JP5766642B2 - Measuring method, sensor chip, and measuring apparatus - Google Patents

Description

  Embodiments described below generally relate to a measurement method, a sensor chip, and a measurement apparatus.

As a sensor chip used in a measuring device for measuring the amount of a substance contained in a sample solution, there is an optical waveguide type sensor chip. In the measurement method using the optical waveguide sensor chip, the amount of the measurement target substance is measured based on the change in absorbance caused by the measurement target substance on the surface of the detection unit.
A sensor chip has been proposed in which a plurality of holding units for holding a sample solution are provided for each detection unit so that highly accurate measurement can be performed even if the amount of reagent is reduced.
However, there is room for improvement with respect to accurately measuring a plurality of types of substances to be measured.

JP 2003-240704 A

  The problem to be solved by the present invention is to provide a measurement method, a sensor chip, and a measurement apparatus that can measure a plurality of types of substances to be measured with high accuracy.

The measurement method according to the embodiment includes a light-transmitting substrate, an optical element portion provided on the substrate, an optical waveguide portion provided on the substrate and the optical element portion, and the optical waveguide. A holding portion having a holding space for holding the sample solution and at least one particle put into the holding space, wherein the optical waveguide portion is unique to the first measurement target substance. The first region in which the first substance that reacts specifically is fixed and the second measurement target substance different from the first measurement target substance specifically reacts with the first region different from the first substance . A second region to which two substances are fixed, the holding unit integrally surrounds the first region and the second region, the particle has a base, and On the surface, a third substance that specifically reacts with the first substance to be measured, and Serial least one of a fourth substance which reacts second analyte and specifically is a measuring method using the sensor chip is fixed.
The measuring method includes a step of introducing a sample solution into the holding space of the holding unit, a step of obtaining an absorbance after the sample solution is introduced, and a step of introducing the sample solution into the holding space of the holding unit. A step of introducing at least one particle, a step of obtaining an absorbance after introducing the at least one particle, an absorbance after introducing the sample solution, and an absorbance after introducing the at least one particle; And a step of obtaining the amount of change in absorbance based on the difference in absorbance and a step of obtaining the amount of the substance to be measured based on the obtained amount of change in absorbance.

(A) is a schematic cross-sectional view of the sensor chip according to the first embodiment, FIG. 1 (b) is a cross-sectional view taken along the line AA in FIG. 1 (a), and FIG. 1 (c) is in FIG. It is BB arrow sectional drawing. FIG. 2A is a schematic diagram for illustrating the particles 9 on which the third substance and the fourth substance are fixed, and FIG. 2B is a schematic diagram for illustrating the particles 9a on which the third substance is fixed. FIG. 2C is a schematic diagram for illustrating the particle 9b on which the fourth substance is fixed. (A), (b) is a schematic cross section for illustrating the base containing a magnetic material. (A) is a schematic diagram for illustrating the measuring apparatus according to the second embodiment, and FIG. 4 (b) is a cross-sectional view taken along the line CC in FIG. 4 (a). (A)-(c) is a schematic process diagram for illustrating about the effect | action of the control part 35. FIG. It is a flowchart for demonstrating about the measuring method which concerns on 3rd Embodiment. (A) is a schematic cross-sectional view of the sensor chip according to the fourth embodiment, (b) is a cross-sectional view taken along the line DD in (a), and (c) is a cross-sectional view taken along the line EE in (a). is there. It is a flowchart for illustrating about the measuring method in the case of using the sensor chip 1a.

Hereinafter, embodiments will be illustrated with reference to the drawings. In addition, in each drawing, the same code | symbol is attached | subjected to the same component and detailed description is abbreviate | omitted suitably.
[First embodiment]
1A is a schematic cross-sectional view of the sensor chip according to the first embodiment, FIG. 1B is a cross-sectional view taken along the line AA in FIG. 1A, and FIG. 1C is FIG. It is a BB arrow sectional view in).
As shown in FIGS. 1A to 1C, the sensor chip 1 is provided with a substrate 2, an optical waveguide unit 3, an optical element unit 4, a detection unit 5, a holding unit 6, and a protection unit 7.

The board | substrate 2 exhibits flat form and is formed from the translucent material. The substrate 2 can be formed from, for example, glass (for example, alkali-free glass) or quartz.
The optical waveguide portion 3 has a higher refractive index than the substrate 2 and is provided on the substrate 2 and the optical element portion 4. The optical waveguide portion 3 is formed of a polymer resin or the like, and can be a film-like body having a substantially uniform thickness of about 3 μm to 300 μm.
Note that the materials and dimensions of the substrate 2 and the optical waveguide portion 3 are not limited to those illustrated, and can be changed as appropriate.

  The optical element portion 4 is provided on the main surface of the substrate 2 on the side where the optical waveguide portion 3 is provided. That is, the optical element unit 4 is provided on the substrate 2. The optical element part 4 illustrated in FIG. 1A functions as a diffraction grating when light is introduced into and emitted from the optical waveguide part 3. Therefore, the optical element portion 4 has a higher refractive index than the substrate 2 and is provided in a lattice shape with a predetermined pitch dimension. For example, the optical element portion 4 may be provided in a lattice shape with a pitch dimension of 1 μm. However, the pitch dimension is not limited to this, and can be changed as appropriate.

The optical element unit 4 may be formed of, for example, titanium oxide (TiO ₂ ), tin oxide (SnO ₂ ), zinc oxide, lithium niobate, gallium arsenide (GaAs), indium tin oxide (ITO), polyimide, or the like. it can. In addition, although the optical element part 4 which functions as a diffraction grating was illustrated, the optical element which can introduce light into the optical waveguide part 3 can be selected suitably. For example, the optical element unit 4 may function as a prism when light is introduced into and emitted from the optical waveguide unit 3.

