WO2018105072A1

WO2018105072A1 - Detection method and detection device

Info

Publication number: WO2018105072A1
Application number: PCT/JP2016/086464
Authority: WO
Inventors: 浩康田中; 勝田　宏
Original assignee: 京セラ株式会社
Priority date: 2016-12-07
Filing date: 2016-12-07
Publication date: 2018-06-14

Abstract

The present invention provides a detection method for a subject to be detected, said subject being contained in a sample. The present invention also provides a detection device for detecting a subject to be detected, said subject being contained in a sample. The detection method is provided with a first detection step for obtaining a detection value on the basis of a first signal value measured in a first measurement step, and a second signal value measured in a second measurement step. Furthermore, the detection device is provided with a first detection unit that obtains a detection value on the basis of a first signal value measured by a first measurement unit, and a second signal value measured by a second measurement unit.

Description

Detection method and detection apparatus

The present invention relates to a detection method and a detection apparatus for a detection target contained in a sample.

A method is known in which a target substance in a sample is detected using a biosensor having a detection element in which an antibody is bound to the surface (see, for example, Patent Document 1 or 2).

Japanese Patent No. 2625577 JP 2001-13142 A

However, the conventional detection method and detection apparatus may not be able to accurately detect the detection target in the sample due to the influence of substances other than the detection target included in the sample. Further, for example, there is a possibility that the signal value of the detection target cannot be accurately detected due to the influence of viscosity and density.

Therefore, detection that can detect the detection target in the sample more accurately by reducing the influence of substances other than the detection target contained in the sample and the influence of the difference in viscosity and density between samples. There is a need for methods and detection devices.

A detection method according to an embodiment of the present invention is a detection method of a detection target contained in a sample, and the sample is supplied to the surface of the detection body in which a primary substance that reacts with the detection target is bound to the surface. A first reaction step in which a primary reactant is formed on the surface of the detection body by a reaction between the detection object and the primary substance; and after the first reaction step, a first liquid is placed on the surface of the detection body. A first supply step for supplying to the substrate, a first measurement step for measuring a first signal value based on a surface state of the detection body after the first supply step, and a substance for signal amplification after the first measurement step To the surface of the detection body, and after the signal amplification process, the signal amplification process of changing the surface state of the detection body by a reaction involving the primary reactant formed in the first reaction process, Measure the second signal value based on the surface condition of the detection object It comprises a second measurement step of, a.

A detection apparatus according to an embodiment of the present invention is a detection apparatus for detecting a detection target contained in a sample, and the sample is combined with the surface of a detection body as a primary substance that reacts with the detection target. A first reaction unit that supplies and forms a primary reactant on the surface of the detection body by a reaction between the detection object and the primary substance, and a first supply unit that supplies a first liquid to the surface of the detection body A first measurement unit that measures a first signal value based on a surface state of the detection body after the first liquid liquid is supplied to the surface of the detection body by the first supply unit, and a signal amplification substance Based on the surface state of the detection body to which the signal amplification substance is supplied, and a signal amplification unit that changes the surface state of the detection body by a reaction involving the primary reactant. A second measuring unit for measuring a second signal value; Provided.

According to the detection method and the detection apparatus according to the embodiment of the present invention, it is possible to reduce the influence of substances other than the detection target contained in the sample by having the above-described configuration. It becomes possible to detect the detection target contained therein more accurately. In addition, since it is possible to reduce the influence of the viscosity and density generated by the sample, it is possible to detect the detection target contained in the sample more accurately.

It is a figure which shows the flowchart of the detection method which concerns on embodiment of this invention. It is a figure which shows the flowchart of the detection method which concerns on embodiment of this invention. It is a block diagram which shows the detection apparatus which concerns on embodiment of this invention. 1 is a perspective view of a biosensor device 200 according to an embodiment of the present invention. It is a disassembled perspective view of the biosensor apparatus 200 which concerns on embodiment of this invention. It is a top view of the detection element 3 which concerns on embodiment of this invention. It is a schematic diagram of a signal value acquired by a detection method according to an embodiment of the present invention. It is a figure which shows the experimental data regarding the detection method which concerns on embodiment of this invention. It is a perspective view of the sample liquid sensor which concerns on other embodiment of this invention. FIG. 10 is a plan view showing a part of the sample liquid sensor of FIG. 9. (A) And (b) is a perspective view which shows the sample liquid sensor apparatus which attached the sample liquid sensor to the reader | leader. It is a perspective view which shows the closed state of the sample liquid sensor apparatus of FIG. FIG. 13 is a schematic cross-sectional view of the sample liquid sensor device of FIG. 12 including the sample liquid sensor shown by the cross section along line AA of FIG. It is a block diagram which shows the structure of the signal processing system of a sample liquid sensor. (A) is a block diagram showing an arrangement example of the reference SAW element and the detection SAW element in the sample liquid sensor, and (b) is a diagram illustrating the SAW phase θref in the reference SAW element and the detection in the arrangement example of (a). FIG. 7C is a graph showing an example of the SAW phase θtest in the SAW element for use, and FIG. 8C is a graph showing the phase difference Δθ (θref−θtest) between θref and θtest shown in FIG. FIG. 16 is an explanatory diagram showing steps (1) to (5) shown in FIG. (A) shows the phase θref of the SAW in the reference SAW element when the detection target in the sample liquid is contained at a higher concentration than the measurement of FIG. 15B and when the density of the specific binding substance is high. And (b) is a graph showing the phase difference Δθ (θref−θtest) between θref and θtest shown in (a). (A) is a block diagram showing an arrangement example of the reference SAW element and the detection SAW element in the sample liquid sensor, and (b) is a diagram illustrating the SAW phase θref in the reference SAW element and the detection in the arrangement example of (a). FIG. 6C is a graph showing another example of the SAW phase θtest in the SAW element for use, and FIG. 8C is a graph showing the phase difference Δθ (θref−θtest) between θref and θtest shown in FIG. (A), (b) is a block diagram which shows the example which has arrange | positioned three or more SAW elements, respectively. It is a schematic plan view which shows the principal part of the sample liquid sensor which concerns on further another embodiment of this invention. It is a schematic sectional drawing which shows the sample liquid sensor which concerns on other embodiment of this invention. It is a perspective view which shows the sample liquid sensor apparatus which concerns on other embodiment of this invention.

<Detection method>
(First embodiment)
FIG. 1 is a flowchart showing a detection method according to the first embodiment of the present invention.
The detection method according to the first embodiment of the present invention is a detection method of a detection target contained in a sample,
A preparation step A1 for preparing a primary substance that is bound to the surface of the detection body and reacts with the detection target;
A first reaction step in which a sample is supplied to the surface of a detection body in which a primary substance that reacts with a detection target is bonded to the surface, and a primary reaction product is formed on the surface of the detection body by a reaction between the detection target and the primary substance. A2 and
After the first reaction step A2, a first supply step A3 for supplying the first liquid to the surface of the detection body;
After the first supply step A3, a first measurement step A4 for measuring a first signal value based on the surface state of the detection body;
After the first measurement step A4, the signal amplification substance is supplied to the surface of the detection body, and the signal changes the surface state of the detection body by the reaction involving the primary reactant formed in the first reaction step A2. Amplification step A5;
After the signal amplification step A5, a second measurement step A6 for measuring a second signal value based on the surface state of the detection body;
A first detection step A7 for obtaining a detection value from the first signal value and the second signal value.
In addition, preparatory process A1 and 1st detection process A7 are not an essential process, and may be provided with either process or both processes.

The preparation step A1 is a step of preparing a primary substance that binds to the surface of the detection body and reacts with the detection target.
The first reaction step A2 is performed by a detection body to which a primary substance is bound, a supply path for supplying a sample to the detection body, a pump for flowing the sample into the supply path, and the like, but the configuration is not limited.
Although 1st supply process A3 is implemented by the supply path for supplying a 1st liquid, a pump, etc., a structure is not limited and may be implemented similarly to 1st reaction process A2.
Although 1st measurement process A4 may be implemented by the apparatus etc. which input the signal into a detection body and acquire the signal value predetermined based on the signal output from a detection body, etc., a structure is not limited.
The signal amplification step A5 is performed by the detection body of the first reaction step A2, the supply path for supplying the signal amplification substance to the detection body, a pump, and the like, but the configuration is not limited, and the reaction unit 20 You may combine.
The second measurement step A6 may be performed by an apparatus including an element that inputs a signal to the detection body and acquires a predetermined signal value based on the signal output from the detection body, but the configuration is not limited. The first measurement step A4 may be performed in the same manner.
The first detection step A7 may be performed by an arithmetic device including an arithmetic element that obtains a detection value from the first signal value and the second signal value, but the configuration is not limited.

Here, in this specification, the “sample” may be, for example, a biological sample itself including blood, urine, saliva, sputum, or the like, or a biological sample diluted with a buffer solution or the like. . A sample other than a biological sample may be used.

Here, when detecting the detection target contained in each sample in the same amount for a plurality of samples, even if the first liquid is supplied to the surface of the detection body in the first supply step A3, A contaminant that is contained and is different from the detection object cannot be completely removed from the surface of the detection object, and there is a possibility that a difference occurs in the signal value for each detection object. However, according to the detection method of the present embodiment, the detection value is obtained from the first signal value and the second signal value, thereby reducing the influence of contaminants or the difference in viscosity and density between samples. Can do. This is because the detection value obtained from the first signal value and the second signal value is not affected by the amount of contaminants remaining on the surface of the detection body. In this way, by reducing the influence of contaminants (residues) between the samples, it becomes possible to detect the detection target contained in the samples more accurately.

Hereinafter, the detection method of this embodiment will be described in order.
Preparatory process A1 of this embodiment prepares the primary substance which is couple | bonded with the surface of a detection body and reacts with a detection target object.

In the present specification, the “detection target” includes, for example, an antigen, an antibody and the like, but is not limited thereto. Hereinafter, the detection target may be described as an antigen.

In this specification, the “detector” includes, for example, an element that outputs a signal value such as a surface acoustic wave element, a QCM (Quartz Crystal Microbalance), an SPR (Surface Plasmon Resonance), and an FET (Field Effect Transistor). However, it is not limited to these.

In the first reaction step A2 of the present embodiment, the sample is supplied to the surface of the detection body in which the primary substance that reacts with the detection target is bound to the surface, and the primary reactant is detected by the reaction between the detection target and the primary substance. It is formed on the surface of the body.

In the present specification, the “primary substance” is not particularly limited as long as it is a substance that specifically reacts with the detection target. For example, when the detection target is an antigen, it binds to the antigen. And an antigen that binds to the antibody when the detection target is an antibody.

In this specification, the “primary reactant” refers to, for example, a capture body in which a primary substance captures a detection target, a complex in which the detection target and the primary substance are bound, and a part of the primary substance is a detection target. This means a dissociated body in which the above-mentioned part is dissociated from the primary substance and includes, but is not limited to, a complex obtained by reacting an antigen and an antibody.

As a modification, as described above, in the first reaction step A2, a primary reactant is formed by combining a detection target and a part of the primary substance and dissociating a part from the primary substance. Is also possible.

In the first supply step A3 of the present embodiment, the first liquid is supplied to the surface of the detection body.

In the present specification, the “first liquid” may be, for example, a buffer solution. Buffers include, for example, phosphate buffer, citrate buffer, borate buffer, HEPES (4- (2-hydroxyethyl) -1-piperidineethanesulfonic acid) buffer, tris (hydroxymethyl) aminomethane) A buffer solution, a MOPS (3-morpholinopropanesulfonic acid) buffer solution, and the like are included, but are not limited thereto, and a known buffer solution may be used as appropriate. The buffer may contain sodium chloride, potassium chloride, magnesium chloride, zinc chloride, and EDTA (ethylenediamine tetraacetic acid), and if necessary, Tween 20 (registered trademark), Triton X-100 (registered trademark), A surfactant such as Brij35 (registered trademark) may be further included. Furthermore, a blocking substance may be mixed in the buffer solution as necessary. Examples of the blocking substance include BSA (bovine serum albumin), casein, polyethylene glycol, MPC (phosphorylcholine methacrylate) polymer, betaine polymer, HEMA (hydroxyethyl methacrylic acid) polymer, and the like. These points are the same in the second liquid and the third liquid described later.

The first measurement step A4 of the present embodiment measures a signal value based on the surface state of the detection body.

