JP2013076642A - Probe for detecting target substance, target substance detection apparatus using the probe, and target substance detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and easy target substance detection method utilizing interaction between substances.SOLUTION: In a target substance detection method, interaction between a sampled target substance and a fixed trapping substance is performed in an inductive capillary including a sampling field for sampling the target substance and a reaction field for fixing the trapping substance for trapping the target substance and performing interaction between both substances to detect emitted light or radioactive information. A probe for detecting a target substance includes an inductive capillary part including a sampling part capable of sampling the target substance by a capillary and a sensitive part for fixing the trapping substance to be bound to the target substance and performing interaction between the substances.

Description

The present disclosure relates to a target substance detection probe, a target substance detection apparatus using the probe, and a target substance detection method.
More specifically, the present disclosure is used as a tool for detecting various target substances, and relates to a target substance detection probe that can detect a target substance by utilizing an interaction between substances. The present disclosure also relates to a target substance detection apparatus using the target substance detection probe. The present disclosure also relates to a target substance detection method that performs an interaction between substances in a capillary tube.

Conventionally, detection methods using interaction between substances such as hybridization of nucleic acid molecules, interaction between proteins, and chemical bonding or dissociation between substances such as antigen-antibody reaction are known.
For example, as an example of a technique that utilizes an electrodynamic effect such as electrophoresis or dielectrophoresis, a technique for detecting double-stranded DNA obtained by hybridization is disclosed (for example, Patent Document 1).
Further, for example, an immunoassay method for detecting a substance to be detected comprising a specific antigen or antibody by utilizing a specific reaction by an antigen-antibody is widely used. Competitive reactions and sandwich reactions are widely used as the mechanism of this type of immunoassay. An ELISA (Enzyme-Linked Immuno-Sorbent Assay) method and an immunochromatographic method using this are known.

For example, Patent Document 2 proposes a simple immunological measurement method that can be quickly and simply determined and a measurement tool used in the measurement method.
A measuring tool is used in which a transparent cover member made of a transparent synthetic resin tube having a large number of grooves formed along the inner longitudinal direction is formed on the outer peripheral surface of a rod-shaped synthetic resin core portion. The sample liquid is sucked up by capillary action using the outer peripheral surface of the core member and the groove on the inner side of the cover member, and the sample liquid is first brought into contact with the labeled antigen or labeled antibody zone formed on the core member to cause an antigen-antibody reaction. After the reaction, the target antigen in the sample liquid is brought into contact with a detection zone made of a specific antigen or antibody, and whether or not a labeled substance attached to the labeled antigen or labeled antibody appears in the detection zone. Or it is confirmed visually whether an antibody exists.

  By the way, in order to detect a target substance, in particular, a substance in a living body, generally, blood is collected and the in-vivo substance in the blood is optically or electrically examined. However, blood collection is an invasive method, and requires a medical qualification for the collector, and is expensive. Furthermore, in actual detection / measurement, purification of blood is necessary, and there is a problem that it takes time and effort to obtain detection / measurement results. In addition, in order to perform continuous measurement, it is necessary to increase the number of samplings, and the burden on the subject is large. Therefore, there is a problem that real-time measurement is impossible. Therefore, in recent years, various methods for reducing invasiveness to living bodies are being developed.

  For example, as a noninvasive detection method, Patent Document 3 proposes a method for easily detecting abnormalities in the digestive system such as stomach cancer and colon cancer. This method uses a collection container consisting of a stool collection device and a liquid container containing a buffer solution prepared in advance so as to prevent the activity of digestive enzymes in the stool. Then, hemoglobin in feces is physically adsorbed on the surface of the collector and separated. Furthermore, the hemoglobin is specifically detected and measured using anti-human enzyme-labeled hemoglobin.

JP 2001-242135 A Japanese Patent Laid-Open No. 10-73593 JP 59-182367 A

Furthermore, there is a need for a method that can easily and easily detect a target substance by utilizing an interaction between substances.
The main object of the present disclosure is to provide a target substance detection probe that can easily and easily detect a target substance by utilizing an interaction between substances. Furthermore, the main object of the present disclosure is to provide a target substance detection apparatus using the target substance detection probe, and a simple and easy target substance detection method using interaction between substances.

  In order to solve the above-described problem, the present disclosure provides an inside of a sensitive capillary having a collection field for collecting a target substance and a reaction field for immobilizing a trapping substance for trapping the target substance and performing an interaction reaction between the substances. Thus, a target substance detection method for detecting the emitted light or radioactive information by performing interaction between substances with the immobilized trap substance on the collected target substance is provided.

  (A) The sample liquid containing the target substance is absorbed into the capillary, the substance is allowed to interact with the target substance with the immobilized trap substance, and then the inside of the capillary is washed; (B) absorbing a solution containing a photolabeled or radioactively labeled detection substance into the capillary to cause the labeled detection substance to interact with the target substance; (C) The target substance detection method including detecting optical information or radioactive information emitted from the bound labeled detection substance is preferable.

  Furthermore, (a) after the reaction of the interaction between the substances with the immobilized trap substance, the capillary is made to absorb the solution containing the detection substance labeled with the enzyme, and the inside of the capillary is washed. And (b) absorbing the solution containing the enzyme substrate in the capillary, and (c) detecting optical information generated by the enzyme substrate being changed, and detecting the target substance. Is preferred.

It is preferable that the immobilized trap substance is a substance in which a trap substance for trapping a target substance is immobilized by absorbing a solution containing the trap substance in a capillary tube.
It is preferred that the enzyme substrate is a dye precursor.
It is preferable that the immobilized trap substance and / or the labeled detection substance is obtained by biotin-avidin crosslinking or biotin-streptavidin crosslinking.
The sample liquid containing the target substance is preferably a body fluid or blood.

The present disclosure includes a sensitive capillary section that includes a collecting section that can collect a target substance with a capillary, and a sensitive section that immobilizes a trapping substance that binds to the target substance and performs an interaction between the substances. A probe for detecting a substance is provided.
The immobilized trap substance is preferably a substance in which a trap substance for trapping a target substance is immobilized by absorbing a solution containing the trap substance in a capillary tube.
The labeled detection substance is preferably a detection substance labeled with an enzyme.
It is preferred that the enzyme substrate is a dye precursor.
It is preferable that the immobilized trap substance and / or the labeled detection substance is obtained by biotin-avidin crosslinking or biotin-streptavidin crosslinking.

The present disclosure provides a target substance detection device including at least the detachable target substance detection probe.
In addition, the present disclosure provides (1) a sensitive capillary section provided with a collecting section capable of collecting a target substance with a capillary, and a sensitive section for immobilizing a trapping substance for trapping the target substance and performing an interaction between the substances. A detachable target substance detection probe comprising: (2) a light irradiation part that irradiates light to the sensitive part of the target substance detection probe; and (3) light irradiation by the light irradiation part There is provided a target substance detection device comprising at least a light detection unit for detecting optical information from the sensitive unit.

  According to the present disclosure, a target substance can be detected simply and easily.