The detection unit 5 includes a detection unit 5a provided in the first region 3a and a detection unit 5b provided in the second region 3b.
The detection units 5a and 5b are made of a substance that specifically reacts with the measurement target substance in the sample solution.
In this case, the first substance 5a fixed to the first region 3a and the second substance 5b fixed to the second region 3b can be different or the same. it can. The first substance 5a and the second substance 5b can be antibodies (primary antibodies), for example, when the substance to be measured in the sample solution is an antigen.
In the following, as an example, a case where the first substance 5a and the second substance 5b are different is illustrated.

The first region 3 a is a part of the main surface 3 c of the optical waveguide portion 3 exposed by the hole 7 a that penetrates the protection portion 7. The second region 3 b is a part of the main surface 3 c of the optical waveguide portion 3 exposed by the hole 7 b that penetrates the protection portion 7.
The first substance 5a is fixed to the first region 3a. The second substance 5b is fixed to the second region 3b.

That is, a first substance 5a that specifically reacts with a measurement target substance 14a (corresponding to an example of a first measurement target substance) described later is fixed to the first region 3a. A second substance 5b that specifically reacts with a measurement target substance 14b (corresponding to an example of a second measurement target substance) to be described later is fixed to the second region 3b.
The first substance 5a and the second substance 5b are fixed by, for example, hydrophobic interaction or chemical bonding between the first substance 5a and the second substance 5b and the main surface 3c of the optical waveguide section 3. Can do.

  For example, the first substance 5 a and the second substance 5 b can be fixed to the main surface 3 c of the optical waveguide portion 3 by performing a hydrophobic treatment with a silane coupling agent. Alternatively, a functional group may be formed on the main surface 3c of the first optical waveguide section 3, and a known linker molecule may be allowed to act to fix the first substance 5a and the second substance 5b by chemical bonding.

  1A to 1C exemplify the case where two regions are provided and different substances are fixed in each of the two regions, the present invention is not limited to this. For example, three or more areas are provided, and different substances can be fixed in each of the three or more areas, or three or more areas can be divided into a plurality of groups and different substances can be fixed in each group. it can.

The holding unit 6 is provided on the upper side of the optical waveguide unit 3 (on the side opposite to the side on which the substrate 2 is provided) and has a holding space (reaction space) 6a for holding the sample solution.
The holding part 6 has a frame shape and is provided so as to surround the first region 3a and the second region 3b. One end of the holding part 6 is provided in a liquid-tight manner on the main surface of the protection part 7, and the other end is provided at a position away from the main surface of the protection part 7. Therefore, the sample solution can be held in the holding space 6 a inside the holding unit 6.

The holding unit 6 is made of a material having high resistance to the sample solution held in the holding space 6a. The holding part 6 can be formed from, for example, a fluororesin.
The holding part 6 may be joined to the protection part 7 or may be formed integrally with the protection part 7.

  Here, the holding space 6a is a space common to the first substance 5a provided in the first region 3a and the second substance 5b provided in the second region 3b. Therefore, the volume of the first substance 5a can be secured twice as much as the case where the specimen solution is held for each substance. In addition, the volume of the second substance 5b can be doubled as compared with the case where the specimen solution is held for each substance.

Therefore, the amount of the measurement target substance 14a to be reacted with the first substance 5a and the amount of the measurement target substance 14b to be reacted with the second substance 5b are increased as compared with the case where the specimen solution is held for each substance. Can do. As a result, a plurality of types of measurement target substances can be measured with high accuracy.
Further, the size of the sensor chip 1 does not increase compared to the case where the specimen solution is held for each substance.

The protection part 7 is provided on the main surface 3 c of the optical waveguide part 3.
A first hole portion 7 a and a second hole portion 7 b that pass through the protection portion 7 are provided in a portion located inside the holding portion 6 of the protection portion 7.
The first hole portion 7a and the second hole portion 7b define a first area 3a and a second area 3b, respectively.

  The shape and size of the first hole portion 7a and the second hole portion 7b are not particularly limited, but the length in the traveling direction of the light propagating through the optical waveguide portion 3 is increased. Is preferred. If the length in the traveling direction of the light propagating through the inside of the optical waveguide portion 3 is increased, the number of places where near-field light such as an evanescent wave (described later) is generated can be increased. Therefore, measurement sensitivity can be improved.

  Further, a separation portion 7c can be provided between the first hole portion 7a and the second hole portion 7b. In this case, the separation part 7c is not necessarily provided. For example, the first hole 7a and the second hole 7b may be one hole, and the first region 3a and the second region 3b may be provided inside one hole. However, if the separation part 7c is provided, the workability at the time of sequentially fixing the first substance 5a and the second substance 5b can be improved.

  The protection part 7 is made of a material having a lower refractive index than the material forming the optical waveguide part 3. Further, the protection part 7 is formed of a material having high resistance to the sample solution held in the holding space 6a. The protection part 7 can be formed from a fluororesin or the like, for example.

Further, when the measurement is performed using the sensor chip 1, the particles 9, 9a, 9b are used.
As will be described later, a plurality of particles 9, 9a, 9b are charged into the holding space 6a.
FIG. 2A is a schematic diagram for illustrating the particles 9 on which the third substance and the fourth substance are fixed, and FIG. 2B is a diagram for illustrating the particles 9a on which the third substance is fixed. FIG. 2C is a schematic diagram for illustrating the particle 9b to which the fourth substance is fixed.