In the first measurement step A4, after the time when the signal value is stabilized due to the fluctuation of the signal value based on the surface state of the detection body due to the supply of the first liquid to the surface of the detection body in the first supply step A3. Will be implemented. For a while after the first liquid starts to be supplied, impurities are not sufficiently removed from the surface of the detection body, and the signal value based on the surface state of the detection body varies. If the contaminants are all removed from the surface of the detection body or are removed to the extent that the acquired signal value is not affected by supplying the first liquid, the signal value based on the surface state of the detection body has a small fluctuation. It will be in a stable state. By performing the first measurement step A4 in a state where the signal value is stable, it becomes possible to obtain a base signal value with reduced variations, excluding the influence of impurities, and such a base signal value By using, it becomes possible to measure a more accurate signal value of the detection target in the detection step described later. In the following embodiments, signal values can be acquired at the same timing.

Here, the stable state is a state in which the fluctuation of the signal value is small. For example, the signal value is continuously obtained from the first supply step A3, and the inflection point in the signal value change curve is determined and determined. What is necessary is to measure after the inflection point.

In this embodiment, a surface acoustic wave element may be formed on the surface of the detection body, and the value of the phase characteristic of the surface acoustic wave element may be used as the signal value based on the surface state of the detection body.

Further, in the present embodiment, the signal value based on the surface state of the detection body is measured by a method selected from a QCM (Quartz Crystal Microbalance) sensor, an SPR (Surface Plasma Resonance) sensor, and an FET (Field Effect Transistor) sensor. It may be a value.

In the signal amplification step A5 of the present embodiment, a signal amplification substance is supplied to the surface of the detection body, and the surface state of the detection body is changed by a reaction involving the primary reactant formed in the first reaction step A2. is there.

In the present specification, the “signal amplification substance” is not particularly limited as long as it is a substance that changes the surface state of the detection body. For example, the labeled substance that specifically reacts with the primary reactant is used. Secondary antibodies, labeled peptides, labeled ligands, labeled aptamers and the like are included. The label is not particularly limited as long as it changes the surface state of the detection body, but includes a protein such as streptavidin, biotin, an enzyme, a fluorescent substance, and nanoparticles such as metal particles.

In the present specification, the “reaction involving the primary reactant formed in the first reaction step” is particularly limited as long as the surface state of the detection body changes depending on the amount of the primary reactant. However, the binding reaction between the primary reactant formed in the first reaction step A2 and the signal amplification substance, the enzyme reaction between the primary reactant formed in the first reaction step A2 and the signal amplification substance, Including the reduction reaction between the primary reactant formed in the reaction step A2 and the signal amplification substance, the primary substance and the signal amplification substance may directly react with each other, and the primary substance and the signal amplification substance may react with each other. You may react indirectly through a substance.

In the present embodiment, an additional reaction step of supplying at least one additional reactant to the surface of the detection body may be further provided between the first measurement step A4 and the signal amplification step A5. The at least one additional reactant may include a plurality of additional reactants, and the additional reactants may be supplied one by one.

When an additional reaction step is performed, the signal amplification substance may not specifically react with the primary reactant, for example, streptavidin, 3,3′diaminobenzidine, 3-amino-9-ethylcarbazole , 4-chloro-1-naphthol, and substrates such as 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium, and combinations of chloroauric acid and hydroxylamine hydrochloride, and silver nitrate and iron sulfate A combination of a metal ion and a reducing agent such as a combination of

In the present specification, the “additional reactant” includes, for example, biotin, streptavidin, biotin-labeled antibody, peroxidase-labeled antibody, enzyme-labeled antibody such as alkaline phosphatase-labeled antibody, and nanoparticle-labeled antibody such as metal particle-labeled antibody, Peroxidase labeled streptavidin, and enzyme labeled streptavidin such as alkaline phosphatase labeled streptavidin, and nanoparticle labeled streptavidin such as metal particle labeled streptavidin and the like.

The additional reactant should be selected according to the selected signal amplification substance. For example, when the signal amplification substance is streptavidin, the additional reaction substance is a biotin-labeled antibody. For example, when the signal amplification substance is 3,3 'diaminobenzidine, the additional reaction substance is a biotin-labeled secondary antibody and peroxidase-labeled streptavidin. For example, if the signal amplification material is 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium, the additional reactants are a biotin-labeled secondary antibody and alkaline phosphatase-labeled streptavidin. For example, if the signal amplification material is chloroauric acid and hydroxylamine hydrochloride, the additional reactants are biotin-labeled secondary antibody and Au particle-labeled streptavidin.

In the additional reaction step, the additional reactant may be repeatedly supplied to further amplify the change in the surface state of the detection body. For example, when the signal amplification substance is streptavidin and the additional reaction substance is a biotin-labeled antibody, in the additional reaction step, the biotin-labeled secondary antibody is supplied first, the second is supplied with streptavidin, The biotin-labeled antibody is supplied to 3, the streptavidin is supplied to the fourth, the biotin-labeled antibody is supplied to the fifth, and then the streptavidin is supplied in the signal amplification step A5.

In the present embodiment, a precursor reaction step of supplying at least one precursor reactant to the surface of the detector may be further provided between the first reaction step A2 and the first supply step A3. The at least one precursor reactant may include a plurality of precursor reactants, and the precursor reactants may be supplied one by one.

When the precursor reaction step is performed, the signal amplification substance may not specifically react with the primary reactant, for example, streptavidin, 3,3′diaminobenzidine, 3-amino-9-ethylcarbazole , 4-chloro-1-naphthol, and substrates such as 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium, and combinations of chloroauric acid and hydroxylamine hydrochloride, and silver nitrate and iron sulfate A combination of a metal ion and a reducing agent such as a combination of

In the present specification, the “precursor reactant” means, for example, biotin, streptavidin, biotin-labeled antibody, peroxidase-labeled antibody, enzyme-labeled antibody such as alkaline phosphatase-labeled antibody, nanoparticle-labeled antibody such as metal particle-labeled antibody, Peroxidase labeled streptavidin, and enzyme labeled streptavidin such as alkaline phosphatase labeled streptavidin, and nanoparticle labeled streptavidin such as metal particle labeled streptavidin and the like.

The precursor reactant should be selected according to the signal amplification material selected. For example, when the signal amplification substance is streptavidin, the precursor reaction substance is a biotin-labeled antibody. For example, when the signal amplification substance is 3,3 'diaminobenzidine, the precursor reaction substance is a biotin-labeled secondary antibody and peroxidase-labeled streptavidin. For example, if the signal amplification material is 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium, the additional reactants are a biotin-labeled secondary antibody and alkaline phosphatase-labeled streptavidin. For example, when the signal amplification substance is chloroauric acid and hydroxylamine hydrochloride, the precursor reaction substances are biotin-labeled secondary antibody and Au particle-labeled streptavidin.

In the precursor reaction step, the precursor reactant may be repeatedly supplied to further amplify the change in the surface state of the detector. For example, when the signal amplification substance is streptavidin and the precursor reaction substance is a biotin-labeled antibody, in the precursor reaction step, a biotin-labeled secondary antibody is first supplied, and secondly streptavidin is supplied. 3 is supplied with a biotin-labeled antibody, fourth is supplied with streptavidin, fifth is supplied with a biotin-labeled antibody, and after passing through the first supply step A3 and the first measurement step A2, in the signal amplification step A5, streptavidin Is supplied.

The signal amplification substance, the additional reaction substance, and the precursor reaction substance may be diluted with a buffer solution or the like. The buffer for diluting the signal amplification substance, the additional reactant, and the precursor reactant (hereinafter also referred to as “reaction liquid”) is selected according to their properties, and the concentration, composition, pH, etc. It may be optimized.

In the signal amplification step A5, the signal amplification substance can be combined with the detection target substance among the primary reactants.

In the present embodiment, a second supply step of supplying the second liquid to the surface of the detection body may be further provided after the signal amplification step A5 and before the second measurement step A6.
According to this, the signal amplification substance that did not react with the primary reactant from the second signal value by removing the signal amplification substance that did not react with the primary reactant in the signal amplification step A5 from the surface of the detector. Can be reduced, and the accuracy of the signal value related to the detection target measured in the second measurement step A6 can be improved.

In the present specification, the “second liquid” may be, for example, a buffer solution.

In the present embodiment, the first liquid and the second liquid can be the same type of liquid. Also, the first liquid and the reaction liquid used in the subsequent reaction process, and the second liquid and the reaction liquid used in the subsequent reaction process may be the same type of liquid. Depending on the object to be detected, the concentration, pH, composition, etc. of the first liquid may be optimized in order to facilitate the removal of contaminants from the surface of the detection body in the first supply step A3. Depending on the detection object, the concentration, pH, composition, etc. of the second liquid may be optimized in order to facilitate the removal of contaminants from the surface of the detection body in the second supply step.

The first liquid may be the same type of liquid as the reaction liquid or may be a different type of liquid. The second liquid may be the same type of liquid as the reaction liquid or a different type of liquid. By making the first liquid and the second liquid different from the reaction liquid, it is possible to optimize the removal of contaminants by the first substance, as well as the signal amplification substance and the additional reaction substance. , And reactions with precursor reactants can be optimized.

If the first liquid and the second liquid and the reaction liquid used in the subsequent reaction step are the same type of liquid, fluctuations in the signal value when the liquid is switched can be reduced and acquired. Variations in signal values can be suppressed.

Here, the same type of liquid means that, for example, in a buffer solution, the same compound type such as a phosphate compound having a buffering action is used, and the same amount of liquid is the same type of liquid. That's it.

The second measurement step A6 of the present embodiment measures a signal value based on the surface state of the detection body.

This step may be performed in the same manner as the first measurement step A4 described above.

In the first detection step A7 of the present embodiment, a detection value is obtained from the first signal value and the second signal value.

In the present embodiment, the second signal value may be larger than the first signal value. Here, the magnitude of the signal value may be determined by, for example, the absolute value of the difference from the signal value before the first reaction step A2.

In the present embodiment, a sample that further includes a contaminant made of a substance different from the detection target may be used.

In this case, in the first reaction step A2, a contaminant different from the detection target adheres to the surface of the detection body, and then the first liquid is supplied to the surface of the detection body in the first supply step A3. In some cases, it cannot be completely removed from the surface of the body and remains. Therefore, the first signal value is affected by a different contaminant from the detection target remaining on the surface of the detection body.

In that case, in order to reduce the influence of the residue of this foreign substance, the signal value derived from the foreign substance remaining on the surface of the detection body is obtained by obtaining the detection value from the first signal value and the second signal value. Can be removed from the second signal value, the more accurate signal value of the detection target can be measured.

In one embodiment of the present invention, a second reaction step may be provided as one aspect of the signal amplification step A5. In the second reaction step, a secondary substance that reacts with the primary reactant is supplied to the surface of the detector, and a secondary reactant is formed on the surface of the detector by the reaction between the primary reactant and the secondary substance.

The second reaction step is performed by the detection body of the first reaction step, a supply path for supplying a secondary substance to the detection body, a pump, and the like, but the configuration is not limited and is the same as the first reaction step May be implemented.

In the present specification, the “secondary substance” is not particularly limited as long as it is a substance that specifically reacts with the primary reactant, and includes, for example, a secondary antibody and the like. An aspect may be sufficient.

As the secondary substance, for example, a labeled secondary antibody labeled with biotin, enzyme, nanoparticles, metal nanoparticles or the like can be used. Since the signal value derived from the secondary substance can be amplified by using the labeled secondary antibody in which the secondary substance is labeled, the detection target can be detected with higher sensitivity.

In this specification, the “secondary reactant” means, for example, a capturing body in which the primary reactant captures the secondary substance, a complex of the primary reactant and the secondary substance, and the like, and is not limited thereto. Although it is not a thing, the composite_body | complex of an antigen, a primary antibody, and a secondary antibody is included.

Note that the molecular weight of the secondary reactant may be larger than the molecular weight of the primary reactant. According to this, a detection target can be detected with high sensitivity by obtaining a large signal value in a second measurement step A6 described later.

In the second reaction step, it is also possible to form a secondary reactant by binding a secondary substance to a detection target among primary reactants.