It is a schematic conceptual diagram which shows typically 1st Embodiment of the probe 1 for target substance detection concerning this indication. It is a schematic conceptual diagram which shows typically 2nd Embodiment of the probe 1 for target substance detection concerning this indication. It is a schematic conceptual diagram which shows typically 3rd Embodiment of the probe 1 for target substance detection concerning this indication. It is a schematic conceptual diagram which shows typically the cross-sectional shape of the sensitive capillary part 11 of the probe 1 for target substance detection concerning this indication. It is a schematic conceptual diagram which shows typically 4th Embodiment of the probe 1 for target substance detection concerning this indication, and 1st Embodiment of the target substance detection apparatus 10. FIG. It is a schematic conceptual diagram which shows typically 5th Embodiment of the probe 1 for target substance detection concerning this indication, and 2nd Embodiment of the target substance detection apparatus 10. FIG. It is a schematic conceptual diagram which shows typically 6th Embodiment of the probe 1 for target substance detection concerning this indication, and 3rd Embodiment of the target substance detection apparatus 10. FIG. It is a schematic conceptual diagram which shows typically the production method of the sensitive capillary part 11 of the probe 1 for target substance detection concerning this indication, and the detection method of a target substance. It is a schematic conceptual diagram which shows typically the production method of the sensitive capillary part 11 of the probe 1 for target substance detection concerning this indication, and the detection method of a target substance. It is a schematic conceptual diagram which shows typically the production method of the sensitive capillary part 11 of the probe 1 for target substance detection concerning this indication, and the detection method of a target substance. It is a figure which shows the example of a sampling of a bodily fluid (gingival crevicular fluid). This is the tip of the sensitive capillary section 11 of the target substance detection probe 1 according to the present disclosure and has a shape that can handle a small amount of gingival crevicular fluid. It is the result at the time of measuring TNF- (alpha) by ELISA method using the target substance detection probe 1 concerning this indication. It is a figure which shows the result of capillary ELISA by the flow system using the probe 1 for target substance detection concerning this indication. It is a mimetic diagram showing typically target substance detection device 10 provided with probe 1 for target substance detection concerning this indication.

DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly. The description will be given in the following order.
1. Probe for target substance detection (1) Sensitive capillary part (2) Sampling part (collecting field)
(3) Sensitive part (reaction field)
2. 2. Target substance detection device Target substance detection method

<1. Target substance detection probe>
1 to 3 and 5 to 7 are schematic conceptual diagrams schematically showing the first to sixth embodiments of the target substance detection probe 1 according to the present disclosure. FIG. 4 is a schematic conceptual diagram schematically illustrating a cross-sectional shape of the target substance detection probe 1 according to the present disclosure.

The target substance detection probe 1 according to the present disclosure is a tool for collecting and detecting the target substance X, preferably one that can be attached to and detached from a measuring device or the like, and one that can be used as a disposable. Thereby, detection accuracy can be improved and it is preferable for infectious disease prevention.
The target substance detection probe 1 includes at least a sensitive capillary section 11 including a collecting section 13 that collects the target substance X and a sensitive section 12 that causes an interaction (reaction) between the target substance X and the substance. It is to be prepared.
Further, the target substance detection probe 1 may be provided with a support part for fixing or supporting the sensitive capillary part 11 as necessary. In addition, when performing various measurements using the target substance detection probe 1, a support unit for connecting to devices such as the light measurement system 16, a radioactive measurement system (not shown), the differential pressure mechanism 18 and the like may be provided. Good.
As such a support part, when performing an optical measurement, the support part 14 and the optical measurement holder 15 etc. which are shown to FIGS. 5-7 can be mentioned, for example.
The support portion 14 and the support portion 15 for supporting the target substance detection probe 1 may be connected to an optical waveguide 17 (optical fiber), and if necessary, ferrules (optical connector members) F (F1, F2) and It may be composed of a sleeve S or the like.

  Since the target substance detection probe 1 of the present disclosure is a tool that uses a capillary tube, it is easy to collect a sample liquid containing the target substance, and at least the amount collected can be measured. For this reason, if the probe 1 for substance detection of this indication is used, it is possible to measure a target substance minimally or non-invasively. In addition, since the target substance detection probe 1 of the present disclosure is integrated with the collection field and the reaction field, it can be measured immediately after the sample is collected, and therefore, alteration of the sample can be reduced. It is also possible to detect with high accuracy.

Here, the “target substance” in the present disclosure is not particularly limited as long as it is a substance capable of causing an interaction between substances. For example, proteins, peptides, glycoproteins, nucleic acid molecules, hormones, etc .; their precursors: their degradation substances. Furthermore, the thing which can be extract | collected from the internal / external tissue (For example, on skin tissue, the inside of a structure | tissue, etc.) of a biological body is desirable.
The target substance is preferably an in-vivo product, and among them, an inflammatory mediator is preferable. Inflammatory mediators are physiologically active substances released from leukocytes, mast cells, macrophages and the like that have infiltrated damaged tissues and inflammatory sites.
For example, analgesics such as bradykinin, serotonin, histamine; brostanoids such as prostaglandins and leukotrienes; cytokines such as interleukin 1, interleukin 6, TNF-α, platelet activating factor, lysosomal enzyme; reactive oxygen and NO, etc. Free radicals (substances) and the like.

In addition, it is known that a physiologically active substance called an inflammatory mediator is released in tissues exhibiting inflammation as a pathological condition, including inflammatory diseases in gingiva such as periodontitis, which is an index of the pathological condition. Therefore, the detection test for inflammatory mediators is used for diagnosis of pathological conditions.
As typical inflammatory mediators, cytokines that are glycoproteins involved in cell-cell interactions are known.
Inflammatory cytokines (inflammatory cytokines) include interleukin (IL), tumor necrosis factor (TNF), and the like. For example, see Reference 1: Yanagida, Murakami, Izumi et al., “The Periodontology”, Chapter 5 (2009, Nagasue Shoten).
If an antibody that specifically binds to a cytokine is used, it can be detected by antibody antigen reaction. It is known that the detected cytokine varies depending on the cause of inflammation, and the cause can be diagnosed by specifying the type of cytokine.
In addition, “TNF-α”, an inflammatory cytokine, is produced from monocytes and macrophages by stimulation of bacterial cell components, has an action of promoting vascular endothelial cell activation and vascular permeability, and is resistant to insulin. There is also an action to increase. From this, it is a substance attracting attention not only from periodontal disease but also from the relation with diabetes.

Here, in the present disclosure, “interaction between substances” broadly means non-covalent bonds, covalent bonds, chemical bonds including hydrogen bonds or dissociation between substances, such as hybridization of nucleic acid molecules, between proteins. This includes a wide range of chemical bonds or dissociations between substances, such as interactions between antigens and antigen-antibody reactions. “Hybridization” means formation of complementary strands (double strands) between complementary base sequence structures.
In the present disclosure, the “nucleic acid chain” means a polymer (nucleotide chain) of a nucleoside phosphate ester in which a purine or pyrimidine base and a sugar are glycoside-bonded. Furthermore, in the present disclosure, the “nucleic acid chain” means an oligonucleotide, a polynucleotide, a DNA in which purine nucleotides and pyrimidine nucleotides are polymerized (full length or a fragment thereof), cDNA obtained by reverse transcription (c probe DNA), Widely includes RNA, polyamide nucleotide derivatives (PNA) and the like.
In the present disclosure, the “protein-protein interaction” means a protein-protein, protein-peptide, peptide-peptide interaction. As this interaction, for example, a protein or the like specifically binds to a portion present in a three-dimensional structure of a protein or the vicinity thereof.
In the present disclosure, “antigen-antibody reaction” includes binding of an antigen (for example, an epitope) and an antibody.