As shown in FIG. 2A, the particle 9 includes a granular base 12 and a third substance 13 a and a fourth substance 13 b fixed to the surface of the base 12.
The third substance 13a and the fourth substance 13b are different.
For example, when the substance to be measured in the sample solution is an antigen, the third substance 13a and the fourth substance 13b can be antibodies (secondary antibodies).
The fixation of the third substance 13a and the fourth substance 13b can be performed, for example, by physical adsorption or chemical bonding via a carboxyl group, an amino group, or the like.

In addition, as shown in FIG. 2B, only the third substance 13a may be a particle 9a fixed on the surface of the base 12.
In addition, as shown in FIG. 2C, only the fourth substance 13b may be a particle 9b fixed on the surface of the base 12.

That is, the particles have a base 12, and the surface of the base 12 specifically reacts with a third substance 13 a that specifically reacts with a measurement target substance 14 a described later and a measurement target substance 14 b described below. At least one of the fourth substances 13b is fixed.
In this case, the first substance 5a and the third substance 13a react specifically with the measurement target substance 14a, and the second substance 5b and the fourth substance 13b react specifically with the measurement target substance 14b.

  If only the third substance 13a is the particle 9a fixed on the surface of the base 12, or only the fourth substance 13b is the particle 9b fixed on the surface of the base 12, only the measurement target substance to be measured is obtained. Since it can be made to react, it becomes possible to perform a highly accurate measurement.

  On the other hand, if the third substance and the fourth substance are the particles 9 fixed on the surface of the base portion 12, the number of particles 9 introduced into the specimen solution can be reduced. For example, when measuring two types of substances to be measured, it is necessary to introduce the particles 9a and 9b into the sample solution. In this case, since the ratio of the two types of substances to be measured cannot be known in advance, the introduction amount of at least one of the particles 9a and the particles 9b may be excessive. On the other hand, if the third substance and the fourth substance are the particles 9 fixed on the surface of the base portion 12, they are automatically allocated according to the ratio of the two types of measurement target substances. Therefore, the number of particles 9 introduced into the sample solution can be reduced.

In this case, a first substance 5a (for example, a primary antibody) fixed to the first region 3a or a second substance 5b (for example, a primary antibody) fixed to the second region 3b, and the base 12 The third substance 13a (for example, a secondary antibody) or the fourth substance 13b (for example, a secondary antibody) immobilized on the surface is an antigen antibody via a measurement target substance (for example, an antigen) that matches each of them. Bind by reaction. Thereby, the particles 9, 9a, 9b are bound to the first region 3a or the second region 3b by the antigen-antibody reaction.
In addition, although the case where the 3rd substance 13a and the 4th substance 13b differ was illustrated, the 3rd substance 13a and the 4th substance 13b can be made the same.

The diameter dimension of the base 12 can be 0.05 μm or more and 200 μm or less. In this case, if the diameter of the base 12 is 0.2 μm or more and 20 μm or less, the light scattering efficiency can be increased. For this reason, it is possible to perform highly accurate measurement.
The base 12 can be formed from, for example, a polymer material.
Further, the base 12 may include a magnetic material.

FIGS. 3A and 3B are schematic cross-sectional views for illustrating a base including a magnetic material.
As shown in FIG. 3A, the base 12b may have a configuration in which the magnetic nanoparticles 12a are wrapped with a polymer material.
Moreover, as shown in FIG.3 (b), the base 12d shall have the core 12c and the shell 12e provided so that the core 12c might be covered.
The core 12c can be formed from a polymer material. The shell 12e is formed of a polymer material and can include the magnetic nanoparticle 12a.

Alternatively, the base may be a particle itself made of a magnetic material.
Examples of the magnetic material include various ferrites such as γ-Fe2O3. 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 nm or less. On the other hand, in order for light scattering to occur, the size of the particles needs to be several hundred nm or more. Therefore, as the particles 9, 9 a, 9 b, for example, the magnetic nanoparticles 12 a illustrated in FIGS. 3A and 3B are preferably covered with a polymer material or the like.

In addition, the refractive index of polymer materials is often about 1.5 to 1.6, and the refractive index of ferrites is about 3.0. In this case, when the particles 9, 9 a, 9 b are near the surface of the optical waveguide portion 3, the higher the refractive index, the easier it is to scatter light.
Therefore, as illustrated in FIG. 3B, if the magnetic nanoparticle 12a is provided near the surface of the base 12d, the sensitivity of measurement can be improved.

  In this case, as illustrated in FIG. 3A, if the magnetic nanoparticle 12a is simply a base 12b wrapped with a polymer material, the magnetic nanoparticle 12a may be dispersed throughout the base 12b. Therefore, in order to improve the sensitivity of the measurement, as illustrated in FIG. 3B, the core-shell type base 12d is formed, and the magnetic nanoparticles 12a are included in the shell 12e at a high density. It is preferable.

Note that the combination of the substance to be measured and the first substance 5a, the second substance 5b, the third substance 13a, and the fourth substance 13b is not limited to a combination of an antigen and an antibody.
For example, when the measurement target substance is sugar, the first substance 5a to the fourth substance 13b can be lectins. When the substance to be measured is a nucleotide chain, the first substance 5a to the fourth substance 13b can be complementary nucleotide chains. When the measurement target substance is a ligand, the first substance 5a to the fourth substance 13b can be receptors for the first substance 5a to the fourth substance 13b.