As an embodiment for carrying out the second reaction step as one aspect of the signal amplification step A5, the detection target is an antigen, a surface acoustic wave element is used as a detection body, a primary antibody is used as a primary substance, and a first liquid is used. An example in which a buffered solution is used and a labeled secondary antibody is used as a secondary substance is shown below.
That is, one example of the first embodiment is a method for detecting an antigen contained in a sample,
A preparation step for preparing a primary antibody against an antigen, which is bound to the surface of the surface acoustic wave device;
A sample is supplied to the surface of the surface acoustic wave device and reacted with a primary antibody against the antigen to form a primary complex of the antigen and the primary antibody contained in the sample on the surface of the surface acoustic wave device. A reaction process;
A first supply step of supplying a buffer solution to the surface of the surface acoustic wave device after the first reaction step;
A first measurement step of measuring a first signal value based on the surface state of the surface acoustic wave device after the first supply step;
After the first measurement step, a labeled secondary antibody against the antigen is supplied to the surface of the surface acoustic wave device, and the secondary complex of the primary complex and the labeled secondary antibody is transferred to the surface of the surface acoustic wave device. A second reaction step to be formed on;
A second measurement step of measuring a second signal value based on the surface state of the surface acoustic wave device after the second reaction step;
The detection method may include a first signal value measured in the first measurement process and a first detection process for obtaining a detection value from the second signal value measured in the second measurement process.

(Second Embodiment)
FIG. 2 is a flowchart showing a detection method according to the second embodiment of the present invention.
The detection method according to the second embodiment of the present invention is a detection method of a detection target contained in a sample,
A preparation step B1 for preparing a primary substance that is bound to the surface of the detection body and reacts with the detection target;
A first reaction step B2 in which a sample is supplied to the surface of a detection body to which a primary substance that reacts with a detection target is bound, and a primary reaction product is formed on the surface of the detection body by a reaction between the detection target and the primary substance; ,
After the first reaction step B2, a first supply step B3 for supplying the first liquid to the surface of the detection body;
After the first supply step B3, a first measurement step B4 for measuring a signal value based on the surface state of the detection body;
After the first measurement step B4, a secondary substance that reacts with the primary reactant is supplied to the surface of the detector, and a secondary reactant is formed on the surface of the detector by the reaction between the primary reactant and the secondary substance. A second reaction step B5;
After the second reaction step B5, a tertiary substance that reacts with the secondary reactant is supplied to the surface of the detector, and a tertiary reactant is formed on the surface of the detector by the reaction between the secondary reactant and the tertiary substance. 3 reaction steps B6,
After the third reaction step B6, a third measurement step B7 for measuring a signal value based on the surface state of the detection body;
A second detection step B8 for obtaining a detection value by subtracting the signal value measured in the first measurement step B4 from the signal value measured in the third measurement step B7.
Note that the preparation step B1 and the second detection step B8 are not essential steps, and may include either or both steps.

In the present embodiment, the first reaction step B2, the first supply step B3, and the first measurement step B4 are the same as those in the first embodiment, and thus description thereof is omitted.

Although 2nd reaction process B5 is implemented by the detection body of 1st reaction process B2, and the supply path for supplying a secondary substance to this detection body, a pump, etc., a structure is not limited.
The third reaction step B6 is performed by the detection body of the second reaction step B5, a supply path for supplying a tertiary substance to the detection body, a pump, and the like, but the configuration is not limited.
The third measurement step B7 may be performed by an apparatus including an element that inputs a signal to the detection body and acquires a predetermined signal value based on the signal output from the detection body, but the configuration is not limited. The first measurement step B4 may be performed in the same manner.
The second detection step B8 may be performed by an arithmetic unit including an arithmetic element that obtains a detection value from the signal value measured in the first measurement step B4 and the signal value measured in the third measurement step B7. The configuration is not limited.

In the present embodiment, the second reaction step B5 is performed as one aspect of the additional reaction step, and after the second reaction step B5, the third reaction step B6 is performed as one aspect of the signal amplification step. In the third reaction step B6, a tertiary substance that reacts with the secondary reactant is supplied to the surface of the detector, and a tertiary reactant is formed on the surface of the detector by the reaction between the secondary reactant and the tertiary substance. . According to this, since the signal value derived from the secondary substance can be amplified by using the tertiary substance, the detection target contained in the sample can be detected with higher sensitivity.

In the present specification, the “tertiary substance” is not particularly limited as long as it is a substance that specifically reacts with the secondary substance. For example, the “tertiary substance” is a labeled secondary antibody in which the secondary substance is labeled with biotin. In some cases, a label detection reagent such as streptavidin that reacts specifically with the label is included.

Here, as in the above-described example, when the sample further includes a contaminant made of a substance different from the detection target, the first liquid is supplied to the surface of the detection body in the first supply step B3. When the contaminants cannot be completely removed from the surface of the detection body, the signal value measured in the first measurement step B4 is affected by the contaminants remaining on the surface of the detection body. Therefore, in order to reduce the influence of impurities remaining on the surface of the detection body, a detection value is obtained from the signal value measured in the first measurement step B4 and the signal value measured in the third measurement step B7. Thus, the signal value derived from the impurities remaining on the surface of the detection body can be reduced from the signal value measured in the third measurement step B7. This is because the difference between the signal value measured in the first measurement step B4 and the signal value measured in the third measurement step B7 is not substantially affected by the amount of contaminants remaining on the surface of the detection body. It is.

In addition, when detecting a detection object in each sample for a plurality of samples, if the viscosity or density differs among the plurality of samples, the first liquid is supplied to the surface of the detection body in the first supply step B3. However, the contaminants different from the detection target cannot be completely removed from the surface of the detection object, and in each sample, a different amount of contamination remains on the surface of the detection object. There may be differences in values. However, the contaminants remaining on the surface do not substantially affect the difference between the signal value measured in the first measurement step B4 and the signal value measured in the third measurement step B7. By obtaining a detection value from the signal value measured in the first measurement step B4 and the signal value measured in the third measurement step B7, it is possible to reduce the influence of contaminant residues between the samples.

Thus, by reducing the influence of the residue of impurities between the samples, it becomes possible to detect the detection target contained in the sample more accurately.

In this embodiment, the secondary substance can be labeled with biotin, and the tertiary substance can contain streptavidin. Thereby, since the signal value derived from the secondary substance can be amplified, the detection target can be detected with higher sensitivity.

In addition, in this embodiment, you may make it further provide the 2nd supply process which supplies a 2nd liquid to the surface of a detection body between 2nd reaction process B5 and 3rd reaction process B6. As a result, the secondary material that has not reacted with the primary reactant in the second reaction step B5 can be removed, and therefore the influence of the secondary material that has not reacted with the primary reactant is determined after the third reaction step B6. And the accuracy of the signal value measured in the third measurement step B7 can be improved.

In this embodiment, an intermediate measurement step for measuring a signal value based on the surface state of the detection body may be further provided between the second reaction step B5 and the second supply step. As a result, the secondary substance that did not react with the primary reactant in the second reaction step B5 can be removed, so that the influence of the secondary substance that did not react with the primary reactant can be eliminated, and the intermediate measurement step It is possible to improve the accuracy of the signal value measured by.

In the present embodiment, a third detection step for obtaining a detection value from the signal value measured in the intermediate measurement step and the signal value measured in the third measurement step B7 may be further provided. Thereby, the signal value generated by the third reaction step B6 can be measured.

In the present embodiment, a third supply step for supplying the third liquid to the surface of the detection body may be further provided between the third reaction step B6 and the third measurement step B7. As a result, the tertiary substance that did not react with the secondary reactant in the third reaction step B6 can be removed, so that the influence of the tertiary substance that did not react with the secondary reactant is measured in the third measurement step B7. The signal value measured in the third measurement step B7 can be improved.

In the present specification, the “third liquid” may be, for example, a buffer solution. Examples of the buffer solution include, but are not limited to, a phosphate buffer solution and the like.

In the present embodiment, the first liquid, the second liquid, and the third liquid can be the same type of liquid. The first liquid, the reaction liquid used in the subsequent reaction process, the second liquid, the reaction liquid used in the subsequent reaction process, and the third liquid, and the reaction liquid used in the subsequent reaction process Can be the same type of liquid. Depending on the detection target, the concentration, pH, composition, and the like of the first liquid may be optimized in order to promote the removal of impurities from the surface of the detection body in the first supply step B3. Depending on the detection object, the concentration, pH, composition, etc. of the second liquid may be optimized in order to facilitate the removal of contaminants from the surface of the detection body in the second supply step. Depending on the object to be detected, the concentration, pH, composition, etc. of the third liquid may be optimized in order to promote the removal of contaminants from the surface of the detection body in the third supply step.

The first liquid may be the same type of liquid as the reaction liquid or a different type of liquid. The second liquid may be the same type of liquid as the reaction liquid or may be a different type of liquid. The third liquid may be the same type of liquid as the reaction liquid or a different type of liquid. By making the first liquid, the second liquid, the third liquid, and the reaction liquid different types of liquids, it is possible to optimize the removal of contaminants, and the signal amplification substance and the additional reaction It is possible to optimize the reaction by the substance and the precursor reactant.

If the first liquid, the second liquid, the third liquid, and the reaction liquid used in the subsequent reaction step are the same type of liquid, it is possible to reduce fluctuations in the signal value when the liquid is switched. And variation in signal values to be acquired can be suppressed.

As an example of performing the second reaction step B5 as one aspect of the additional reaction step and performing the third reaction step B6 as one aspect of the signal amplification step, the detection target is an antigen, and the surface acoustic wave is used as the detection body. An example using a device, using a primary antibody as a primary substance, using a buffer solution as a first liquid, using a labeled secondary antibody as a secondary substance, and using a labeled detection reagent as a tertiary substance is shown below.
That is, one example of the third embodiment is a method for detecting an antigen contained in a sample,
A preparation step for preparing a primary antibody against an antigen, which is bound to the surface of the surface acoustic wave device;
A sample is supplied to the surface of the surface acoustic wave device and reacted with a primary antibody against the antigen to form a primary complex of the antigen and the primary antibody contained in the sample on the surface of the surface acoustic wave device. A reaction process;
A first supply step of supplying a buffer solution to the surface of the surface acoustic wave device after the first reaction step;
A first measurement step of measuring a signal value based on the surface state of the surface acoustic wave device after the first supply step;
After the first measurement step, a labeled secondary antibody against the antigen is supplied to the surface of the surface acoustic wave device, and the secondary complex of the primary complex and the labeled secondary antibody is transferred to the surface of the surface acoustic wave device. A second reaction step to be formed on;
After the second reaction step, a third reaction step of supplying a labeled detection reagent for the labeled secondary antibody to the surface of the surface acoustic wave device to form a tertiary complex of the secondary complex and the labeled detection reagent. When,
After the third reaction step, a third measurement step of measuring a signal value based on the surface state of the surface acoustic wave device;
The detection method may include a second detection step of obtaining a detection value from the signal value measured in the third measurement step from the signal value measured in the first measurement step.

(Third embodiment)
The detection method according to the third embodiment of the present invention is a detection method of a detection target contained in a sample,
A preparation step for preparing a primary substance that is bound to the surface of the detection body and reacts with the detection target;
A first reaction step in which a sample is supplied to the surface of a detection body in which a primary substance that reacts with a detection target is bonded to the surface, and a primary reaction product is formed on the surface of the detection body by a reaction between the detection target and the primary substance. When,
A first supply step for supplying the first liquid to the surface of the detection body after the first reaction step;
After the first supply step, the signal amplification substance is supplied to the surface of the detection body, and the surface state of the detection body is changed by the reaction involving the primary reactant formed in the first reaction step. A signal acquisition step of acquiring a first signal value and subsequently acquiring a second signal value;
A calculation step of calculating a detection value from the first signal value and the second signal value.

In the present embodiment, the preparation step, the first reaction step, and the first supply step are the same as those in the first embodiment, and thus description thereof is omitted.

In the signal acquisition step, after the first supply step, the signal amplification substance is supplied to the surface of the detection body, and the surface state of the detection body is changed by the reaction involving the primary reactant formed in the first reaction step. It is a step of acquiring a first signal value and subsequently acquiring a second signal value while changing. In the signal acquisition step, a signal amplification substance is used as in the first embodiment. In the first supply step, the first liquid is supplied to the surface of the detection body, impurities are removed, and the signal value based on the surface state of the detection body is stabilized, and then the signal amplification substance is supplied to the surface of the detection body. . When the signal amplification substance is supplied to the surface of the detection body, the surface state of the detection body starts to change due to the reaction between the primary reactant formed on the surface of the detection body and the signal amplification substance. A first signal value based on the surface state of the detection body is obtained after a predetermined time has elapsed since the signal amplification substance was supplied to the surface of the detection body. After a predetermined time has elapsed since the first signal value was acquired, a second signal value based on the surface state of the detection body is acquired.