By the way, when performing a series of operations of collecting and measuring a small amount of sample by a conventional method, a complicated operation is required to obtain a measurable state, and an operation time for obtaining a measurement result is required. There is a problem that it becomes long. In addition, there is a problem that the stability of measurement accuracy tends to be lowered.
For example, in a normal ELISA (ELISA), if the amount of the sample liquid is very small, the number of processing steps increases and sensitivity decreases. More specifically, as a method of collecting a very small amount of specimen (for example, gingival crevicular fluid), a technique of sucking with a filter paper is adopted, but an operation for extracting a target substance from the filter paper is necessary. Will be diluted.
In addition, if the collected sample liquid is to be introduced into the measurement system, an operation for separating it as a pre-treatment is required. At this time, a loss occurs, and the smaller the amount, the higher the possibility of measurement difficulty.
Thus, the conventional method collects a minute amount (for example, gingival crevicular fluid) with a conventional collection tool, prepares a target substance from the collected material, and further prepares the preparation with a measuring device or a measuring instrument (for example, It was to measure with Eliza etc.).

On the other hand, the present inventor has intensively studied, a collection field for collecting the target substance in the capillary, and a reaction field for immobilizing the trapping substance capable of trapping the target substance and performing an interaction between the substances. It was found that the above-mentioned problems can be solved by providing the organically.
That is, the present inventor has radically changed the conventional method (idea) as described above, and has completed the contents of the present technology with the unusual idea of performing collection and reaction in the same place.

  Furthermore, the measurement target of the present technology is not particularly limited, and includes in vivo tissues and the like. Examples of the in vivo tissue include body fluid and blood. Examples of the body fluid include gingival crevicular fluid (exudate), semen, secretion, saliva, urine and the like.

  In recent years, it has become clear that various in vivo substances exist in the gingival crevicular fluid. Moreover, since it can be collected non-invasively, a technique for diagnosing, treating, and managing various diseases by measuring the target in vivo substance contained in the gingival crevicular fluid as a target substance Has been developed. However, since the in-vivo substance is collected from a very narrow site called the gingival crevice, the amount of gingival crevicular fluid that can be collected is small. For this reason, it is difficult to secure the amount necessary for detection, and it is also difficult to maintain the stability of measurement accuracy. For this reason as well, there has been a fact that a technique for measuring in-vivo substances in the gingival crevicular fluid in real time has not been established.

On the other hand, if this technique is used, it can be measured even with gingival crevicular fluid containing a very small amount of target substance, and can also be measured non-invasively.
The “gingival crevicular fluid” is a fluid that exists in the gingival crevice and contains in vivo substances from the tooth tissue. Specific examples include gingival crevicular fluid (GCF: fluid containing plasma components and tissue fluid components exuded from the gingiva) and blood (including white blood cell components, red blood cell components, plasma, etc.) bleeding from the gums. The gingival crevicular fluid also contains inflammatory mediators and the like.
In general, a “gingival sulcus” refers to a groove at the boundary between a tooth and a gingiva in a tooth tissue.

The sensitive capillary section 11 of the present disclosure includes a collection unit (collection field) 13 that collects a target substance, and a sensitivity unit (reaction field) that immobilizes a trapping substance that binds to the target substance and performs an interaction reaction between the substances. ) 12.
The size and shape of the sensitive capillary section 11 are not particularly limited and can be designed to suit the purpose. For example, a size and shape that facilitates the collection of a sample by the collection unit 13 described later and allows the reaction to be performed by the sensitive unit 12 described later are suitable.

  Moreover, the cross-sectional shape (form) of the short direction of the said sensitive capillary part 11 has the example schematic diagram of FIG. 4, for example. Specific examples include an elliptical shape, a square shape, a rectangular shape, a circular shape, a crescent shape, and a rugby ball shape. The short diameter W of the sensitive capillary section 11 is preferably 0.5 mm or less, more preferably 0.2 mm or less.

  Moreover, the part (collection part 13) of the front-end | tip direction of the sensitive capillary part 11 may be diagonally cut shape or cone shape. Furthermore, a tapered shape (see, for example, FIGS. 7 and 12) is preferable because a capillary action can be easily obtained and even a small amount of sample can be easily measured. More specifically, it is preferable to have a tapered shape (see, for example, a tapered shape 110 in FIG. 7) such that the diameter and the cross-sectional area gradually increase from the tip of the sensitive capillary portion 11 toward the other end. At this time, it is preferable that the minor axis W of the tip of the tapered shape (structure) is 0.15 mm or less. This is because it is possible to prevent gingiva and damage from being added when inserting into a living tissue, particularly the gingival crevice.

Further, it is preferable to provide one or more vent holes 11H in the sensitive capillary section 11 (see, for example, FIG. 7). This can be used as an air vent when forming the sensitive part 12. For example, using the vent 11H in the capillary tube, the sensitive membrane preparation solution is sucked up to the height of the vent 11H from the tip 11A to form a sensitive membrane, and the first sensitive portion 12 is obtained. Then, from the other end 11 </ b> B, a sensitive film forming film solution different from the first sensitive film is formed in the same procedure to form the second sensitive part 12. In this way, it is also possible to form a plurality of sensitive parts 12 (see, for example, sensitive part 12a and sensitive part 12b in FIG. 3).
Further, by closing the vent hole 11H, it is possible to suck up the sensitive membrane preparation solution beyond the height of the closed vent hole 11H. For this reason, it is also possible to produce a plurality of sensitive portions 12 having different properties by providing a plurality of vent holes 11H and adjusting the opening and closing of each vent hole.
In addition, as shown in FIG. 7, it is preferable to provide the said vent hole 11H in the position of about half to 3/4 from the front end A between the front end 11A and the other end 11B.
The hole diameter (diameter) of the vent hole 11H is not particularly limited, but is preferably 0.01 to 0.2 mm, more preferably 0.05 to 0.1 mm.

The material used for the sensitive capillary part 11 is not particularly limited. Among these, the use of a flexible material is preferable because damage to the living tissue can be reduced when it comes into contact with the living tissue. Examples of the material having flexibility include resin, metal, ceramic, paper, cloth such as felt material, and the like.
Further, the sensitive capillary tube portion 11 may be formed by a known manufacturing method such as injection molding, etching, cutting, machining, or the like using the above material.