[Second Embodiment]
Next, the measurement apparatus according to the second embodiment is illustrated.
FIG. 4A is a schematic view for illustrating the measuring apparatus according to the second embodiment, and FIG. 4B is a cross-sectional view taken along the line CC in FIG.
In the present embodiment, the particles 9 illustrated in FIG. 2A are used. Further, the base 12 of the particle 9 includes a magnetic material.

As shown in FIG. 4, the measuring device 30 includes a sensor chip 1, a light projecting unit 31, a light receiving unit 32, a magnetic field application unit 33, a magnetic field application unit 34, a control unit 35, and a calculation unit 36.
The light projecting unit 31 emits light toward the sensor chip 1. The light projecting unit 31 can be, for example, a light emitting diode that emits light having a center wavelength of about 635 nm.
The light receiving unit 32 receives the light from the sensor chip 1 and converts it into an electrical signal corresponding to the intensity of the light. The light receiving unit 32 can be, for example, a photodiode.
A pair of light projecting unit 31 and light receiving unit 32 is provided for each of the first region 3a and the second region 3b. For this reason, it is possible to determine the intensity of light, and thus the amount and concentration (for example, antigen concentration, etc.) of the substance to be measured for each region.

The magnetic field application units 33 and 34 generate a magnetic field and apply the generated magnetic field to the sensor chip 1 to change the position of the particle 9 according to the magnetic field.
The magnetic field application units 33 and 34 are not necessarily required, but if at least one of the magnetic field application unit 33 and the magnetic field application unit 34 is provided, the measurement accuracy can be improved. Details regarding the operation of the magnetic field application units 33 and 34 and the effect of providing the magnetic field application units 33 and 34 will be described later.

The magnetic field application unit 33 is provided on the side opposite to the side where the optical waveguide unit 3 is present when viewed from the particle 9.
For example, in the case of the measuring device 30, the magnetic field application unit 33 is provided in the upward direction of the sensor chip 1.
The magnetic field application unit 34 is provided on the side where the optical waveguide unit 3 is present when viewed from the particle 9.
For example, in the case of the measuring apparatus 30, the magnetic field application unit 34 is provided in the lower direction of the sensor chip 1.
The “upward direction” is the upward direction in the gravity direction, and the “downward direction” is the downward direction in the gravity direction.
In this case, the magnetic field application unit 33 can move the particles 9 in a direction away from the optical waveguide unit 3, and the magnetic field application unit 34 can move the particles 9 in a direction closer to the optical waveguide unit 3.

The magnetic field application units 33 and 34 may have, for example, permanent magnets or electromagnets.
When the magnetic field application units 33 and 34 have permanent magnets such as ferrite magnets, the magnetic field strength can be adjusted by changing the magnetic force of the permanent magnets or the distance from the sensor chip 1.

  In this case, the distance between the permanent magnet and the sensor chip 1 is such that the thickness dimension of the spacer provided between the permanent magnet and the sensor chip 1 is changed, or an actuator such as a linear motor is used. It can be changed by changing the position relative to 1.

Further, in the case where the magnetic field application units 33 and 34 have electromagnets, the magnetic field strength can be adjusted by changing the value of the current flowing through the coil.
In this case, in order to dynamically adjust the magnetic field strength, it is preferable to adjust the magnetic field strength by an electric current value using an electromagnet.
Further, if the magnetic field application units 33 and 34 have electromagnets, the magnetic field can be applied in a pulsed manner.
In addition, what was illustrated to Fig.4 (a) is a case where the magnetic field application parts 33 and 34 have an electromagnet.

  The control unit 35 controls the magnetic field strength of the magnetic field applied by both or one of the magnetic field application unit 33 and the magnetic field application unit 34. In this case, for example, as shown in FIG. 4A, a common control unit 35 and a changeover switch 35 a can be provided for the magnetic field application unit 33 and the magnetic field application unit 34. Independent control units may be provided for the magnetic field application unit 33 and the magnetic field application unit 34, respectively. In addition, a control unit that controls the magnetic field strength at the same time for the magnetic field application unit 33 and the magnetic field application unit 34 may be provided. Alternatively, the control unit 35 may be configured to control the magnetic field strength as needed so that the magnetic field strength is dynamically adjusted to an appropriate magnetic field strength.

Further, the control unit 35 may control the timing of applying the magnetic field in each of the magnetic field application unit 33 and the magnetic field application unit 34. For example, the magnetic field application unit 33 and the magnetic field application unit 34 may 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).
In addition, although the case where the magnetic field application part 33 and the magnetic field application part 34 were provided was illustrated, either one may be provided.

The calculating unit 36 calculates the amount of the measurement target substance based on the output from the light receiving unit 32. For example, the calculation unit 36 calculates the absorbance from the relationship between the output from the light receiving unit 32 and the absorbance obtained in advance through experiments and the output from the light receiving unit 32.
When calculating the absorbance change amount in the calculation unit 36, first, the absorbance immediately after the introduction of the sample solution is calculated. Next, a magnetic field is applied by the magnetic field application unit 33, and the particles 9 adsorbed on the first region 3a and the second region 3b are moved upward without passing through the measurement target substance. Then, the absorbance after moving the particles 9 upward is calculated. Next, the absorbance change amount is calculated from the difference between the absorbance immediately after the sample solution is introduced and the absorbance after the particles 9 are moved upward.
Then, the calculation unit 36 calculates the amount of the measurement target substance from the relationship between the amount of the measurement target substance and the absorbance change amount obtained in advance by performing an experiment or the like, and the calculated absorbance change amount. At this time, the calculation unit 36 can also calculate the concentration (for example, antigen concentration) of the measurement target substance in the sample solution.
Further, when the first substance 5a and the second substance 5b are different and the third substance 13a and the fourth substance 13b are different, it is possible to measure the amounts of different kinds of measurement target substances at a time. it can.
In addition, when the first substance 5a and the second substance 5b are the same, and the third substance 13a and the fourth substance 13b are the same, the amount of one kind of measurement target substance is determined at a time. Multiple measurements can be made. Therefore, an average value such as the amount of the substance to be measured can be measured at a time.