In the present embodiment, the first signal value may be acquired after a predetermined time has elapsed since the signal amplification substance was supplied to the surface of the detection body, and may be acquired, for example, after 1 second to 30 seconds. The second signal value may be acquired after a predetermined time has elapsed since the first signal value was acquired. For example, the second signal value is acquired after 1 to 10 minutes. The second signal value may be acquired in a state in which the change in the surface state of the detection body due to the signal amplification substance has converged.

In the present embodiment, it is also possible to acquire the second signal value by determining in advance the time from when the first signal value is acquired until the second signal value is acquired. According to this, the second signal value increases as the amount of the detection target contained in the sample increases, and the detection target contained in the sample can be detected with high accuracy in a short time. The detected value can be a difference value obtained by subtracting the first signal value from the second signal value.

In the present embodiment, the second signal value that is the end point of the measurement is determined in advance, and the time from when the first signal value is acquired until the second signal value reaches a predetermined value is used as the detection value. It is also possible to detect the detection target contained in the sample by measuring. According to this, as the amount of the detection target contained in the sample increases, the time until the second signal value reaches a predetermined value is shortened, and the detection target contained in the sample can be accurately detected in a short time. Can be detected.

In the present embodiment, the slope (differential value depending on time) of the change (increase) of the signal value may be calculated as the detected value from the first signal value and the second signal value. According to this, as the amount of the detection target contained in the sample increases, the inclination becomes steeper, and the detection value can be obtained with high accuracy in a short time. The time for calculating the differential value can be shorter than the time for obtaining the difference value.

<Detection device>
Next, an example of the detection apparatus 100 is shown. FIG. 3 is a block diagram illustrating an example of a detection device. The detection apparatus 100 is used for measuring a signal value in a sample liquid sensor including a supply unit 10 that supplies various liquids and a reaction unit 20 that performs not only a reaction for signal amplification but also a precursor reaction and an additional reaction. The measurement unit 30 is configured by connecting a detection unit 40 that is a calculation device for calculation, and a display unit 50 that is a display for displaying detection results.

In the detection apparatus 100 of the present embodiment, each supply process such as the first supply process and the second supply process described above is performed by repeatedly using one supply unit 10. Similarly, each reaction step such as the first reaction step and the second reaction step described above is performed by repeatedly using one reaction unit 20, and each measurement step described above uses one measurement unit 30 repeatedly. And each above-mentioned detection process is each implemented by using one detection part 40 repeatedly. In this embodiment, the display unit 50 is not an essential component, and may be configured so that the detection result can be output from the detection unit 40 to the outside. The electrical connection between the measurement unit 30 and the detection unit 40 and between the detection unit 40 and the display unit 50 may be wired connection using a signal cable or the like. The wireless connection used may be used.

4 is a perspective view of the sample liquid sensor 200, FIG. 5 is an exploded perspective view of the sample liquid sensor 200, and FIG. 6 is a plan view of the detection element 3. FIG.

The sample liquid sensor 200 includes a substrate 1, a flow path structure 2, and a detection element 3. As shown in FIG. 4, the flow path structure 2 is disposed on the substrate 1 via the detection element 3 and the support member 4. The channel structure 2 has an inlet 14 that is an inlet for a liquid sample on one end side in the longitudinal direction, and a channel that communicates with the inlet 14 is formed therein. The board | substrate 1 is flat form, for example, is a resin substrate, a ceramic substrate, etc., and has provided the wiring conductor etc. in the surface layer or the inner layer.

The detection element 3 is mounted on one end side of the upper surface of the substrate 1. Terminals 6 electrically connected to the detection element 3 are provided on both sides of the detection element 3. The terminal 6 is connected to a device, an arithmetic device, or the like.

The detection element 3 is a surface acoustic wave element, and includes a piezoelectric substrate 7, a first IDT (Inter Digital Transducer) electrode 8, a second IDT electrode 9, and a detection unit 13. The piezoelectric substrate 7 is made of a single crystal substrate having piezoelectricity such as lithium tantalate. The first IDT electrode 8 has a pair of comb electrodes. Each comb electrode has two bus bars facing each other and a plurality of electrode fingers extending from each bus bar to the other bus bar side. The pair of comb electrodes are arranged such that a plurality of electrode fingers mesh with each other. The second IDT electrode 9 is configured in the same manner as the first IDT electrode 8. The first IDT electrode 8 and the second IDT electrode 9 constitute a transversal IDT electrode.

The first IDT electrode 8 is for generating a predetermined surface acoustic wave, and the second IDT electrode 9 is for receiving the SAW generated by the first IDT electrode 8. The first IDT electrode 8 and the second IDT electrode 9 are made of, for example, aluminum or an alloy of aluminum and copper.

The detection unit 13 is provided between the first IDT electrode 8 and the second IDT electrode 9. The detection unit 13 has a two-layer structure of, for example, chromium and gold formed on chromium. A primary substance that reacts with the detection target is bonded to the surface of the metal film of the detection unit 13. When the sample is supplied to the detection unit, the detection target in the sample reacts with the primary substance to form a primary reactant.

Suppose that the first IDT electrode 8, the second IDT electrode 9, and the detection unit 13 are one set, the detection element 3 is provided with two sets. For example, one detection unit 13 can measure a sample, and the other detection unit 13 can measure a reference value. For example, the other detection unit 13 is not coupled with a primary substance that reacts with the detection target.

In the detection element 3 using such SAW, first, a signal having a predetermined voltage is applied to the first IDT electrode 8 from the outside. In the first IDT electrode 8, the surface of the piezoelectric substrate 7 is excited, and SAW having a predetermined frequency is generated. Part of the generated SAW propagates toward the detection unit 13, passes through the detection unit 13, and is received by the second IDT electrode 9. In the detection unit 13, a primary reaction product is formed according to the amount of the detection target, and the mass of the detection unit 13 is increased by the amount of the primary reaction product. When the phase of the SAW passing through the detection unit 13 changes as the mass increases, a voltage corresponding to the change is generated in the second IDT electrode 9. The difference between the phase of the signal applied to the first IDT electrode 8 and the phase of the signal output from the second IDT electrode 9 is measured as a phase change.

A support member 4 is further mounted on the upper surface of the substrate 1, and the support member 4 supports the flow path structure 2. The flow path structure 2 is arranged so as to cover at least a part of the detection element 3. The flow path structure 2 includes, for example, a first adhesive layer 19, a first hydrophilic sheet 22, a second adhesive layer 23, and a second hydrophilic sheet 24.

The first adhesive layer 19 is a frame having a through hole 19h, and a part of the detection element 3 is exposed through the through hole 19h. A first hydrophilic sheet 22 is laminated on the first adhesive layer 19. The first hydrophilic sheet 22 has a through hole 22h similar to the through hole 19h, and the first adhesive layer 19 and the first hydrophilic sheet 22 are laminated so that the through holes communicate with each other. A second adhesive layer 23 is laminated on the first hydrophilic sheet 22. The second adhesive layer 23 has a through hole 23h extending in the longitudinal direction constituting the flow path. One end of the through hole 23h extends to a position overlapping the through hole 22h. A second hydrophilic sheet 24 is laminated on the second adhesive layer 23. Near the both ends of the second hydrophilic sheet 24, an inflow port 14 and an exhaust port 18 each including a through hole are provided. The inflow port 14 and the exhaust port 18 are formed at a position overlapping the through hole 23h.

The schematic diagram shown in FIG. 7 shows that a precursor reaction step is performed using a biotin-labeled secondary antibody and alkaline phosphatase-labeled streptavidin as precursor reactants, and 5-bromo-4-chloro-3-indolyl as a signal amplification substance. It is an example of the signal value which performed the signal amplification process using the phosphate / nitro blue tetrazolium, and acquired the signal value in the SAW element.

The steps performed in the schematic diagram shown in FIG. 7 are described below. First, the 1st reaction process and the 1st supply process were performed, and the primary reactant was formed. Next, the first precursor reaction step was performed to react the biotin-labeled secondary antibody with the primary reactant. After the first precursor reaction step, a second supply step of supplying 10 mM PBS (10 mM Phosphate, 137 mM Sodium Chloride, 2.7 mM Potassium Chloride, 0.005% Tween 20 (registered trademark), pH 7.4) is performed, and is free. The biotin-labeled secondary antibody was removed. After the second supply step, a second precursor reaction step was performed to react with alkaline phosphatase-labeled streptavidin. After the second precursor reaction step, a third supply step of supplying 10 mM PBS (10 mM Phosphate, 137 mM Sodium Chloride, 2.7 mM Potassium Chloride, 0.005% Tween 20 (registered trademark), pH 7.4) is performed, and free. Alkaline phosphatase labeled streptavidin was removed. After the third supply step, the first measurement step was performed to obtain the first signal value. After the first measurement step, a signal amplification step was performed, and 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium was reacted. After the signal amplification step, a second measurement step was performed to obtain a second signal value. Finally, the detection value was obtained by subtracting the first signal value from the second signal value.

Hereinafter, examples of the detection method according to the above-described embodiment will be described.
In this example, a biological sample was used to detect a detection target contained in the biological sample.

As biological samples, two different types of samples (biological samples A and B) each containing the same amount of antigen were prepared. That is, the biological sample A and the biological sample B have different viscosities and contained substances, but contain the same amount of antigen.

As the detection body, a surface acoustic wave element (SAW element) is used, and an antibody that is a primary substance (hereinafter simply referred to as “antibody”) is prepared in advance on the surface of the SAW element. Here, the antibody is a substance that binds to the detection target.

As Example 1, first, as a first reaction step, a biological sample is supplied to the surface of the SAW element, and a primary reaction in which an antigen as an object to be detected (hereinafter simply referred to as “antigen”) reacts with an antibody. Formed.

Next, in the first supply step, the buffer solution 10 mM PBS as the first liquid
(10 mM Phosphate,
137 mM Sodium Chloride,
2.7 mM Potassium Chloride,
1 mM MgCl ₂ , 0.005% Tween 20 (registered trademark), pH 7.4) was supplied to the surface of the SAW element.

Next, in the first measurement step, the phase change between the input signal and the output signal was obtained as a signal value in the SAW element.

Next, in the signal amplification step, a biotin-modified secondary antibody is supplied to the surface of the SAW element as a signal amplification substance, and the primary reaction product obtained by reacting the primary reactant with the biotin-modified secondary antibody and the biotin-modified secondary antibody. A complex with the antibody was formed.

Next, in the second measurement step, the phase change between the input signal and the output signal was obtained as a signal value in the SAW element.

Next, in the first detection step, the detection value was obtained by subtracting the first signal value measured in the first measurement step from the second signal value measured in the second measurement step.

The signal values for each biological sample obtained as described above are shown as a graph in FIG.

As shown in FIG. 8, in Example 1, the detected value of biological sample A and the detected value of biological sample B each containing the same amount of antigen were equivalent.

From this, according to the detection method according to the embodiment of the present invention, the influence due to the difference between the biological samples is reduced, and it is possible to accurately detect the detection target (antigen) contained in each biological sample. I found out.

On the other hand, as a comparative example, the value of the phase change measured in the second measurement step was acquired as the detection value without performing the first measurement step as described above.

In such a comparative example, the detection value of the biological sample A and the detection value of the biological sample B each containing the same amount of antigen are different, and the detection target included in each biological sample due to the influence of the difference between the biological samples. It was found that the product (antigen) could not be detected accurately (see FIG. 8).

In Example 2, without using a biological sample, the same amount of antigen as in Example 1 was added to a buffer solution of 10 mM PBS (10 mM Phosphate,
137 mM Sodium Chloride,
2.7 mM Potassium Chloride,
The detection value obtained in the same manner as in Example 1 was obtained by using a sample diluted with 1 mM MgCl ₂ , 0.005% Tween 20 (registered trademark), pH 7.4) as a sample. It was equivalent to 1.
This also proved that the detection target (antigen) contained in the biological sample can be detected accurately in Example 1.

Next, an example of a specific method for measuring the first signal value in the reaction steps other than the first reaction step for supplying the sample among the above-described reaction steps will be described. Note that the present method described in the specimen liquid measurement method described later may be used when the sensor chip has a configuration in which a plurality of SAW elements are positioned along the flow direction of the specimen liquid.