(2) Collection unit (collection site)
A sampling section 13 is provided in the sensitive capillary section 11, and a sampling field is formed inside the sensitive capillary section 11. It is possible to absorb the sample liquid containing the above-mentioned target substance from the distal end portion of the collection unit 13 into the capillary (collection field portion) by capillary action or differential pressure. Thereby, it becomes possible to guide the target substance to the sensitive part 12 which is a reaction field described later.
The size or shape (form) of the sampling part 13 is, for example, such that the short side or the short diameter W of the sampling part is preferably 0.5 mm or less, more preferably 0.2 mm, and even more preferably 0.15 mm or less. Is preferred. If the vicinity of the end where the target substance is collected by contact with the specimen liquid containing the target substance is thin, it is easy to collect the target substance and the amount of specimen to be collected can be reduced. Further, it is preferable to make it thin, because not only the differential pressure but also the capillary action can be used, and the amount of sample to be collected can be reduced.
Thus, by narrowing the vicinity of the tip of the collection part 13 (capillary tip 11A), the gingival sulcus of a healthy person is 0.2 mm wide and 2 mm deep, so the sensitive capillary part 11 is inserted into the gingival sulcus. can do. It is also possible to reduce irritation and damage to the gingiva. Therefore, it becomes easy to collect a highly non-invasive gingival crevicular fluid.

  Further, the distal end portion of the collection unit 13 that comes into contact with a living tissue or the like may be rounded, for example, like the capillary distal end 11A in FIG. In addition, it is preferable to provide a flexible covering portion (not shown) such as a nonwoven fabric, a woven fabric, or a foam at the distal end portion of the collection portion 13, and it is non-absorbable in order to suppress loss of the sample liquid. It is desirable to use Thereby, even if the tip of the collection unit 13 comes into contact with the living tissue, it is difficult to cause irritation or damage, and thus it is easy to directly collect. By collecting directly, it is easy to grasp the state of the subject at that time, and the amount of the sample may be small.

(3) Sensitive part (reaction field)
The sensitive capillary part 11 is provided with a sensitive part 12, and a reaction field for interaction between substances is provided inside the sensitive capillary part 11. In this reaction field, a trapping substance TA capable of trapping the target substance X by the interaction between substances is fixed, and a reaction is caused by the interaction between various substances.
The sensitive portion 12 functions as a region or space that provides a reaction field for interaction between various substances such as hybridization, protein-protein interaction, and antigen-antibody reaction. Furthermore, the sensitive part 12 preferably functions as a region or space that provides a detection field for detecting the target substance X trapped by this interaction.
The sensitive part 12 includes one or more trapping substances TA that can capture the target substance X by interaction. It is possible to interact with the target substance X by this trapping substance TA, and to detect optical information or radioactive information optically or radioimmunometrically emitted by a reaction due to the interaction between substances. is there. Examples of the reaction for detection at this time include a competitive reaction and a sandwich reaction. Further, it is possible to detect a plurality of optical information or radioactive information.

The position (range) of the sensitive part 12 may be set as appropriate, and the sensitive part 12 may be provided with one or more sensitive regions (sensitive films).
For example, as shown in FIG. 1, the range of the sensitive unit 12 may be substantially the same range as the sampling unit 13.
In addition, as shown in FIG. 2, the sampling part 132 near the tip of the sensitive capillary part 11 is not formed as a sensitive part without forming the sensitive part 12, and the sensitive part 12 is formed in the sampling part 131 in the other end direction. Also good. Alternatively, the sensitive part 12 may be formed near the center of the sensitive capillary part 11.
Further, as shown in FIG. 3, a plurality of sensitive portions 12 may be formed. For example, the sensitive portion 12a may be formed in the distal direction, and a different sensitive portion 12b may be formed in the other end direction. The sensitive portions may be adjacent to each other, or a certain range of non-sensitive portion regions may be provided so as not to be adjacent. The vent 11H may be used as described above when forming the plurality of sensitive regions and non-sensitive regions.

The immobilized trap material T is obtained by immobilizing the trap material TA on the inner wall surface of a capillary tube (sensitive capillary portion 11).
The trapping substance TA used for the immobilized trapping substance T may be any substance that can trap the target substance X by the interaction between the substances as described above. For example, when the target substance X is an antigen or an antibody, the trap substance TA may be an antibody or an antigen that performs an antigen-antibody reaction with the target substance X, and may be appropriately determined according to the target substance X (epitope etc.). . The trapping material TA can be freely produced by a commonly used method according to the target material, or a commercially available product may be obtained and used.
Moreover, you may use the kind of trap substance TA used for formation of the sensitive part 12 1 type or in combination of 2 or more types as needed. For example, a plurality of sensitive portions 12 may be used according to the type of trapping material TA to be used. For example, a plurality of different types of trapping materials TA may be used for one sensitive portion 12.

  The immobilizing agent for immobilizing the trapping substance TA on the inner wall surface of the capillary and the immobilizing method thereof are not particularly limited. For example, a method using a photocuring resin is simple and suitable.

A suitable example of the fixing agent and the immobilizing method of the present disclosure will be described.
For example, a trapping substance TA is mixed in an aqueous solution (an immobilized aqueous solution) containing a water-soluble compound having various photoreactive groups and a water-soluble polymer chain, thereby preparing a solution for preparing a sensitive film. After this mixed aqueous solution (sensitive membrane preparation solution) is introduced and adhered to the inner wall surface of the capillary tube by capillary action or differential pressure, the moisture is dried, and then light (ultraviolet rays) is irradiated and immobilized. According to this method, the capillary is less likely to be clogged with respect to the inner wall surface of the capillary, and can be reliably fixed while controlling the capacity (volume) of the sensitive portion 12.

As the water-soluble polymer, a nonionic polymer is preferable, and examples thereof include a vinyl polymer of polyethylene glycol mono (meth) acrylate.
Examples of the water-soluble compound having a photoreactive group include an azide group (—N ₃ ), particularly a diazide compound having two such azide groups (for example, a compound having a structure in which two phenylazide groups are linked). It is done. Further, water-soluble diazide compound, _{e.g., N 3 -Ph-R-Ph} -N 3 and _{N 3 -CH 2 -CH 2 -O- (} CH 2 -CH 2 -O) n-CH 2 -CH 2 -N ₃ etc. are mentioned. As R, —CH ₂ —, —O—, —SO ₂ —, —S—S—, —CH═CH—, —CH═CH—CO—, —CH═CH—CO—CH═CH—, -CH = CH- and the like are preferable. Moreover, 1-1000 are preferable as said n, and 1-500 is more preferable. Specific examples of the compound include alkali metal salts of 4,4′-diazidostilbene-2,2′-disulfonic acid (such as sodium).

  Note that the azide group releases nitrogen molecules upon irradiation with light and generates nitrogen radicals, which can bind not only to functional groups such as amino groups and carboxyl groups, but also to carbon atoms constituting organic compounds. Since it is possible, it has the property that it can form a supply bond with most organic compounds.

The inner wall surface (inner wall portion 113) of the capillary tube (sensitive capillary portion 11) is preferably formed of a solid phase having a hydrophilic group such as an alcoholic hydroxyl group. This is because it is easy to form the sensitive portion 12 on the inner wall surface of the capillary tube. Examples of such a solid phase include glass, quartz, polyalcoholic resin, surface-modified resin, and a solid phase provided with a hydrophilic group such as a silane coupling agent.
Further, the inner wall surface (inner wall portion 113) of the capillary tube (the sensitive capillary tube portion 110) may be monoporous or porous, and is preferably formed in a form having an increased surface area. By increasing the surface area, it becomes possible to cause the reaction part 12 provided on the surface to contain more trapping material TA.