Next, the operation of the measurement apparatus 30 according to the second embodiment will be illustrated.
Light emitted from the light projecting unit 31 and incident on the incident-side optical element unit 4 through the substrate 2 is diffracted by the optical element unit 4 and propagates while reflecting inside the optical waveguide unit 3. Near-field light such as evanescent waves is generated in the first region 3a and the second region 3b by the light propagating while reflecting the inside of the optical waveguide portion 3. Near-field light is generated at the interface when the light is totally reflected at the interface between the optical waveguide section 3 and the holding space 6a. The distance that the near-field light reaches (seepage distance) is about a fraction of the wavelength from the surface of the optical waveguide portion 3 (the surfaces of the first region 3a and the second region 3b).

  Near-field light is absorbed or scattered by the particles 9 in the vicinity of the surfaces of the first region 3a and the second region 3b. At this time, near-field light is absorbed and scattered according to the amount of the particles 9.

Thereafter, the light is diffracted by the optical element section 4 on the emission side and emitted toward the outside. The light emitted toward the outside is received by the light receiving unit 32 and converted into an electrical signal corresponding to the intensity of the light.
These steps are performed for each of the first region 3a and the second region 3b.

  The output from the light receiving unit 32 is input to the calculation unit 36, and the amount of the measurement target substance and the like are calculated for each of the first region 3a and the second region 3b.

Next, the operation of the control unit 35 will be further illustrated.
5A to 5C are schematic process diagrams for illustrating the operation of the control unit 35. FIG. In FIGS. 5A to 5C, the state in the holding space 6a is represented in order to avoid complication.
First, as shown in FIG. 5A, the specimen solution is introduced into the holding space 6a, and the particles 9 are put into the specimen solution. Note that the sample solution includes a measurement target substance 14a (for example, a substance derived from influenza A virus) and a measurement target substance 14b (for example, a substance derived from influenza B virus).

It is also possible to introduce the particles 9 into the specimen solution and introduce it into the holding space 6a. However, in order to further improve the measurement accuracy, it is preferable to introduce the sample solution first and then introduce the particles 9. If the particles 9 are put into the sample solution, there is a possibility that a plurality of measurement target substances 14a and 14b are bound to one particle 9 due to an antigen-antibody reaction. Therefore, the sample substance is introduced first, the measurement target substance 14a is bound to the first substance 5a fixed to the first area 3a by the antigen-antibody reaction, and the second substance fixed to the second area 3b is obtained. The substance 14b to be measured is bound to 5b by an antigen-antibody reaction. After that, if the particles 9 are put into the specimen solution, one particle 9 can be bound to one measurement target substance 14a bound to the first substance 5a by the antigen-antibody reaction by the antigen-antibody reaction. . Similarly, one particle 9 can be bound to one measurement target substance 14b bound to the second substance 5b by antigen-antibody reaction by antigen-antibody reaction.
Therefore, the accuracy of measurement can be further improved.

  Next, as shown in FIG. 5 (b), the particles 9 settle (sedimentation) toward the first region 3a and the second region 3b by gravity. At this time, for example, the first substance 5a fixed to the first region 3a and the third substance 13a fixed to the surface of the base 12 are bound by the antigen-antibody reaction via the measurement target substance 14a. Further, for example, the second substance 5b fixed to the second region 3b and the fourth substance 13b fixed to the surface of the base 12 are bound by the antigen-antibody reaction via the measurement target substance 14b. Thereby, a part of particle | grains 9 is couple | bonded with the 1st area | region 3a and the 2nd area | region 3b by antigen antibody reaction.

  Note that the control unit 35 may control the magnetic field application unit 34 to attract the particles 9 downward. In this case, the particles 9 are attracted to the first region 3 a and the second region 3 b by natural sedimentation due to gravity and suction by the magnetic field application unit 34. Therefore, the measurement time can be shortened.

  Next, as shown in FIG. 5C, the magnetic field application unit 33 is controlled by the control unit 35 to attract the particles 9 upward. Then, the particles 9 adsorbed on the first region 3a and the second region 3b are moved upward without passing through the measurement target substances 14a and 14b. That is, the particles 9 simply adsorbed on the first region 3a and the second region 3b (particles 9 that may cause noise during measurement) are removed.

  At this time, by setting the magnetic field intensity applied by the magnetic field application unit 33 to an appropriate value, the antigen-antibody reaction couples to the first region 3a and the second region 3b via the measurement target substances 14a and 14b. The particles 9 are not peeled off, but only the adsorbed particles 9 are removed.

Further, the control unit 35 can control the magnetic field application units 33 and 34 to alternately and repeatedly apply a magnetic field that attracts the particles 9 upward and a magnetic field that attracts the particles 9 downward.
If the magnetic field that attracts the particles 9 upward and the magnetic field that attracts the particles 9 downward are applied alternately, the amount of movement of the particles 9 can be increased. Can be increased. Therefore, measurement sensitivity can be improved.

  In the present embodiment, since the particles 9 are moved using a magnetic field, a manual stirring operation or a stirring mechanism having a pump or the like is not required, and a simple and small measuring apparatus can be realized. For example, if the application of the magnetic field by the control unit 35 is automated, the measurement can be performed by only one operation in which the measurer introduces the sample solution into the sensor chip 1.