<Sample fluid sensor>
FIG. 9 shows a sample liquid sensor 303 according to this embodiment. The sample liquid sensor 303 includes a base 343 and a cover 345 superimposed thereon, and a flow path 335 is formed between the base 343 and the cover 345. In addition, a sensor chip 332 (corresponding to the detection element 3 described above) is formed on the top surface of the base 343 at the tip of the flow path 335, and an external terminal for transmitting data from the sensor chip 332 on the bottom of the base 343. 331 is formed. The shape of the sample fluid sensor 303 is generally plate-shaped as a whole, and for example, its planar shape is a rectangle.

The flow path 335 is formed, for example, so as to extend linearly in the longitudinal direction of the sample liquid sensor 303 (x direction, a direction from a portion exposed from the reader 305 to a portion sandwiched by the reader 305). Both ends of the flow path 335 communicate with the outside of the sample liquid sensor 303. One end is an inflow port 339 for taking in the sample liquid, and the other end is an exhaust port 341 for exhausting the flow path 335 when the sample liquid flows into the flow path 335. The inflow port 339 and the exhaust port 341 are preferably open on the upper surface of the sample liquid sensor 303.

The flow path 335 is configured to guide the sample liquid dropped onto the inflow port 339 (in contact with the inflow port 339) toward the exhaust port 341 by a capillary phenomenon. For example, the height (thickness, z direction) of the flow path 335 is set to be relatively low, and the wettability of at least one of the bottom surface and the ceiling surface is set to be relatively high.

The height of the flow path 335 in the z direction is not particularly limited, but is 50 μm to 0.5 mm, preferably about 50 μm, from the viewpoint of reducing the amount of the sample liquid. Note that when the diluted sample solution such as blood is used as the sample solution, the amount of the sample solution is not necessarily reduced. In addition, the contact angle (wetting property) of the sample liquid (which may be represented by water) on the bottom surface and the ceiling surface of the flow path 335 is less than 90 °, and preferably less than 60 °.

In the present embodiment, the base 343 has an insulating property. As a material of the base 343, for example, resin or ceramic can be used. In addition, the base 343 may be a multi-layer board such as having a ground layer as a shield inside. The planar shape of the base 343 is the same as the planar shape of the entire sample liquid sensor 303, for example.

The planar shape of the outer shape of the cover 345 is, for example, generally the same as the planar shape of the entire specimen liquid sensor 303. On the lower surface of the cover 345, a groove for forming the flow path 335 is formed between the cover 345 and the base 343. The cover 345 is formed with the above-described inflow port 339 and exhaust port 341 so as to penetrate the cover 345 vertically. The cover 345 is bonded to the base 343 with an adhesive, for example. The cover 345 is made of an insulating material such as resin or ceramic. Note that the entire cover 345 may be integrally formed of the same material. Further, the cover 345 may be configured by stacking a plurality of layered members made of the same material or different materials. For example, the cover 345 may be configured by a layered member in which a slit to be the flow path 335 is formed, and a layered member that is superimposed on the layered member and forms the ceiling surface of the flow path 335.

At least one of the base 343 and the cover 345 is made of a highly hydrophilic material or subjected to a hydrophilic treatment so that the wettability of the inner surface of the flow path 335 is increased at least in a region constituting the flow path 335. It is preferable that a hydrophilic film is attached. For example, the base 343 may be attached with a hydrophilic film in a region overlapping with the flow path 335. In this case, the hydrophilic film may be regarded as a part of the base 343. Further, for example, in the case where the cover 345 is configured by stacking layered members as described above, the upper layered member closing the slit may be configured by a hydrophilic film.

The sample liquid sensor 303 as a whole preferably does not have flexibility. For example, at least one of the base 343 and the cover 345 may not have flexibility.

FIG. 10 is a plan view showing the sensor chip 332 of the sample liquid sensor 303 with the cover 345 removed from the sample liquid sensor 303.

The sample liquid sensor 303 has a sensor chip 332 for detecting a detection target included in the sample liquid passing through the flow path 335 mounted on the upper surface of the base 343. The sensor chip 332 is a sensor unit that substantially converts a signal according to the sample liquid, and the base 343 and the cover 345 function as a package that contributes to an improvement in the handleability of the sensor chip 332 and the like. Although not particularly illustrated, at least one of the lower surface of the cover 345 and the upper surface of the base 343 is formed with a recess for housing the sensor chip 332.

The sensor chip 332 includes a piezoelectric substrate 353 and at least two

SAW elements

350A and 350B located on the upper surface of the piezoelectric substrate 353 along the flow direction of the specimen liquid. The

SAW elements

350A and 350B include a first IDT electrode 355A that generates a surface acoustic wave (SAW) on the main surface of the piezoelectric substrate 353, a second IDT electrode 355B that is positioned in the SAW propagation path, and that receives the SAW. A plurality of chip pads 357 provided for input of an electrical signal to the 1 IDT electrode 355A or output of an electrical signal from the second IDT electrode 355B, and a sensitive unit 359 for changing the SAW according to the property or component of the sample liquid have. As the sensitive part 359, for example, an insulating film (an oxide film, a nitride film, etc.) can be used in addition to a metal film described later.

The piezoelectric substrate 353 is made of, for example, a single crystal substrate having piezoelectricity such as lithium tantalate (LiTaO ₃ ) single crystal, lithium niobate (LiNbO ₃ ) single crystal, or quartz. The planar shape and various dimensions of the piezoelectric substrate 353 may be set as appropriate. As an example, the thickness of the piezoelectric substrate 353 is 0.3 mm to 1.0 mm. The piezoelectric substrate 353 is disposed so that its main surface is parallel to the base 343.

The first IDT electrode 355A and the second IDT electrode 355B (hereinafter simply referred to as “IDT electrode”, which may not be distinguished from each other) include a conductor layer positioned on the upper surface of the piezoelectric substrate 353. The first IDT electrode 355A and the second IDT electrode 355B are opposed to each other with the flow path 335 interposed therebetween. Each IDT electrode 355 has a pair of comb electrodes. Each comb electrode has a bus bar and a plurality of electrode fingers extending from the bus bar. The pair of comb electrodes are arranged so that the plurality of electrode fingers mesh with each other. The first IDT electrode 355A and the second IDT electrode 355B are spaced apart from each other in the SAW propagation direction, and constitute a transversal IDT electrode.

It is possible to design the frequency characteristics using parameters such as the number of electrode fingers of the IDT electrode 355, the distance between adjacent electrode fingers, and the cross width of the electrode fingers. As the SAW excited by the IDT electrode 355, there are Rayleigh waves, Love waves, leaky waves, and the like, and any of them may be used. The sensor chip 332 uses a love wave, for example.

In the SAW propagation direction (y direction), an elastic member for suppressing SAW reflection may be provided outside the first IDT electrode 355A and the second IDT electrode 355B. The SAW frequency can be set, for example, within a range of several megahertz (MHz) to several gigahertz (GHz). Especially, if it is several hundred MHz to 2 GHz, it is practical, and downsizing of the piezoelectric substrate 353 and thus downsizing of the sensor chip 332 can be realized.

The chip pad 357 is connected to the IDT electrode 355 via the chip wiring 356. The chip pad 357 and the chip wiring 356 are made of a conductor layer located on the upper surface of the piezoelectric substrate 353, for example, like the IDT electrode 355. The chip pad 357 connected to the first IDT electrode 355A is located on the opposite side of the first IDT electrode 355A from the second IDT electrode 355B, and the chip pad 357 connected to the second IDT electrode 355B is the first IDT of the second IDT electrode 355B. It is located on the side opposite to the electrode 355A. From the viewpoint of reducing the arrangement range of the plurality of chip pads 357 in the x direction while ensuring a large area of each chip pad 357, the chip pad 357 is opposed to the first IDT electrode 355A and the second IDT electrode 355 in the opposing direction (y (Direction), the IDT electrode 355 overlaps.

The IDT electrode 355, the chip wiring 356, and the chip pad 357 are made of, for example, gold, aluminum, an alloy of aluminum and copper, or the like. These electrodes may have a multilayer structure. In the case of a multilayer structure, for example, the first layer is made of titanium or chromium, and the second layer is made of aluminum or an aluminum alloy. These thicknesses are, for example, 100 nm to 300 nm.

The upper surface of the piezoelectric substrate 353 is covered with a protective film (not shown) from above the IDT electrode 355 and the chip wiring 356. The protective film contributes to suppressing oxidation of the IDT electrode 355 and the chip wiring 356. The protective film is made of silicon oxide, aluminum oxide, zinc oxide, titanium oxide, silicon nitride, silicon, or the like. In the sample liquid sensor 303, silicon dioxide (SiO ₂ ) is used as a protective film. For example, the protective film is formed over the entire upper surface of the piezoelectric substrate 353 so that the chip pad 357 is exposed. The thickness of the protective film (height from the upper surface of the piezoelectric substrate 353) is, for example, 200 nm to 10 μm, which is larger than the thickness of the IDT electrode 355.

The sensitive part 359 is located between the first IDT electrode 355A and the second IDT electrode 355B on the piezoelectric substrate 353 or the protective film. The sensitive part 359 is located in the flow path 335. As the sensitive part 359, for example, a metal film having a two-layer structure of titanium and gold formed on titanium or chromium and gold formed on chromium can be given.

Of the two

SAW elements

350A and 350B, one SAW element 350A does not have a specific binding substance that binds to the detection target contained in the sample liquid on the surface of the sensitive portion 359, and the other SAW element 350B is specific. Has a binding substance. Hereinafter, the SAW element 350A having no specific binding substance may be referred to as a reference SAW element 350A, and the SAW element 350B having a specific binding substance may be referred to as a detection SAW element 350B. By comparing the two, the change in SAW due to the binding between the sample liquid and the specific binding substance can be measured as described later. Further, for each SAW element, different types of specific binding substances may be immobilized on the metal film of the sensitive portion 359, and different properties or components may be measured for the sample liquid. Both the reference SAW element 350A and the detection SAW element 350B may have a specific binding substance. In that case, the density of specific binding substances in both

SAW elements

350A and 350B is made different. Specifically, the density of the specific binding substance in the reference SAW element 50A may be set lower than the density of the specific binding substance in the detection SAW element 350B.

The two

SAW elements

350A and 350B have the formula: t · V <L (where t is the data read interval time from the two

SAW elements

350A and 350B, V is the flow rate of the sample liquid, L is the two SAW elements 350A, A distance of 350 B). That is, the above formula means that the distance through which the sample liquid flows between the first data reading and the second data reading is smaller than the distance between the

SAW elements

350A and 350B, for example. As a result, it is possible to measure the detection target contained in the sample liquid described later.

The detection SAW element 350B and the reference SAW element 350A may have different densities of specific binding substances. Specifically, the density of the specific binding substance in the reference SAW element 350A may be lower than that of the detection SAW element 350B. Specific binding substances include aptamers consisting of nucleic acids and peptides. The aptamer is immobilized on the surface of the sensitive part 359.

When the sample liquid comes into contact with the sensitive part 359 to which the aptamer is immobilized, a specific target substance in the sample liquid is combined with an aptamer corresponding to the target substance, and the weight of the sensitive part 359 changes. As a result, the phase characteristics of the SAW propagating from the first IDT electrode 355A to the second IDT electrode 355B change. Therefore, the properties or components of the sample liquid can be examined based on the change in the phase characteristics and the like.

The SAW element including the combination of the first IDT electrode 355A, the second IDT electrode 355B, and the sensitive portion 359 may be provided in an appropriate number in the flow channel direction of the flow channel 335 (the flow direction of the sample liquid). A method of mounting the sensor chip 332 on the base 343 may be appropriate. In the present embodiment, the mounting method of the sensor chip 332 is surface mounting using the bonding wire 365. When the sample liquid sensor has a sensor chip, the sensor chip mounting method is not limited to surface mounting using wire bonding. For example, flip chip mounting using bumps may be used, or lead insertion mounting in which leads are inserted into a substrate may be used.

In the sensor chip 332, IDT electrodes 355 (first IDT electrode 355A and second IDT electrode 355B) are connected to a chip pad 357 via a chip wiring 356, and the chip pad 357 is connected to an external terminal 331 by a bonding wire 365. .

<Sample fluid sensor device>
Next, the sample liquid sensor device 301 equipped with the sample liquid sensor 303 will be described with reference to FIGS. FIG. 11A is a perspective view showing the sample liquid sensor device 301 (hereinafter simply referred to as “device 301”) in a closed state. FIG. 11B is a perspective view showing a part of the apparatus 301 in an open state and a state before the sample liquid sensor is attached. FIG. 12 is a perspective view showing the device 301 in a closed state.