Further, the trapping material TA may be directly immobilized with the immobilizing agent, such as the trapping material TA-fixed material 2-inner wall surface. Thereby, it is easy to stably fix the trapping material TA to the inner wall surface.
Alternatively, the trapping substance TA may be immobilized using a crosslinked body (composite crosslinked body) C, such as trapping substance TA-crosslinked body C-inner wall surface. By using the cross-linked body, it becomes easy to dispose the trapping substance in a direction in which the target substance passing through the capillary tube is easily trapped (interaction between substances is likely to occur). In addition, since a cross-linked body is used to bond firmly, there is also an advantage that it can be prevented from falling off during cleaning.
Further, the trapping substance TA-crosslinked body C-fixed product 2-inner wall surface may be immobilized by combining a crosslinked body and a fixing agent.
Moreover, it is preferable to use the some partially crosslinked body which forms the composite crosslinked body C at the point which preparation is easy. For example, trapping substance TA-crosslinked body a-crosslinked body b-inner wall surface; trapping substance TA-crosslinked body a-crosslinked body b-fixed material 2-inner wall surface, and the like. The “-crosslinked product a-crosslinked product b-” may be “-crosslinked product b-crosslinked product a-” and is not particularly limited.

  As a method for forming the trap substance TA-crosslinked body (composite crosslinked body) C-, for example, a method using a crosslinking reagent used for enzyme labeling in enzyme immunoassay can be used. Examples thereof include a method using glutaraldehyde, a maleimide compound, a pyridyl disulfide compound, a biotin / avidin compound, a biotin / streptavidin compound, a method using a NaKaue method by periodate oxidation, and the like. Of these, methods using biotin / avidin compounds and biotin / streptavidin compounds are desirable.

The labeled detection substance Y is a substance that can detect optical information or radioactive information optically or radioimmunometrically with a measuring device or the like due to the interaction between the target substance X and the substance. is there.
The detection substance YA used for the labeled detection substance Y may be any substance that can interact between the target substance X and the substance, such as the immobilized trap substance T described above. For example, when the target substance X is an antigen or an antibody, the detection substance YA includes an antibody or an antigen that performs an antigen-antibody reaction with the target substance X, and may be appropriately determined according to the target substance X (epitope or the like). The detection substance YA can be prepared by a commonly used method according to the target substance, or a commercially available product may be obtained and used. The type of the substance for detection YA may be used alone or in combination of two or more as desired.
Examples of the labeled detection substance Y include those obtained by photolabeling and / or radioactively labeling the detection substance YA. This photolabel enables light detection, such as one that emits a light component (fluorescence, light absorption, etc.) or can generate one that emits light.
Examples of the labeled detection substance Y include isotope-labeled substances, fluorescently-labeled substances, cross-linked substances, and enzyme-labeled substances.

When labeling, different types of detection substances YA may be used as necessary. At this time, different types of detection substances YA may be labeled with different types of labels M, respectively. Thereby, it becomes possible to detect a plurality of target substances. In addition, when one target substance has a plurality of parts that interact with each other, detection accuracy can be further improved.
For labeling detection of labeled M-crosslinked product C-detecting material YA; labeled M-composite crosslinked product C-detecting material YA, etc. obtained by a method using a crosslinking reagent such as the above-mentioned “trap material” Substance Y may be used. As an example, a partially cross-linked substance-detecting substance YA binds to a target substance X that is trapped, and a partial cross-linked substance-label (for example, POD) binds to the detecting substance YA-partial cross-linked substance. Cross-linked substance-detecting substance-target substance-immobilized trap substance. Since the substance to be bound is small, it becomes possible to bind efficiently. This is preferable because detection accuracy is also improved. In addition, since a cross-linked body is used to bond firmly, there is also an advantage that it can be prevented from falling off during cleaning.

The radioisotope used for radiolabeling is not particularly limited. For example, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like can be mentioned.
The fluorescent dye used for the fluorescent label is not particularly limited. Examples thereof include fluorescein, rhodamine, phycobilin, acridine, coumarin, cyanine, Alexa Fluor (registered trademark), Cy dye (registered trademark), ruthenium (II) complex, and lanthanoid complex.
Examples of the dye used for labeling the change in absorbance include ortho-dianisidine.

The enzyme used for enzyme labeling is not particularly limited.
For example, alkaline phosphatase, peroxidase, β-D-galactosidase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidase, glucose oxidase, invertase, acetate kinase, glucose dehydrogenase, pyruvate kinase, uricase (Lactate oxidase), urease, lactate oxidase, fructosyl. Examples include amino acid oxidase, sarcosine oxidase, and cholesterol oxidase. These may be used alone or in combination. Peroxidase is preferable. These can be purchased and used on the market.

In addition, as the enzyme substrate, any of the aforementioned enzyme substrates can be used. As this enzyme substrate, a dye precursor is preferred. As the dye precursor, for example, an enzyme that changes from a dye precursor to a substance that emits light or changes absorbance is preferable.
For example, in the case of alkaline phosphatase, p-nitrophenyl phosphate and the like can be mentioned.
For example, in the case of peroxidase, 10-acetyl-3,7-dihydroxyphenoazine (ADHP), o-dianisidine, 5-aminosalicylic acid, o-phenylenediamine, tetramethylbenzidine (3,3'5, 5'-tetramethylbennizine) and the like.
Of these, it is convenient to optically measure the dye produced from the dye precursor by the action of the enzyme and detect and quantify the target substance X. For example, a substance that becomes a fluorescent substance or a substance that changes the absorbance from a dye precursor by peroxidase in the presence of hydrogen peroxide is preferable. Of these, it is convenient to carry out with peroxidase-ADHP and peroxidase-orthodianisidine.

ADHP becomes resorufin (Resorufin) by peroxidase in the presence of hydrogen peroxide, and this resorufin serves as a fluorescence source, so that the fluorescence intensity can be measured.
In addition, the absorbance of orthodianisidine (reduced) is changed by peroxidase in the presence of hydrogen peroxide to orthodianisidine (oxidized), so this change in absorbance can be measured by visible light spectroscopy. And

  For example, when different types of detection substances YA are used, different labels may be separately labeled. By using different labels, even when the type of target substance X is different, it is possible to detect at different detection wavelengths.

<2. Target Substance Detection Device 10>
The target substance detection apparatus 10 according to the present disclosure includes at least the target substance detection probe 1 according to the present disclosure.
As described above, the target substance detection probe 1 immobilizes the collection unit 13 that can collect the target substance X in the capillary section and the trapping substance TA that binds to the target substance, and performs an interaction between the substances. And a sensitive capillary part 11 provided with a sensitive part 12. And this sensitive capillary part 11 may be any of the 1st-6th embodiment as shown to FIGS. 1-3, 5-7, However, It is not limited to the illustration of these embodiment.
Moreover, it is suitable that the front-end | tip of the sensitive capillary part 11 is a taper shape. Since the target substance detection probe 1 overlaps with the configuration of the target substance detection probe 1 described above, a detailed description is omitted here.