  Furthermore, if particles 9 having a base including a superparamagnetic material that quickly loses magnetization when application of the magnetic field is stopped are used, even if the particles 9 aggregate due to magnetization when the magnetic field is applied, the magnetic field It can be redispersed by stopping the application of.

  Even if the particles 9 aggregate when the magnetic field is applied, the application of the magnetic field is stopped before the aggregates of the particles 9 reach the vicinity of the first region 3a and the second region 3b. Aggregates of 9 can be redispersed. Therefore, the particles 9 can reach the first region 3a and the second region 3b in a dispersed state. Accordingly, it is possible to prevent an increase in measurement noise due to aggregation of the particles 9.

  Further, in order to further improve the redispersibility when the application of the magnetic field is stopped, the surface of the base 12 of the particle 9 may have a positive or negative charge. Alternatively, a dispersing agent such as a surfactant can be added to the sample solution.

[Third embodiment]
Next, a measurement method according to the third embodiment will be exemplified.
The measurement method according to the third embodiment can be performed using the measurement apparatus 30 described above. FIG. 6 is a flowchart for illustrating the measurement method according to the third embodiment.

First, the sample solution is introduced into the holding space 6a of the holding unit 6 (step S1).
Next, the absorbance after the sample solution is introduced is determined (step S2).
The absorbance after the sample solution is introduced can be calculated based on the output from the light receiving unit 32 as described above.
In addition, the light absorbency calculated | required in step S2 is an light absorbency in an initial state (state before the particle | grains 9 couple | bond with the 1st area | region 3a or the 2nd area | region 3b).
Therefore, the “absorbance after introducing the sample solution” in step S2 is not only the absorbance before the at least one particle 9 is introduced, but also the absorbance immediately after the at least one particle 9 is introduced in step S3. Is also included.
In addition, in FIG. 6, the case where the light absorbency before at least 1 particle | grain 9 is thrown in is illustrated as an example.
Next, at least one particle 9 is put into the sample solution introduced into the holding space 6a of the holding unit 6 (step S3).
Next, the absorbance after the introduction of at least one particle 9 is obtained (step S4).
The absorbance after the introduction of at least one particle 9 can be calculated based on the output from the light receiving unit 32 as described above.
Next, the amount of change in absorbance is obtained from the difference between the absorbance after introducing the sample solution and the absorbance after introducing at least one particle 9 (step S5).
Next, based on the obtained amount of change in absorbance, the amount of the substance to be measured is obtained (step S6).
For example, the amount of the measurement target substance is obtained from the relationship between the amount of the measurement target substance and the absorbance change amount obtained in advance and the obtained absorbance change amount.
As described above, the amount of the substance to be measured in the sample solution can be measured.

In step S1, the sample solution and at least one particle 9 can be mixed and introduced into the holding space 6a. At this time, at least one particle 9 can be put into the sample solution and mixed, or the sample solution can be put into at least one particle 9 and mixed.
In this case, in step S2, the absorbance immediately after the sample solution mixed with at least one particle 9 is introduced into the holding space 6a is obtained.
And step S3 is abbreviate | omitted and step S4 is performed after predetermined time progress.
However, as described above, in order to further improve the accuracy of measurement, it is preferable to introduce the sample solution first and then introduce at least one particle 9 thereafter.
In addition, since the content of the process illustrated above can be the same as that of what was mentioned above, detailed description is abbreviate | omitted.

[Fourth Embodiment]
7A is a schematic cross-sectional view of a sensor chip according to the fourth embodiment, FIG. 7B is a cross-sectional view taken along the line DD in FIG. 7A, and FIG. 7C is FIG. It is an EE arrow sectional view in).
As shown in FIGS. 7A to 7C, the sensor chip 1a is provided with a substrate 2, an optical waveguide unit 3, an optical element unit 4, a detection unit 15, a holding unit 6, and a protection unit 7.
The detection unit 15 includes a detection unit 15a provided in the first region 3a and a detection unit 15b provided in the second region 3b.
The detection units 15a and 15b are formed of a substance that has a film shape and specifically reacts with the measurement target substance in the sample solution.
In this case, the first substance 15a fixed to the first region 3a and the second substance 15b fixed to the second region 3b can be different or the same. it can.

The detectors 15a and 15b include, for example, a color former, a film-forming body that holds the color former, a substrate for the enzyme that is the measurement target substance, a reagent that is used when performing elementary reactions, a color development reaction accelerator, and the like. be able to.
The film forming body may have, for example, a porous structure and hold a color former or the like in the pores in the porous structure. Examples of the film forming material include carboxymethyl cellulose (CMC) and polyethylene glycol (PEG).
The detection units 15a and 15b generate, for example, a precursor solution in which a color developing agent, a film-forming polymer, and the like are homogeneously mixed in a mixed solvent of water and a water-soluble organic solvent (for example, alcohol). It can be formed by drying into a shape.

  When a peroxide such as hydrogen peroxide is generated as a reaction product, the color former can be, for example, a benzidine color former. Examples of benzidine-based color formers include 4-chloro-1-naphthol, 3,3′-diaminobenzidine, 3,3 ′, 5,5′-tetramethylbenzidine (hereinafter referred to as TMBZ as appropriate), and TMBZ. Examples thereof include hydrochloride (3,3 ′, 5,5′-tetramethylbenzidine.2HCl.2H2O) and the like. In this case, as the color former, TMBZ having low solubility in water and extremely low toxicity to a living body can be used.