The apparatus 301 includes a reader 305 to / from which the sample liquid sensor 303 is attached / detached. The reader 305 includes a first part 307 (for example, a fixing portion) and a second part 309 (for example, a fixed part) that are connected so as to be capable of transition (relatively movable) between the open state illustrated in FIG. 11 and the closed state illustrated in FIG. A movable part) and a connection component 302 including a connection terminal 321, a positioning pin 323, and a terminal holding member 329 (contact unit) located on the upper surface of the first portion 307. Both the first part 307 and the second part 309 constitute the outer shape of the reader 305.

In the reader 305, the connection terminal 321 located on the upper surface of the first part 7 is closed by the external terminal 331 of the sample liquid sensor 303 that is detachably sandwiched between the upper surface of the first part 307 and the lower surface of the second part 309. Connected in state. The reader 305 inputs an electrical signal from the connection terminal 321 to the external terminal 331 and receives an electrical signal output from the sample liquid sensor 303. At this time, the sample liquid sensor 303 that has aspirated and accommodated the sample liquid changes the input electric signal according to the property or component of the sample liquid and outputs it.

As shown in FIGS. 9 and 10, the external terminal 331 is connected to the lower surface side of the sample liquid sensor 303 so as to come into contact with the connection terminal 321 on the first part 307 when the sample liquid sensor 303 is sandwiched between the readers 305. It is provided on the first part 307 side). The number and arrangement of the external terminals 331 are, for example, the external terminals 331 arranged along the flow path 335 at both ends in the width direction of the flow path 335 in the present embodiment. It is set appropriately according to the internal circuit configuration and the like.

In the open state, the second part 309 is farther from the first part 307 than in the closed state (FIG. 11A), and in the closed state, the second part 309 overlaps the upper surface of the first part 307 and faces the first part 307 ( FIG. 12). Therefore, when the reader 305 is opened, the surfaces of the first part 307 and the second part 309 facing each other are exposed to the outside. By placing the specimen liquid sensor 303 on the exposed upper surface of the first part 307 and displacing (moving) the second part 309 to make it close, the specimen liquid sensor 303 has the first part 307 and the second part. 309 and is attached to the reader 305. Further, when removing the sample liquid sensor 303 from the reader 305, the above procedure may be reversed.

As shown in FIGS. 11 and 12, the transition method (opening / closing mechanism) between the closed state and the open state of the first part 307 and the second part 309 is connected so as to be rotatable around the rotation axis, so-called It may be foldable. For example, as shown in FIG. 12, a first convex portion 309 a is formed at one end of the first portion 307 so as to protrude in the direction (z direction) facing the second portion 309 in the closed state. On the other hand, a notch 309b in which the first convex portion 309a is accommodated is formed at one end of the second portion 309. In other words, a pair of second convex portions 309c constituting the notch 309b is formed at one end of the second portion 309. A hinge member (not shown) is inserted in the y direction through the first convex portion 309a and the second convex portion 309c, so that the first portion 307 and the second portion 309 are rotated about the rotation axis parallel to the y direction. They are connected to each other in a rotatable manner. As such an opening / closing mechanism, a known cellular phone or a notebook personal computer opening / closing mechanism may be used. Note that the opening / closing mechanism of the first part 307 and the second part 309 is not limited to the case where one end is connected or fixed using a hinge member or the like as described above, and the two parts that exist independently are fitted. A method of matching may be used. According to this, for example, after the sample liquid sensor 303 is placed on the upper surface of the first part 307, the second part 308 is fitted to the first part 307 from above to be in a closed state. . In addition, in a state where the upper surface of the first part 307 and the lower surface of the second part 308 are fitted to each other, the specimen liquid sensor 303 can be inserted between the two so as to be in a closed state. is there.

The shape and material of the first part 307 and the second part 309 are not particularly limited, but are preferably small and lightweight so that the user can carry them. For example, the first part 307 and the second part 309 are made of a resin such as polyethylene terephthalate (PET). It is good to be.

As shown in FIG. 11B, the first part 307 includes a connection component 302 for electrically connecting the sample liquid sensor 303 for positioning and fixing the sample liquid sensor 303 and the reader 305. You may have.

The positioning pin 323 protrudes from the upper surface of the first part 307. The positioning pin 323 is provided in the first part 307 as the connection component 302, but may be integrally formed with the first part 307, for example. In this embodiment, the case where two positioning pins 323 having a circular cross section are provided is illustrated, but the present invention is not particularly limited thereto, and the number, arrangement position, cross-sectional shape, diameter, and height of the positioning pins 323 are as follows. May be set appropriately.

On the other hand, the specimen fluid sensor 303 has a positioning hole 303h into which the positioning pin 323 is fitted. Then, the specimen liquid sensor 303 is fitted in the positioning hole 303h with the positioning pin 323, whereby the specimen liquid sensor 303 is moved in the direction along the xy plane (the plane direction along the facing surface of the first part 307 and the second part 309 in the closed state). Positioning with respect to one part 307 is performed. As another example, the specimen liquid sensor 303 may be provided with a downward positioning pin, and a positioning hole may be provided on the opposing surface of the first part 307.

As shown in FIG. 13, the terminal holding member 329 has a plurality of connection terminals 321 at the upper part and a circuit terminal 322 connected to the circuit board 328 at the lower part. Note that the terminal holding member 329 is fixed to the upper surface of the first portion 307 with a screw 333 as shown in FIG.

The terminal holding member 329 is, for example, generally formed in a plate shape. The wiring of the terminal holding member 329 is connected by a connector and a signal line made of an FPC (flexible wiring board) or the like disposed inside the first part 307 through an opening formed in the first part 307, for example. Yes. As shown in FIG. 13, the first portion 307 further includes a circuit board 328 below the terminal holding member 329. The circuit board 328 and the terminal holding member 329 are connected by a circuit terminal 322. As will be described later, the circuit board 328 detects the sample liquid data and transmits / receives the data to / from an external device or the like regarding the detection of the sample liquid data.

The terminal holding member 329 is fixed to the upper surface of the first portion 307 by a screw 333 and is exposed to the outside when the reader 305 is open.

As shown in FIG. 11A, the reader 305 has a temperature adjustment unit 325 on the lower surface of the second portion 309 that can perform at least one of heating and cooling of the sample liquid sensor 303. The temperature adjusting unit 325 is a member including a thermoelectric conversion element such as a Peltier element. The Peltier element includes, for example, a semiconductor, electrodes disposed on both sides thereof, and heat dissipating plates disposed on both sides thereof.

The temperature adjustment unit 325 may include a display unit 326 on the upper surface (the surface opposite to the surface facing the first region 307) in the second region 309 (FIG. 12). It is preferable that the display unit 326 is adjacent to the temperature adjusting unit 325 so as to be able to exchange heat, and is attached to the surface of the second portion 309 so that the user can visually recognize it. An example of the display unit 326 is a liquid crystal display unit. The display unit 326 displays data detected by the sample liquid sensor 303 from the sample liquid. The display unit 326 can cool heat generated by the operation of the display unit 326 adjacent to the cooling surface side of the temperature adjustment unit 325.

FIG. 14 is a block diagram showing the configuration of the signal processing system of the apparatus 301. The device 301 (reader 305) is used by being connected to a personal computer (PC) 101, for example. Although not particularly illustrated, the reader 305 is appropriately provided with a connector conforming to a predetermined standard for connection with the PC 101. The PC 101 is connected to interfaces such as the display unit 103 and the operation unit 105, for example. The display unit 103 and the operation unit 105 may constitute a touch panel.

For example, the PC 101 displays information prompting the user's operation on the display unit 103 and outputs a control signal to the reader 305 based on the user's operation on the operation unit 105. The reader 305 inputs an electrical signal to the sample liquid sensor 303 in accordance with a control signal from the PC 101. Further, the reader 305 performs appropriate processing such as amplification, filtering, or AD (analog / digital) conversion on the electrical signal output from the sample liquid sensor 303 and outputs the processed electrical signal to the PC 101. The PC 101 causes the display unit 103 to display information on the properties or components of the sample liquid based on the electrical signal from the reader.

The reader 305 includes at least a transmission circuit 373 that generates an electrical signal input to the first IDT electrode 355A, a reception circuit 375 that receives an electrical signal output from the second IDT electrode 355B, and the temperature adjustment unit 325 described above. It has a temperature sensor 377, a control unit 379 that performs these controls, and a power supply unit 381 that supplies these electric power.

The transmission circuit 373 is configured by, for example, an IC and includes a high frequency circuit. Then, the transmission circuit 373 generates an AC signal having a frequency and voltage corresponding to the signal from the control unit 379 and inputs the AC signal to the first IDT electrode 355A.

The receiving circuit 375 is configured by, for example, an IC (integrated circuit) or the like, and includes an amplifier circuit, a filter, or an AD conversion circuit. Then, the reception circuit 375 performs an appropriate process on the electrical signal output from the second IDT electrode 355B and outputs it to the control unit 379.

The control unit 379 includes a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), and the like. Then, based on the control signal from the PC 101, the transmission circuit 373 and the reception circuit 375 are driven. In addition, the control unit 379 performs feedback control of the temperature adjustment unit 325 so that the temperature detected by the temperature sensor 377 configured by a resistance type contact temperature sensor such as a thermistor converges to a predetermined target value. Do. The target value is input from the PC 101, for example.

The power supply unit 381 includes an inverter or a converter, converts the power from the commercial power supply or the PC 101 into an appropriate voltage, and transmits a transmission circuit 373, a reception circuit 375, a temperature adjustment unit 325, a temperature sensor 377, and a control unit 379. To supply.

Further, a temperature sensor 377 may be provided. This temperature sensor 377 is constituted by a contact type temperature sensor such as a thermistor, for example, and is provided in the vicinity of the position where the sample liquid sensor 303 is placed, and controls an electric signal according to the ambient temperature. Output to the unit 379.

In the present embodiment, the external terminal 331 is provided on the lower surface side (the first part 307 side) of the sample liquid sensor 303, but the present invention is not limited to such a correspondence. An external terminal may be formed on the upper surface side (the second part 309 side) of the sample liquid sensor 303 so as to contact a connection terminal provided on the lower surface of the second part 309. Further, an external terminal may be formed on the side surface of the sample liquid sensor 303 so as to come into contact with a connection terminal provided in the first part 307 or the second part 309.

<Measurement method of sample liquid>
Next, a method for measuring a specific detection target contained in the sample liquid using the apparatus 301 equipped with the above-described sample liquid sensor 303 will be described.
FIG. 15A shows that in the embodiment shown in FIG. 10, the sample liquid flows in the direction indicated by the arrow in the order of the reference SAW element 350A and the detection SAW element 350B. In this case, the reference SAW element 350A and the detection SAW element 350B have the formula: t · V <L (where t is the data reading interval time from both SAW elements, V is the flow rate of the sample liquid, and L is both SAW elements) As described above. Here, the data reading interval time t from both the SAW elements 350 </ b> A and 350 </ b> B can be set by the control unit 379 in the reader 305 by operating the operation unit 105. Further, since the sample liquid flows between the base 343 and the cover 345 by capillary action, the flow rate V of the sample liquid is adjusted by adjusting the height (thickness, z direction) in the flow path 335 or the like. Can be adjusted. The position where the flow velocity V of the sample liquid is measured may be, for example, within the region indicated by the symbol D in FIG. That is, it is in the region from the upstream end of the reference SAW element 350A where the sample liquid reaches first to the downstream end of the next detection SAW element 350B. The flow velocity V is measured by, for example, a method of measuring the distance that the sample liquid flows within a predetermined time by photographing with a high-speed camera, a method of measuring and obtaining the time that the sample liquid flows through the predetermined distance, and the detection SAW element 350B. A method for obtaining the flow rate flowing out from the downstream side of the gas by dividing the flow rate by the cross-sectional area of the flow path is exemplified, and the measurement method is not particularly limited.

As shown in FIG. 15A, the distance L between the two

SAW elements

350A and 350B is one end of one SAW element 350A and one end positioned on the upstream side of the flow of the sample liquid, and the other SAW element 350B. It is the distance to one end which is also located on the upstream side. Here, the one ends of the

SAW elements

350A and 350B are, for example, one end (upstream end) of the sensitive part 359 and the IDT electrode 355 (first IDT electrode) in the

SAW elements

350A and 350B shown in FIG. 355A and the second IDT electrode 355B) may be a portion on the upstream side of one end (upstream end).