Furthermore, the target substance detection device 10 includes at least a light irradiation unit and a light detection unit. Further, if necessary, a dichroic element, a condensing lens, an optical filter, or the like can be provided. Each will be described in detail below.
The target substance detection probe 1 in the target substance detection apparatus 10 may be arranged in a horizontal type (for example, FIGS. 5 and 6) or a vertical type (for example, FIG. 7). And the like can be designed freely.
In addition, since the target substance detection device 10 uses the target substance detection probe 1 having a collection field and a reaction field, it is possible to detect the target substance almost in real time after collecting the target substance.

FIG. 6 is a schematic conceptual diagram schematically showing the first embodiment of the target substance detection device 10 according to the present disclosure.
The target substance detection device 10 according to the present disclosure includes at least the detachable target substance detection probe 1 and a detection unit. The detection unit may be any device that can detect optical information or radioactive information. And in the case of optical information, it is suitable to provide the light measurement system 16 comprised from a light irradiation part and a light detection part. The light irradiation unit irradiates light to the sensitive unit 12 of the target substance detection probe 1. The light detection unit detects light information from the sensitive unit 12 by light irradiation by the light irradiation unit.

Further, the light measurement system 15 and the light measurement holder 15 and the light measurement system 16 are connected to the light measurement system 16 so that the sensitive part 12 of the target substance detection probe 1 can be detected more easily. An optical waveguide 17 is preferably provided.
The method of forming the optical waveguide 17 is not particularly limited as long as it can transmit light to the sensitive part 12 and / or transmit optical information generated from the front sensitive part, and can be freely designed. For example, an optical waveguide can be formed using a rod, an optical waveguide using an optical fiber can be formed, or an optical waveguide can be formed using a light guide plate.

  In addition, it is preferable to provide the differential pressure mechanism 18 when creating the target substance detection probe 1 and when performing interaction between substances. In addition, it is preferable that a hollow support portion 14 (for example, a columnar shape or the like) is provided on the other end 11B of the sensitive capillary portion 11 and connected to a differential pressure mechanism. This makes it possible to easily flow various solutions into the capillaries of the target substance detection probe.

FIG. 5 is a schematic conceptual diagram schematically showing the second embodiment of the target substance detection device 10 of the present disclosure.
When an optical fiber is used, optical detection is possible by forming a main body using the ferrule (optical connector member) F1 and the sleeve S and connecting the ferrule F1 and the ferrule F2 on the measuring device side with the sleeve S. is there.
In addition, in 2nd Embodiment, although the sleeve S is provided in the said target substance detection probe 1 side, it is not limited to this embodiment, The sleeve S is provided or connected to the ferrule F2 by the side of a measuring device. The sleeve S may be designed so as to be used only when
In addition, although the sensitive capillary part 11 may be any of 1st-6th embodiment as shown to FIGS. 1-3 and 5-7, it is not limited to the illustration of these embodiment. The tip of the sensitive capillary section 11 is preferably tapered.

FIG. 7 is a schematic conceptual diagram schematically showing the third embodiment of the target substance detection device 10 of the present disclosure.
Regarding the arrangement configuration of the optical waveguide 17 with respect to the sensitive capillary portion 11, the optical waveguide 17 is not arranged in series with respect to the upper end surface 111 of the sensitive capillary portion 11 but with respect to the side body portion 112 of the sensitive capillary portion 11. It may be designed to be connected. It is preferable that the sensitive portion 12 (the sensitive film 21) is formed on the inner wall surface of the side body portion 112, preferably the inner wall surface near the connecting portion. Further, a slidable light measurement holder as shown in the first embodiment may be provided. Thereby, it becomes easy to acquire the optical information emitted from the sensitive unit 12.
Further, it is preferable to provide a differential pressure mechanism as shown in the first embodiment on the capillary other end 11B. Thereby, it is possible to easily suck up various solutions, clean the capillaries, and the like.

An example of the third embodiment of the target substance detection device 10 will be described below.
A hole 152 is formed in the vicinity of one end of a PFA (fluororesin) tube 151, and the upper portion of the sensitive capillary section 11 is attached to the hole 152 and fixed with an adhesive or the like. For example, the inner diameter of the PFA tube is designed to be 0.5 mm and the outer diameter is designed to be 1.0 mm. An optical waveguide 17, for example, an optical fiber 171 (for example, a diameter of 0.25 mm) is provided inside the PFA tube 151. One end portion of the optical fiber is arranged in contact with the side body portion 112 of the sensitive capillary tube portion 11, and light is transmitted through the optical fiber.
Specifically, measurement light L1 (for example, fluorescence excitation light) is incident on the sensitive part 12 (sensitive film 21), or light L2 such as fluorescence or light emitted from a fluorescent source or the like existing in the sensitive part 12 is used. Are transmitted to an optical measurement system (not shown). The sensitive part 12 (sensitive film 21) is formed on the inner wall 113 of the sensitive capillary part 11 as a film.
Moreover, the optical connector member F and the optical measurement holder 15 for fixing the optical connector member F may be provided, and the holder and the PFA tube 151 may be bonded with an adhesive or the like.
In addition, although the said sensitive capillary part 11 may be any of the 1st-6th embodiment as shown to FIGS. 1-3 and 5-7, it is not limited to the illustration of these embodiment. The tip of the sensitive capillary section 11 is preferably tapered.

  The light irradiation unit is a device for irradiating light (for example, fluorescence excitation light) to the sensitive unit 12 provided in the target substance detection probe 1.

  The type of light emitted from the light irradiating part is not particularly limited, but light having a constant light direction, wavelength, and light intensity is desirable in order to reliably generate fluorescence and scattered light from the sensitive part 12. As an example, a laser, LED, a mercury lamp, etc. can be mentioned. When the laser is used, the type is not particularly limited, but one or more of argon ion (Ar) laser, helium-neon (He-Ne) laser, die laser, krypton (Kr) laser, etc. , Can be used in any combination.

  The light detection unit is a device for detecting optical information emitted from the sensitive unit 12 by light irradiation by the light irradiation unit.

  The type of the light detection unit that can be used in the target substance detection device 10 according to the present invention is not particularly limited as long as optical information can be detected, and a known light detector can be freely selected and adopted. it can. For example, fluorescence measuring instrument, scattered light measuring instrument, transmitted light measuring instrument, reflected light measuring instrument, diffracted light measuring instrument, ultraviolet spectroscopic measuring instrument, infrared spectroscopic measuring instrument, Raman spectroscopic measuring instrument, FRET measuring instrument, FISH measuring instrument and others Various spectrum measuring devices, so-called multi-channel photodetectors in which a plurality of photodetectors are arranged in an array, etc., can be used alone or in combination of two or more.

In addition, the target substance detection device 10 can include a dichroic element (not shown). By providing this dichroic element, only necessary optical information can be selected and detected.
The dichroic element is not particularly limited, and a known dichroic element can be freely selected and used according to the purpose. For example, a dichroic mirror, a duplexer, etc. can be used.