When NADH (reduced nicotinamide adenine dinucleotide) or the like is produced as a reaction product, the color former can be, for example, a tetrazolium salt.
In addition, when the enzyme that is the measurement target substance is ALT (GPT), the substrate can be, for example, L-alanine, α-ketoglutaric acid, or the like.
When the enzyme as the measurement target substance is ALT (GPT), the reagent can be, for example, pyruvate oxidase.
When a peroxide such as hydrogen peroxide is generated as a reaction product, the color development reaction accelerator can be, for example, peroxidase (POD).
When NADH (reduced nicotinamide adenine dinucleotide) or the like is produced as a reaction product, the color development reaction accelerator can be, for example, diaphorase. However, it is not necessarily limited to the exemplified color former, film forming body, substrate, reagent, color reaction accelerator, etc., and can be appropriately changed.

In the case of the sensor chip 1 described above, the near-field light is absorbed or scattered by the particles 9 in the vicinity of the surfaces of the first region 3a and the second region 3b. At this time, near-field light is absorbed and scattered according to the amount of the particles 9, and the amount of the measurement target substance can be obtained using this.
On the other hand, in the case of the sensor chip 1a according to the present embodiment, the color formers of the detection units 15a and 15b develop color by reacting with the measurement target substance in the sample solution, and approach according to the color development of the color former. The field light will be absorbed. Therefore, the amount of the measurement target substance can be obtained using this.

In this case, in the measurement device using the sensor chip 1a, it is not necessary to provide the magnetic field application unit 33, the magnetic field application unit 34, and the control unit 35 provided in the measurement device 30 described above. That is, in the measuring apparatus using the sensor chip 1a, at least the sensor chip 1a, the light projecting unit 31, the light receiving unit 32, and the calculating unit 36 may be provided.
Further, the operation of the measuring device 30 can be the same as that described above except that the absorption and scattering of near-field light by the particles 9 and the absorption of near-field light by coloring of the color former are different.
Therefore, details regarding the measuring apparatus using the sensor chip 1a are omitted.

In the measurement method using the sensor chip 1a, it is not necessary to introduce the particles 9 as in the measurement method described above.
Therefore, measurement can be performed according to the following procedure.
FIG. 8 is a flowchart for illustrating the measurement method when the sensor chip 1a is used.
First, the sample solution is introduced into the holding space 6a of the holding unit 6 (step S11).
Next, the absorbance after the sample solution is introduced is determined (step S12).
Next, after the sample solution is introduced, the absorbance after a lapse of a predetermined time is obtained (step S13).
Next, an absorbance change amount is obtained from the difference between the absorbance after the sample solution is introduced and the absorbance after a predetermined time has elapsed (step S14).
Next, based on the obtained amount of change in absorbance, the amount of the substance to be measured is obtained (step S15).
Here, a plurality of types of reagents may be required to be used when the elementary reaction described above is performed. In this case, all the reagents can be fixed to the detection units 15a and 15b, but the reagents react with each other, cannot be physically fixed to the detection units 15a and 15b, and some reagents react with the measurement target substance first. It may be necessary to let them.
Therefore, it may be necessary to mix the reagent and the sample solution in advance, or to introduce the reagent into the introduced sample solution.
When the reagent and the sample solution are mixed in advance, a step of mixing the sample solution and at least one reagent may be further provided.
In the step of introducing the sample solution into the holding space 6a of the holding unit 6 (step S11), the sample solution mixed with at least one reagent may be introduced into the holding space 6a of the holding unit 6.
When mixing the sample solution and at least one reagent, the sample solution can be mixed with at least one reagent, or the sample solution can be mixed with at least one reagent. it can.
In addition, when the reagent is introduced into the introduced sample solution, a step of introducing the reagent into the holding space 6a of the holding unit 6 after introducing the sample solution into the holding space 6a of the holding unit 6 may be further provided. Good.
Then, after the introduction of the sample solution, in the step of obtaining the absorbance after the lapse of a predetermined time (step S13), the absorbance after the lapse of the predetermined time may be obtained after the introduction of the reagent.
In addition, the light absorbency calculated | required in step S12 is a light absorbency in an initial state (state before reaction in the 1st area | region 3a or the 2nd area | region 3b occurs).
Therefore, the “absorbance after introducing the sample solution” in step S12 includes not only the absorbance before the at least one reagent is introduced, but also the absorbance immediately after the at least one reagent is introduced.
When measurement is performed by reacting multiple types of reagents, the reaction order may affect the measurement accuracy. Therefore, it is preferable to appropriately select the measurement method described above according to the purpose of measurement and the reagent used.
The amount of the measurement target substance is calculated from the calculation of the absorbance based on the output from the light receiving unit 32, the relationship between the amount of the measurement target substance obtained in advance and the change in absorbance, and the obtained change in absorbance. Obtaining and the like can be performed in the same manner as the measurement method described above, and a detailed description thereof will be omitted.

According to the embodiment illustrated above, it is possible to realize a sensor chip, a measuring apparatus, and a measuring method that can accurately measure a plurality of types of measurement target substances.
As mentioned above, although several embodiment of this invention was illustrated, these embodiment is shown as an example and is not intending limiting the range of invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, changes, and the like can be made without departing from the spirit 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 equivalents thereof. Further, the above-described embodiments can be implemented in combination with each other.