As shown in FIG. 15A, when the sample liquid flowing in the flow path 335 reaches the surface of the sensitive part 359 of the

SAW elements

350A and 350B, mass is added to the sensitive part 359. As a result, the phase θ of the SAW propagating from the first IDT electrode 355A to the second IDT electrode 355B is delayed and changes to minus. FIG. 15B shows an example of the change over time of the phase θref of the reference SAW element 350A and the phase θtest of the detection SAW element 350B, and FIG. 15C shows the positions of θref and θtest. The change over time of the phase difference Δθ (θref−θtest) is shown. Hereinafter, the steps (1) to (5) shown in FIG. Note that the regions represented by the steps (1) to (5) are schematically indicated by arrows in FIG.

Stage (1): This is an initial state in which the sample liquid has not reached either the reference SAW element 350A or the detection SAW element 350B. At this stage, since no mass is added to the SAW element, neither θref nor θtest is changed (θref = θtest = 0). Therefore, the phase difference Δθ is not changed.

Stage (2): A state in which the sample liquid reaches the upstream end of the reference SAW element 350A and then reaches the downstream end of the reference SAW element 350A. At this stage, since the mass of the sample liquid is added to the reference SAW element 350A, θref changes to a negative value, but since the mass of the sample liquid is not added to the detection SAW element 350B, θtest does not change. . Therefore, the phase difference Δθ changes to minus.

Stage (3): A state in which the sample liquid passes through the reference SAW element 350A and passes through the gap C between the reference SAW element 350A and the detection SAW element 350B. At this stage, the sample liquid has not reached the detection SAW element 350B, and the mass of the sample liquid added to the reference SAW element 350A has reached the maximum (the entire upper surface of the reference SAW element 350A is the sample surface). It is covered with liquid), and there is no change in θref. Accordingly, in the time region between the time point (A) when the mass addition of the sample liquid to the reference SAW element 350A reaches the maximum (A) and the time point (B) when the mass of the sample liquid is added to the detection SAW element 350B. The phase difference Δθ is a minimum value P (extreme value).

Stage (4): A state in which the sample liquid reaches the upstream end of the detection SAW element 350B and then reaches the downstream end of the detection SAW element 350B. At this stage, since the mass addition of the sample liquid to the reference SAW element 350A reaches the maximum due to the continuous inflow of the sample liquid, θref does not substantially change. On the other hand, since the mass of the sample liquid is added to the detection SAW element 350B, θtest changes to minus. Therefore, the phase difference Δθ changes to the plus side. When the inflow of the sample liquid further proceeds and the addition of the mass of the sample liquid to the detection SAW element 350B reaches the maximum, the phase difference Δθ becomes zero.

Stage (5): As in the above stage (4), the mass addition due to the inflow of the sample liquid into the detection SAW element 350B has reached a maximum, and the detection target (receptor) in the sample liquid And a specific binding substance are bound to each other. At this stage, since the mass of the receptor is added in addition to the mass of the specimen liquid, the phase θtest in the detection SAW element 350B changes to a negative value larger than the phase θref in the reference SAW element 350A. Therefore, the phase difference Δθ changes to plus. It should be noted that even after mass addition due to the inflow of the sample liquid into the detection SAW element 350B reaches the maximum, the sample liquid may flow continuously or intermittently and flow out from the downstream end of the detection SAW element 350B. . Alternatively, the flow of the sample liquid may be stopped when the addition of mass due to the flow of the sample liquid into the detection SAW element 350B reaches the maximum. In any case, the phase difference Δθ in step (5) continues to change while the binding reaction continues between the detection target (receptor) in the sample liquid and the specific binding substance in the detection SAW element 350B. Thereafter, when the above-described binding reaction in the detection SAW element 350B reaches saturation, the phase difference Δθ becomes a constant value.

Here, the concentration of the detection target in the sample liquid, which is the above-described receptor, is proportional to the amount of binding of the detection target to the specific binding substance possessed by the detection SAW element 350B, and thus the negative change amount of θtest. For example, when the concentration is higher than the concentration of the detection target in the sample liquid shown in FIGS. 15B and 15C, or when the density of the specific binding substance is high, FIG. As shown in b), the negative change in θtest in step (5) is larger than the negative change in θtest shown in FIG.

15 (c) and 17 (b), the phase difference Δθ has a minimum value P (extreme value) at a certain time. As is clear from FIGS. 15B and 17A, this minimum value P is the time when the sample liquid reaches the detection SAW element 350B and the mass of the sample liquid is added to the detection SAW element 350B. Is shown.

The concentration of the detection target in the sample liquid can be determined by the amount of binding of the detection target to the specific binding substance of the detection SAW element 350B. This amount of coupling can be determined from the amount of change in the phase difference Δθ within a certain time, the slope of the phase difference Δθ over a certain time, and the like. Therefore, the point in time when the mass of the sample liquid is added to the detection SAW element 350B, that is, the minimum value P of the phase difference Δθ or the later can be set as the starting point for measuring the detection target. Here, the value after the minimum value P can be appropriately selected according to the movement of the output signal. However, the time after the minimum value P and when the value returns to a value close to the initial value before the introduction of the sample liquid (for example, The time until the phase difference Δθ returns to zero) may be used. The measurement of the detection target is preferably started from the point in time when the sample liquid flows into the downstream SAW element (detection SAW element 350B in this embodiment), and according to this, the upstream SAW element (this embodiment) In the embodiment, it is possible to accurately measure the signal change itself in the downstream SAW element (the detection SAW element 350B in this embodiment) without being substantially affected by the signal from the reference SAW element 350A). . That is, in the detection SAW element 350B on the downstream side, it is possible to accurately measure a signal change based on the binding reaction that occurs between the detection target in the sample liquid and the specific binding substance.

As described above, in order to cause an extreme value (maximum value or minimum value) to appear in the temporal change of the phase difference Δθ, both the

SAW elements

350A and 350B may have a relationship of the formula: t · V <L. . On the other hand, when both

SAW elements

350A and 350B do not have the relationship of the formula: t · V <L, an extreme value (maximum value or minimum value) cannot appear or is difficult. There is.

In order to measure the concentration or the like of the detection target, for example, by using a sample liquid containing the detection target of a known concentration, the amount of change in the phase difference Δθ within a certain time period or the phase difference Δθ at a certain time is measured. A slope or the like is measured, and a calibration curve is created with the amount of change or slope as the horizontal axis and the vertical axis as the concentration to be detected. Next, with respect to the actual sample liquid, the amount of change in the phase difference Δθ, the slope of the phase difference Δθ at a certain time, and the like can be measured, and the concentration and the like of the detection target can be measured from the calibration curve.

In FIG. 15B, when the mass addition of the sample liquid to the reference SAW element 350A reaches the maximum (A), and when the mass of the sample liquid is added to the detection SAW element 350B (B). And are relatively close. On the other hand, when the distance L between both SAW elements is large in the above equation (for example, when the distance C between both

SAW elements

350A and 350B is large as shown in FIG. 18A), as shown in FIG. 18B. In some cases, time point (B) appears later than time point (A). This can also occur when the flow rate V of the sample liquid is small. In such a case, as shown in FIG. 18C, the minimum value P has a certain time region. In the present embodiment, any time point of the minimum value P having such a constant time region may be set as the starting point. This is because there is only a time lag between the case where the starting point is the right end of the fixed time region and the case where the starting point is the left end. In FIGS. 18B and 18C, (1) to (5) indicate the stages shown in FIG.

As described above, instead of flowing the sample liquid in the order of the reference SAW element 350A and the detection SAW element 350B, the sample liquid flows in the order of the detection SAW element 350B and the reference SAW element 350A. The arrangement of the reference SAW element 350A and the detection SAW element 350B may be reversed. In such an arrangement, if the change over time of the phase difference Δθ (= θref−θtest) is calculated from the measured values of θref and θtest, a local maximum value (extreme value) appears instead of a local minimum value. Therefore, the maximum value or the subsequent value may be used as a starting point for measuring the detection target.

The number of SAW elements is not limited to two, that is, the reference SAW element 350A and the detection SAW element 350B, and three or more reference SAW elements and the detection SAW elements are arranged in the flow direction of the sample liquid. It may be. Specifically, for example, three or more SAW elements have at least one SAW element having a specific binding substance that binds to a detection target contained in the sample liquid, and have no specific binding substance or specific binding. And at least one other SAW element whose material is less than the at least one SAW element described above. As described above, the three or more SAW elements may have different types of specific binding substances. In addition, among the three or more SAW elements, SAW elements having specific binding substances may have different specific binding substances or may have different specific binding substances. Here, when the density of specific binding substances is different among a plurality of SAW elements, the same detection target may be measured. Further, when a plurality of SAW elements have different specific binding substances, they may measure the same detection target, or may measure different detection targets. For example, RS, human metaneum, adeno, influenza, etc. are mentioned as different detection targets, and a plurality of detection targets (viruses) including these can be measured simultaneously.

19A and 19B show an example of the arrangement of three or more such SAW elements. In FIG. 19A, a reference SAW element 501A that does not have a specific binding substance and

detection SAW elements

501B and 502B are arranged in this order in the flow direction of the sample liquid indicated by an arrow. The two

detection SAW elements

501B and 502B may have different or the same specific binding substance densities. The

detection SAW elements

501B and 502B may have different specific binding substances. In FIG. 19B, the reference SAW element 501A, the detection SAW element 501B, the reference SAW element 502A, and the detection SAW element 502B are arranged in this order in the flow direction of the sample liquid indicated by the arrow. These reference SAW elements and detection SAW elements may have different detection targets or may be the same.

In the arrangement of the SAW elements shown in FIGS. 19A and 19B, the reference SAW element for measuring the detection target and the detection SAW element may have a relationship of the above formula: t · V <L. As long as it is necessary and has this relationship, the distance L between adjacent SAW elements may be the same or different. Specifically, for example, in FIG. 19A, the reference SAW element 501A and the detection SAW element 501B are adjacent to each other, while the reference SAW element 501A and the detection SAW element 502B are adjacent to each other. And have no relationship. In any case, if the relationship is set so as to have the relationship of the above formula: t · V <L, as described above, the extreme value or the subsequent value is used as the signal starting point from the sample liquid sensor, and the subsequent change with time By using this to measure the detection target, measurement (concentration measurement, etc.) of the detection target contained in the sample liquid becomes easy and accurate. Further, even when the plurality of SAW elements have three or more SAW elements positioned at different intervals, similarly, the plurality of SAW elements may be set so as to have the relationship of the above formula: t · V <L.

As described above, according to the sample liquid measurement method according to the present embodiment, the reference SAW element and the detection SAW element have the relationship of the above formula. Therefore, the extreme value (maximum value or minimum value) appears in the difference between the phase θref of the surface acoustic wave in the reference SAW element and the phase θtest of the surface acoustic wave in the detection SAW element, that is, the change over time of the phase difference Δθ. To do. This extreme value represents a point in time when the sample liquid flowing through one SAW element reaches the other SAW element. Therefore, measurement of the detection target contained in the sample liquid (concentration measurement) is performed by measuring the detection target using the extreme value or subsequent time as a signal starting point from the sample liquid sensor and using the change over time of the extreme value or subsequent time. Etc.) is easy and accurate.

FIG. 20 shows a sample liquid sensor according to still another embodiment of the present invention. The sample liquid sensor 330 includes a frame body 370 in which the sensor chip 332 ′ surrounds the reference SAW element 350′A and the detection SAW element 350′B. The sample liquid sensor 330 is formed between the base 343 and the cover 345 in the above-described embodiment, and does not have a flow path 335 that allows the sample liquid to flow between them using a capillary phenomenon. Instead, a frame body 370 for preventing the sample liquid from flowing out is provided on the sensor chip 332 ′. The frame body 370 is not particularly limited as long as it is a member that can prevent the flow of the sample liquid, and examples thereof include a resin material.

The specimen liquid is dropped at a dropping position 340 shown in FIG. 20, for example. Then, it may flow from the dropping position 340 toward the reference SAW element 350′A and the detection SAW element 350′B, and flow into both SAW elements 350′A and 350′B in this order. In order to cause the sample liquid to flow from the dropping position 340 as described above, for example, a method of providing an inclination to the sensor chip 332 ′ or using the surface tension of the surface of the sensor chip 332 ′ can be employed.

Note that the inflow of the sample liquid may be in the order of the detection SAW element 350′B and the reference SAW element 350′A in the same manner as in the above-described embodiment. Similarly to the above-described embodiment, the sample liquid may be sequentially introduced into three or more SAW elements. Since others are the same as that of the above-mentioned embodiment, detailed description is abbreviate | omitted.