  In addition, although not shown in the drawing, the target substance detection apparatus 10 collects excitation light between the light irradiation unit and the sensitive unit 12 for the purpose of collecting the excitation light from the light irradiation unit onto the sensitive unit 12. A lens can be placed. Although this condensing lens is not essential, if the condensing lens for excitation light is provided, it is possible to irradiate the sensitive part 12 with excitation light more accurately.

  Although not shown in the drawing, the target substance detection device 10 receives light between the sensitive unit 12 and the photodetecting unit for the purpose of concentrating optical information emitted from the sensitive unit 12 on the photodetecting unit. A condensing lens can be arranged. The light receiving condensing lens is not essential, but if a light receiving condensing lens is provided, the signal of optical information such as fluorescence can be further enhanced. As a result, it is possible to improve the SN ratio.

  In the target substance detection device 10, although not shown, an excitation optical filter can be disposed between the light irradiation unit and the sensitive unit 12. This excitation optical filter is not essential, but if an excitation optical filter is provided, the sensitive portion 12 can be selectively irradiated with excitation light having a desired wavelength.

  Further, in the target substance detection device 10, although not shown, a light receiving optical filter can be arranged between the sensitive unit 12 and the light detection unit. Although this light receiving optical filter is not essential, if a light receiving optical filter is provided, light having a desired wavelength is selectively received from optical information such as fluorescence emitted from the sensitive portion 12. be able to.

<3. Target substance detection method>
The target substance detection method of the present disclosure will be described below with reference to FIGS. At this time, since the technical contents of the target substance detection probe 1 and the target substance detection apparatus 10 overlap with the target substance detection probe 1 and the target substance detection apparatus 10 described above, description thereof will be omitted as appropriate. .

8 to 10 disclose schematic illustrations of the target substance detection method of the present disclosure, which will be described below with reference to this. 8 to 10 are schematic views including a method for forming the sensitive portion 12.
In the target substance detection method of the present disclosure, in the sensitive capillary (part) 11, the collected target substance X is interacted with the immobilized trap substance T, and the emitted light or radioactive information is obtained. To detect. The sensitive capillary unit 11 immobilizes a collection site (collection unit 13) that collects the target substance X and a trapping substance TA that traps the target substance X, and a reaction field (sensitive) that reacts between the substances. Part 12).
The immobilized trap substance T is obtained by immobilizing the trap substance TA for trapping the target substance X by absorbing a solution containing the trap substance TA in the capillary tube.

The target substance detection method of the present disclosure can be performed by the following [Procedure A] to [Procedure D].
[Procedure A] As shown in FIGS. 8 (1), (2) and 10 (1), the target substance is trapped by absorbing the solution containing the trapping substance TA in the capillary tube (sensitive capillary section 11). The trapping material TA to be fixed is immobilized on the inner wall surface of the capillary tube.
At this time, it is desirable to use the immobilizing agent, and it is desirable to further use a mixed solution containing the trapping material TA and the immobilizing agent. Then, light irradiation or the like is performed, and the fixed object 2 and the immobilized trap substance T are formed as the sensitive film 21 on the inner wall surface of the capillary (the sensitive capillary part 11). This part becomes the sensitive part 12. Since the range of the sensitive part 12 and the sampling part 13 and the manufacturing method thereof are as described above, they are omitted.
Thereafter, the inside of the capillary tube may be washed with a solution or the like.

In [Procedure A], as shown in FIG. 9 (1), a method using a crosslinked product C may be used. In addition, since the composite crosslinked body C is formed of a plurality of partially crosslinked bodies, any order in which each partially crosslinked body is used may be first.
The mixed solution containing the (partial) crosslinked body Ca that becomes the composite crosslinked body C and the immobilizing agent is absorbed into the capillary tube (sensitive capillary section 11) to immobilize the crosslinked body Ca. Thereafter, a solution containing the trapping substance TA combined with the (partial) crosslinked body Cb which becomes the crosslinked body Ca and the composite crosslinked body C is absorbed into the capillary tube (sensitive capillary section 11).
As shown in FIG. 9 (2), the partially crosslinked body Ca and the partially crosslinked body Cb form a composite crosslinked body C, whereby the trapping material TA is immobilized and becomes the immobilized trapping material T.
Thereafter, the inside of the capillary tube may be washed with a solution or the like.

[Procedure B] As shown in FIG. 8 (3), FIG. 9 (3) and FIG. 10 (2), the sample liquid containing the target substance X is absorbed into the capillary (sensitive capillary section 11), Interaction between substances is performed on the target substance X by the immobilized trap substance T.
Thereafter, the inside of the capillary tube may be washed with a solution or the like.

[Procedure C] As shown in FIGS. 8 (4) and 9 (4), a solution containing a detection substance YA labeled with light or a radioactive label is absorbed into the capillary (sensitive capillary section 11), The labeled detection substance Y is allowed to interact between the target substance X and the substance.
Thereafter, if necessary, the inside of the capillary tube may be washed with a solution or the like.

Moreover, in [Procedure C], as shown in FIGS. 10 (3) to (5), a method using a crosslinked product C may be used. In addition, since the composite crosslinked body C is formed of a plurality of partially crosslinked bodies, the order in which the partially crosslinked bodies are used may be any first.
As shown in FIG. 10 (3), a solution containing the substance for detection YA is absorbed into the capillary tube, and the substance for detection YA interacts with the target substance X to bond them. Thereafter, the inside of the capillary tube may be washed with a solution or the like.
As shown in FIG. 10 (4), the solution containing the crosslinked body Cb is absorbed, and the crosslinked body Cb is bonded to the detection substance YA. Thereafter, the inside of the capillary tube may be washed with a solution or the like.
As shown in FIG. 10 (5), a solution containing the crosslinked body Ca having the label M is absorbed into the capillary, and the partially crosslinked body Ca is bonded to the partially crosslinked body Cb. As a result, the labeled detection substance Y composed of the labeled M-complex crosslinked body C-detection substance YA is obtained. This is a state where the labeled detection substance Y and the target substance X are bound.
Thereafter, the inside of the capillary tube may be washed with a solution or the like.

[Procedure D] Light or radioactive information emitted from the bound labeled detection substance is detected by a measuring device or the like.
Moreover, as shown in FIG. 8 (5), it is suitable to detect by the following procedures.
In the [Procedure C], after the reaction of the interaction between the substances is performed, the solution containing the substance for detection Y labeled with the enzyme M is absorbed into the capillary tube (sensitive capillary part 11). At this time, you may utilize the method using the above-mentioned crosslinked body. Thereafter, the inside of the capillary tube may be washed with a solution or the like.
Furthermore, the solution containing the substrate Z of the enzyme M is absorbed into the capillary tube. Then, optical information (luminescent material Za) generated when the enzyme substrate Z changes is detected. For this reason, it is preferable that the enzyme substrate Z is a dye precursor that becomes the luminescent material Za.

  Further, the immobilized trap substance T and / or the labeled detection substance Y is preferably obtained by biotin-avidin crosslinking or biotin-streptavidin crosslinking.

