the first substance can specifically bind to the glycated hemoglobin A plurality of first magnetic fine particles;
A first magnetic field application unit provided above the first optical waveguide and capable of moving at least one of the plurality of first magnetic fine particles from the first sensing area by a magnetic force;
A first light source capable of making light incident on the first optical waveguide and generating near-field light in the first sensing area;
A first light receiving element capable of receiving light emitted from the first optical waveguide;
An optical waveguide type measuring system.   2. The optical waveguide measurement system according to claim 1, wherein the first magnetic fine particles include a plurality of fine particles having superparamagnetism.   The optical waveguide type measurement system according to claim 1, wherein the first magnetic fine particles include a plurality of fine particles having superparamagnetism and a material covering them.   2. The optical waveguide type measurement system according to claim 1, wherein the first magnetic fine particles include a plurality of fine particles having superparamagnetism and a polymer material covering them. The first magnetic fine particles have a core and a shell covering the core,
2. The optical waveguide measurement system according to claim 1, wherein the shell includes a plurality of superparamagnetic fine particles.   6. The optical waveguide measurement system according to 5, wherein a refractive index of the superparamagnetic fine particles is higher than a refractive index of the core.   7. The optical waveguide measurement system according to claim 5, wherein the core is made of a polymer material.   The optical waveguide measurement system according to claim 5, wherein the shell is made of a polymer material.   The optical waveguide type measurement system according to any one of claims 1 to 8, wherein the glycated hemoglobin is HbA1c (hemoglobin A1C) in which glucose (Glc) is bonded to the N-terminal of the β chain of HbA1 (hemoglobin A1).   The optical waveguide measurement system according to claim 9, wherein the first substance is a monoclonal antibody whose antigen is a glycated peptide at the N-terminal of the β chain of HbA1c.   The optical waveguide type according to claim 10, wherein the second substance is a monoclonal antibody whose antigen is the HbA1c other than the glycated peptide at the N-terminal of the β chain of the HbA1c or the β subunit of the HbAlc other than the glycated peptide. Measuring system.   The optical waveguide measurement system according to claim 10 or 11, wherein the glycated peptide is a fructosyl peptide.   The first magnetic field application unit can apply a magnetic field that moves at least one of the plurality of magnetic fine particles in a direction away from the first optical waveguide. Optical waveguide measurement system. A second magnetic field application unit below the first optical waveguide;
The said 2nd magnetic field application part can apply the magnetic field which moves at least 1 of these magnetic fine particles in the direction approached to the said 1st optical waveguide, It is any one of Claims 1-13 Optical waveguide measurement system.   The optical waveguide measurement system according to claim 14, wherein the first magnetic field application unit and the second magnetic field application unit can alternately apply a magnetic field to the dispersion medium. An optical waveguide type measurement system according to any one of claims 1 to 15,
A second optical waveguide having a second sensing area on which a third substance capable of specifically binding to hemoglobin is immobilized;
A plurality of superparamagnets having immobilized thereon a fourth substance capable of specifically binding to the hemoglobin at a location different from a location where the third substance can specifically bind to the hemoglobin. Second magnetic fine particles;
A second magnetic field application unit capable of moving at least one of the plurality of magnetic fine particles from the second sensing area by a magnetic force;
A second light source capable of making light incident on the second optical waveguide and generating near-field light in the second sensing area;
A second light receiving element capable of receiving light emitted from the second optical waveguide;
Another optical waveguide type measurement system having
An optical waveguide type measurement system.   The optical waveguide type measurement system according to claim 16, wherein the third substance and the fourth substance are monoclonal antibodies in which the α subunits of the different hemoglobins are antigens.   The optical waveguide measurement system according to claim 16 or 17, wherein the optical waveguide measurement system and the another optical waveguide measurement system are installed flat. The first substance can specifically bind to glycated hemoglobin, is immobilized on the sensing area on the first optical waveguide,
The second substance can specifically bind to the glycated hemoglobin at a location different from the location where the first substance can specifically bind to the glycated hemoglobin, and has a plurality of superparamagnetic properties. Of the first magnetic fine particles,
In the first dispersion medium, the plurality of first magnetic fine particles on which the second substance is immobilized are dispersed, and further, the glycated hemoglobin is mixed,
Contacting the first dispersion medium with the first material;
A step of generating near-field light in the sensing area in a state where the first dispersion medium is in contact with the first substance, and measuring a light intensity of light emitted from the first optical waveguide as a first light intensity; ,
Applying a magnetic field to the first dispersion medium;
After applying the magnetic field, generating near-field light in the sensing area and measuring the light intensity of light emitted from the first optical waveguide as a second light intensity;
Quantifying the glycated hemoglobin based on a difference between the first light intensity and the second light intensity;
A method for measuring glycated hemoglobin, comprising:

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