The present invention is not limited to the above embodiment, and various changes and improvements can be made. For example, in the above embodiment, the temperature adjustment unit 325 is provided in the second part 309 of the reader 305, but may be provided in the first part 307. As illustrated in FIG. 21, the temperature adjustment unit 325 ′ may be provided in a region between the pair of

connection terminals

321 and 321 rows on the upper surface of the first portion 307. According to this, an electric circuit can be made into a simple structure in the 2nd site | part 309 of the reader | leader 305 by providing the main component in the 1st site | part 307 of the reader | leader 305. FIG. Further, as shown in FIG. 22, the sample liquid sensor 303 ′ may be mounted inside the reader 305 so that it is not exposed to the outside when the reader 305 is closed. According to this, it is possible to reduce the influence of the external environment such as electromagnetic waves during measurement. In the above-described embodiment, the configuration in which no special member is present in the flow path 335 has been described. Instead, a porous member is disposed in a part or all of the flow path 335. May be. This makes it possible to control the flow rate of the sample liquid flowing through the flow path 335. In addition, when providing a porous member in a part inside flow path 335, it can form so that a porous member and the SAW element 350 may contact | connect, for example.

The embodiments shown in FIGS. 9 to 22 and the description thereof are embodiments corresponding to the following aspects of the sample liquid measurement method, sample liquid sensor, and sample liquid sensor device.

(Aspect 1) The piezoelectric substrate has a plurality of SAW elements positioned along the flow direction of the specimen liquid on the upper surface of the piezoelectric substrate, and among the plurality of SAW elements, the reference SAW element and the detection SAW element have the formula: a step of preparing a sensor chip having a relationship of t · V <L (where t is a data reading interval time from both SAW elements, V is a flow rate of the sample liquid, and L is a distance between both SAW elements) , The step of flowing the sample liquid sequentially from one side of either the reference SAW element or the detection SAW element, the phase θref of the surface acoustic wave in the reference SAW element, and the surface acoustic wave in the detection SAW element Calculating a change with time of the phase difference Δθ, which is a difference in the phase θtest, and measuring a detection target starting from an extreme value or a time point after that of the change with time of the phase difference Δθ. Measurement method of body fluids.

(Aspect 2) The detection SAW element has a specific binding substance that specifically binds to a detection target contained in a sample liquid, and the reference SAW element does not have the specific binding substance or The method for measuring a sample liquid according to aspect 1, wherein the density of the specific binding substance is lower than that of the detection SAW element.

(Aspect 3) The method for measuring a sample liquid according to

Aspect

1 or 2, further comprising a step of measuring a change with time of the phase θref and a step of measuring a change with time of the phase θtest.

(Aspect 4) The method for measuring a specimen liquid according to any one of aspects 1 to 3, wherein the specimen liquid is flowed in the order of the reference SAW element and the detection SAW element.

(Aspect 5) Each of the reference SAW element and the detection SAW element includes a first IDT electrode electrode, a second IDT electrode electrode positioned in a propagation path of the surface acoustic wave excited by the first IDT electrode electrode, A sensitive portion positioned between the first IDT electrode electrode and the second IDT electrode electrode, and the sample liquid is supplied to the sensitive portion of the reference SAW element and the sensitive portion of the detection SAW element. The method for measuring a sample liquid according to any one of aspects 1 to 4, wherein the sample liquid is flowed.

(Aspect 6) The sensor chip further includes a flow path in which the sample liquid flows and the reference SAW element and the detection SAW element are positioned in the flow direction, and the reference SAW among the flow paths. 6. The method for measuring a sample liquid according to any one of aspects 1 to 5, further comprising a step of causing the sample liquid to flow from an upstream side of any of the element and the detection SAW element.

(Aspect 7) The sensor chip further includes a frame body that surrounds the reference SAW element and the detection SAW element, and from one side of the reference SAW element and the detection SAW element inside the frame body. 6. The method for measuring a sample liquid according to any one of aspects 1 to 5, further comprising a step of allowing the sample liquid to flow toward the other side.

(Aspect 8) The method for measuring a specimen liquid according to any one of aspects 1 to 7, wherein the measurement of the detection target is performed from a time point until the phase difference Δθ returns from the extreme value to zero.

(Aspect 9) A substrate and a sensor chip positioned on the upper surface of the substrate, wherein the sensor chip is positioned on the upper surface of the substrate, and the specimen liquid is formed on the upper surface of the piezoelectric substrate. A plurality of SAW elements, and two SAW elements of the plurality of SAW elements are expressed by the formula: t · V <L (where t is determined from the two SAW elements) Data reading interval time, V is the flow rate of the sample liquid, and L is the distance between the two SAW elements.)

(Aspect 10) Each of the plurality of SAW elements includes a first IDT electrode electrode, a second IDT electrode electrode positioned in a propagation path of the surface acoustic wave excited by the first IDT electrode electrode, the first IDT electrode electrode, The specimen liquid sensor according to aspect 9, further comprising: a sensitive portion located between the second IDT electrode and the specimen liquid flow path.

(Aspect 11) Of the two SAW elements, one SAW element further has a specific binding substance that binds to a detection target contained in the sample liquid, and the other SAW element has the specific binding substance. The specimen liquid sensor according to

aspect

9 or 10, wherein

(Aspect 12) Each of the two SAW elements further includes a specific binding substance that binds to a detection target contained in the sample liquid, and one SAW element and the other SAW element are formed of the specific binding substance. The specimen liquid sensor according to

aspect

9 or 10, wherein the densities are different.

(Aspect 13) The specimen fluid sensor according to aspect 11 or 12, wherein the specific binding substance includes an aptamer.

(Aspect 14) The sensor chip further includes a cover that covers the base, and the flow path of the specimen liquid is located between the base and the cover that covers the base. The specimen liquid sensor according to any one of the above.

(Aspect 15) The sensor chip further includes a frame that is positioned on the upper surface of the piezoelectric substrate and surrounds the two sensitive parts of the two SAW elements, and the flow path of the sample liquid is the 14. The specimen liquid sensor according to any one of aspects 9 to 13, which is located inside.

(Aspect 16) The sample liquid sensor according to any one of Aspects 9 to 15, wherein the two SAW elements are positioned adjacent to each other.

(Aspect 17) The sample liquid sensor according to any one of aspects 9 to 15, wherein the two SAW elements are not positioned adjacent to each other.

(Aspect 18) The plurality of SAW elements have three or more SAW elements positioned at equal intervals, and SAW elements adjacent to or not adjacent to each other among the three or more SAW elements are respectively The specimen liquid sensor according to

aspect

9 or 10, wherein the formula: t · V <L.

(Aspect 19) The plurality of SAW elements include three or more SAW elements positioned at different intervals, and the SAW elements adjacent to or not adjacent to each other among the three or more SAW elements are respectively The specimen liquid sensor according to

aspect

9 or 10, wherein the formula: t · V <L.

(Aspect 20) Among the three or more SAW elements, at least one SAW element has a specific binding substance that binds to a detection target contained in the sample liquid, and the other at least one SAW element includes the above-mentioned 20. The analyte liquid sensor according to

aspect

18 or 19, which does not have a specific binding substance.

(Aspect 21) Of the three or more SAW elements, two SAW elements have a specific binding substance that binds to a detection target contained in the sample liquid, and the two SAW elements have the specific binding 20. The specimen liquid sensor according to

aspect

18 or 19, wherein the substance density is different.

(Aspect 22) The three or more SAW elements include a first SAW element having the specific binding substance and a second SAW element having the specific binding substance, and the specific binding substance of the first SAW element and the The specimen liquid sensor according to aspect 20 or 21, which is different from the specific binding substance of the second SAW element.
(Aspect 23) A specimen liquid sensor device comprising: the specimen liquid sensor according to any one of aspects 9 to 22; and a reader to which the specimen liquid sensor is detachably attached.

The present invention is not limited to the above-described embodiment, and the same effect can be obtained even if the detection target is a different type of antigen.

As described above, the present invention is not limited to the configurations shown in the above-described embodiments and modifications, and it goes without saying that improvements and modifications can be made without departing from the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Flow path structure 3 Detection element 4 Support member 6 Terminal 7 Piezoelectric board 8 1st IDT electrode 9 2nd IDT electrode 10 Supply part 13 Detection part 14 Inlet 20 Reaction part 30 Measurement part 40 Detection part 50 Display part 100 Detection apparatus DESCRIPTION OF SYMBOLS 200 Biosensor apparatus 301 Sample liquid sensor apparatus 302 Connection component 303,330 Sample liquid sensor 305 Reader 307 1st site | part 309 2nd site | part 321 Connection terminal 323 Positioning pin 325 Temperature adjustment part 326 Display part 329 Terminal holding member 331 External terminal 332 Sensor Chip 335 Channel 342 Through-hole 343 Base 349 Conductor 350A, 350'A Reference SAW element 350B, 350'B Detection SAW element 370 Frame C Gap L Distance

Claims

A method for detecting a detection target contained in a sample,
A sample is supplied to the surface of a detection body in which a primary substance that reacts with the detection target is bound to the surface, and a primary reaction product is formed on the surface of the detection body by a reaction between the detection target and the primary substance. A first reaction step;
After the first reaction step, a first supply step of supplying a first liquid to the surface of the detection body;
A first measurement step of measuring a first signal value based on a surface state of the detection body after the first supply step;
After the first measuring step, a signal amplification substance is supplied to the surface of the detection body, and the signal changes the surface state of the detection body by a reaction involving the primary reactant formed in the first reaction step. An amplification process;
A second measurement step of measuring a second signal value based on the surface state of the detection body after the signal amplification step;
A detection method comprising:
The detection method according to claim 1, further comprising a second supply step of supplying a second liquid to a surface of the detection body between the signal amplification step and the second measurement step.
The detection method according to claim 1 or 2, further comprising an additional reaction step of supplying at least one additional reactive substance to the surface of the detection body between the first measurement step and the signal amplification step.
The detection method according to claim 3, wherein the at least one additional reactant includes a plurality of additional reactants, and the additional reactants are supplied one by one.
The detection method according to claim 1 or 2, further comprising a precursor reaction step of supplying at least one precursor reactant to the surface of the detection body between the first reaction step and the first supply step.
6. The detection method according to claim 5, wherein the at least one precursor reactant has a plurality of precursor reactants, and the precursor reactants are supplied one by one.
The sample further includes a contaminant different from the detection object,
The contaminant is attached to the surface of the detection body in the first reaction step, and the contaminant remains on the surface of the detection body after the first supply step. The detection method described.
The detection method according to any one of claims 1 to 7, wherein the sample is a biological sample.
The detection method according to any one of claims 1 to 8, wherein in the first reaction step, the detection target and the primary substance are combined to form the primary reaction product.
9. The primary reaction product according to claim 1, wherein, in the first reaction step, the primary reactant is formed by binding the detection target and a part of the primary substance and dissociating the part from the primary substance. The detection method according to any one of the above.
A surface acoustic wave element is formed on the surface of the detection body,
The detection method according to any one of claims 1 to 10, wherein the signal value based on the surface state of the detection body is a value of a phase characteristic of the surface acoustic wave element.
The signal value based on the surface state of the detection object is a value measured by a method selected from a QCM (Quartz Crystal Microbalance) sensor, an SPR (Surface Plasmon Resonance) sensor, and an FET (Field Effect Transistor) sensor. The detection method according to any one of 1 to 10.
The detection method according to any one of claims 1 to 12, wherein the second signal value is larger than the first signal value.
The detection method according to any of claims 1 to 13, wherein the first liquid is a buffer solution.
The detection method according to any one of claims 2 to 14, wherein the first liquid and the second liquid are the same.
16. The detection method according to claim 2, further comprising a first detection step of obtaining a detection value from the first signal value and the second signal value.
An apparatus for detecting a detection target contained in a sample,
A sample is supplied to a primary substance that is coupled to the surface of a detection body and reacts with the detection object, and a first reactant is formed on the surface of the detection body by a reaction between the detection object and the primary substance. A reaction part;
A first supply unit for supplying a first liquid to the surface of the detection body;
A first measurement unit that measures a first signal value based on a surface state of the detection body after the first liquid is supplied to the surface of the detection body by the first supply unit;
Supplying a signal amplification substance to the surface of the detection body, and a signal amplification section that changes the surface state of the detection body by a reaction involving the primary reactant;
A second measurement unit that measures a second signal value based on a surface state of the detection body supplied with the signal amplification substance.