  DESCRIPTION OF SYMBOLS 1 Sensor chip, 2 Substrate, 3 Optical waveguide part, 3a 1st area | region, 3b 2nd area | region, 4 Optical element part, 5 Detection part, 5a 1st substance, 5b 2nd substance, 6 Holding part, 6a Holding space, 7 protective part, 7a first hole part, 7b second hole part, 9 particles, 9a particles, 9b particles, 12 base part, 13a third substance, 13b fourth substance, 14a substance to be measured, 14b Measurement target substance, 30 measuring device, 31 light projecting unit, 32 light receiving unit, 33 magnetic field applying unit, 34 magnetic field applying unit, 35 control unit, 36 computing unit

Claims (13)

A substrate having translucency;
An optical element provided on the substrate;
An optical waveguide provided on the substrate and the optical element; and
A holding unit provided on the optical waveguide unit and having a holding space for holding a sample solution;
At least one particle charged into the holding space;
With
The optical waveguide portion is
A first region in which a first substance that specifically reacts with a first measurement target substance is fixed;
A second region that specifically reacts with a second measurement target substance different from the first measurement target substance and to which a second substance different from the first substance is fixed;
Have
The holding portion integrally surrounds the first region and the second region,
The particle has a base, and a surface of the base has a third substance that specifically reacts with the first measurement target substance, and a second substance that specifically reacts with the second measurement target substance. A measurement method using a sensor chip to which at least one of the four substances is fixed,
Introducing a sample solution into a holding space of the holding unit;
Determining the absorbance after introducing the sample solution;
Introducing at least one particle into the sample solution introduced into the holding space of the holding unit;
Determining the absorbance after the at least one particle is charged;
Obtaining an amount of change in absorbance from the difference between the absorbance after introducing the sample solution and the absorbance after introducing the at least one particle;
A step of determining the amount of the substance to be measured based on the obtained amount of change in absorbance;
Measuring method. The base includes a core and a shell that is provided so as to cover the core, includes a material having superparamagnetism, and has a refractive index higher than that of the core;
The measurement method according to claim 1, wherein at least one of the third substance and the fourth substance is fixed to a surface of the shell. A substrate having translucency;
An optical element provided on the substrate;
An optical waveguide provided on the substrate and the optical element; and
A holding unit provided on the optical waveguide unit and having a holding space for holding a sample solution;
With
The optical waveguide portion is
A first region in which a first substance that specifically reacts with a first measurement target substance is fixed;
A second region that specifically reacts with a second measurement target substance different from the first measurement target substance and to which a second substance different from the first substance is fixed;
Have
The holding unit is a sensor chip that integrally surrounds the first region and the second region. Further comprising at least one particle charged into the holding space;
The particle has a base, and a surface of the base has a third substance that specifically reacts with the first measurement target substance, and a second substance that specifically reacts with the second measurement target substance. The sensor chip according to claim 3 , wherein at least one of the four substances is fixed. The sensor chip according to claim 4 , wherein the base portion includes a magnetic material. The base has a core and a shell provided to cover the core,
Said shell, look contains a material having a superparamagnetic,
The sensor chip according to claim 4 or 5 , wherein at least one of the third substance and the fourth substance is fixed to a surface of the shell .   The sensor chip according to claim 6, wherein the shell has a refractive index higher than a refractive index of the core. The sensor chip according to any one of claims 3 to 7 ,
A light projecting unit for making light incident on the sensor chip;
A light receiving unit that receives light from the sensor chip and converts it into an electrical signal corresponding to the intensity of the light; and
Measuring device. A measurement method using the sensor chip according to any one of claims 5 to 7 ,
Introducing the sample solution into the holding space of the holding unit;
Determining the absorbance after introducing the sample solution;
Introducing at least one particle into the sample solution introduced into the holding space of the holding unit;
Determining the absorbance after the at least one particle is charged;
Obtaining an amount of change in absorbance from the difference between the absorbance after introducing the sample solution and the absorbance after introducing the at least one particle;
A step of determining the amount of the substance to be measured based on the obtained amount of change in absorbance;
Measuring method. A measurement method using the sensor chip according to any one of claims 4 to 7 ,
Mixing the sample solution with at least one particle;
Introducing the sample solution mixed with the at least one particle into the holding space of the holding unit;
Obtaining the absorbance after the sample solution mixed with the at least one particle is introduced into the holding space of the holding unit;
Obtaining the absorbance after elapse of a predetermined time after introducing the sample solution into which the at least one particle has been introduced into the holding space of the holding unit;
Obtaining an amount of change in absorbance from the difference between the absorbance after introducing the sample solution into which the at least one particle has been introduced into the holding space of the holding portion and the absorbance after the lapse of the predetermined time;
A step of determining the amount of the substance to be measured based on the obtained amount of change in absorbance;
Measuring method. A measurement method using the sensor chip according to claim 3 ,
Introducing the sample solution into the holding space of the holding unit;
Determining the absorbance after introducing the sample solution;
A step of obtaining an absorbance after a lapse of a predetermined time after introducing the sample solution;
Obtaining an absorbance change amount from the difference between the absorbance after the sample solution is introduced and the absorbance after the predetermined time has elapsed;
A step of determining the amount of the substance to be measured based on the obtained amount of change in absorbance;
Measuring method. Further comprising the step of mixing the sample solution and at least one reagent,
The measurement method according to claim 11 , wherein in the step of introducing the sample solution into the holding space of the holding unit, the sample solution mixed with the at least one reagent is introduced into the holding space of the holding unit. A step of introducing a reagent into the holding space of the holding unit after introducing the sample solution into the holding space of the holding unit;
The measurement method according to claim 11 , wherein in the step of obtaining the absorbance after a lapse of a predetermined time after introducing the sample solution, the absorbance after the lapse of a predetermined time is obtained after the introduction of the reagent.

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