The method of forming the sensitive part (reaction field) 12 inside the sensitive capillary part 11 (inner wall surface) is not particularly limited, and can be formed using a free method. At this time, it is desirable to form the sensitive portion 12 using a material that does not adversely affect the living body.
For example, a solution containing the trap substance TA is introduced into a capillary tube (sensitive capillary portion 11) by capillary action or differential pressure, and then dried. Thereby, the sensitive part (reaction field) 12 can be formed in a state in which the trap substance TA is directly fixed to the inner wall surface of the capillary (sensitive capillary part 11).

  As a more preferable method for immobilizing the trapping substance TA on the inner wall surface of the capillary (sensitive capillary portion 11), it is desirable to use a fixing agent. More specifically, for example, a solution containing the trapping substance TA and a solution containing a fixing agent are mixed and introduced into the capillary tube by capillary action or differential pressure, followed by drying, light irradiation, and the like. As a result, a sensitive film (sensitive layer) 21 composed of at least the fixed substance 2 and the trapping substance TA is formed on the inner wall surface of the capillary tube (sensitive capillary part 11), and the trapping substance TA is fixed. Become.

Hereinafter, an example of the target substance detection method of the present disclosure will be described in the case where an antigen-antibody reaction (immune reaction) is performed in a capillary tube with reference to FIG. In this case, in particular, explanation will be given by the enzyme-linked immunosorbent assay (ELISA) for the target substance (antigen).
In [Procedure a], an antibody TA that binds to the target substance (antigen) X is immobilized in a capillary tube (sensitive capillary portion 11) as shown in FIG. Thereby, a sensitive film 21 (sensitive part 12) composed of the immobilized trap substance T and the fixed substance 2 is formed.
In [Procedure a], as [Procedure a1], antibody TA may be directly immobilized with a UV fixing agent. Specifically, the antibody is immobilized by absorbing a solution containing the antibody TA and the UV fixing agent with capillary force or differential pressure.
Further, as [Procedure a2], the antibody TA may be immobilized by a method using a biotin-avidin or biotin-streptavidin method and a UV fixing agent. In the case of biotin, an antibody modified with avidin or streptavidin, and in the case of avidin or streptavidin, an antibody modified with biotin will be used.
Specifically, a solution containing one partially crosslinked body of the composite crosslinked body C and the UV fixing agent is absorbed by capillary force or differential pressure, and one partially crosslinked body of the composite crosslinked body C is fixed. Thereafter, a solution containing the antibody TA modified with the other partial cross-linked body of the composite cross-linked body is absorbed by capillary force or differential pressure to fix the antibody.

  As [Procedure b], as shown in FIG. 8 (3), the sample liquid containing the target substance X is absorbed into the capillary (the sensitive capillary section 11) by capillary force or differential pressure.

At this time, a method using the composite crosslinked body C may be used as [Procedure b1].
A solution containing the secondary antibody YA modified with one partial cross-linked body of the composite cross-linked body C is absorbed into the capillary tube by capillary force or differential pressure. This secondary antibody YA binds to the trapped target substance X. The secondary antibody modified with one partial crosslinked body of the composite crosslinked body C is preferably a secondary antibody modified with biotin or a secondary antibody modified with avidin or streptavidin.
Furthermore, the solution containing the enzyme modified with the other partial cross-linked product of the composite cross-linked product C is absorbed into the capillary tube by capillary force or differential pressure. By binding the other partially crosslinked body of this composite crosslinked body C to one partially crosslinked body, a labeled secondary antibody Y having the enzyme as a label M is obtained.
When biotin is used as the secondary antibody YA, an enzyme modified with avidin or streptavidin is used. When avidin or streptavidin is used as the secondary antibody YA, an enzyme modified with biotin is used.

  Further, as [Procedure b2], a solution containing the secondary antibody Y to which the enzyme (label M) is bound may be absorbed into the capillary tube by capillary force or differential pressure.

Furthermore, in [Procedure c], the solution containing the enzyme M substrate Z (including the dye precursor) is absorbed into the capillary tube by capillary force or differential pressure.
Further, in [Procedure d], the dye Za generated from the dye precursor Z by the action of the enzyme M is optically measured, and the target substance X is detected and quantified.
Further, as [Procedure b3], a radioactive substance or the like may be used instead of the dye precursor Z. In such a case, the secondary antibody Y to which a label M such as a radioactive substance is bound is used, the measurement corresponding to the product of the substance is performed, and the target substance X is detected and quantified.

In addition, you may insert the removal process and washing | cleaning process of a solution by capillary force or differential pressure.
A body fluid or blood may be used as the sample liquid containing the target substance X. As this body fluid, gingival crevicular fluid, saliva, and urine may be used. The target substance X is preferably an inflammatory mediator.
It is also preferable to construct a system having the sensitive capillary section 11 of the present disclosure and having a mechanism for differential pressure for performing the above-described ELISA.

Specific examples and the like will be described below, but the present technology is not limited thereto.
[Measurement of signs]
In the case of fluorescence, the capillary tube is irradiated with excitation light and the fluorescence is received.

[1. Verification experiment using reagents]
Assuming that gingival crevicular fluid is collected from gingival tissue causing periodontitis and analyzing inflammatory mediators in it, this technology detects inflammatory cytokines in a small amount of sample liquid. An experiment was conducted to verify that this was possible with a certain capillary ELISA method.
The target substance for ELISA was TNF-α, an inflammatory cytokine. It is produced from monocytes and macrophages by stimulation of bacterial cell components, has the effect of promoting vascular endothelial cell activation and vascular permeability, and also has the effect of increasing insulin resistance, thus periodontal disease. It is a substance that attracts attention not only because of its relationship with diabetes. For example, see Reference 1 (Yanada, Murakami, Izumi et al., “The Periodontology”, Chapter 5 (2009, Nagasue Shoten)).

In the TNF-α detection experiment, the antigen and antibody of a commercially available TNF-α detection kit were used. The reagent kit used was Accukine Human TNF-α ELISA (E8005, Symmansis), which was purchased and used. This is fixed to the well bottom of the well plate. Specifically, a reagent for Coating antibody, antigen TNF-α (standard substance), Detection antibody, Streptavidin-HRP (horseradish peroxidase) is used as a kit. This is a reagent kit for detecting TNF-α by ELISA.
The coating antibody (trap antibody) is an antibody that binds to an antigen through an antigen-antibody reaction. The detection antibody binds avidin and binds to TNF-α by an antigen-antibody reaction. The Streptavidin-HRP (horseradish peroxidase) is streptavidin combined with POD (peroxidase).

This kit is usually used in a well plate having a large number of wells on a single substrate. Coating antibody is fixed to the bottom of the well, and a sample solution containing the target molecule (for example, a TNF-α solution) is injected therein. And it wash | cleans after the coupling | bonding by the antigen antibody reaction (immune reaction) between Coating antibody and a target molecule. Then, a detection antibody (probe antibody) solution is injected, and the target molecule and the detection antibody are further bound by an antigen-antibody reaction. This allows the sandwich complex to form and then washed.
When a Streptavidin-HRP solution is injected here, POD is added to the sandwich complex by binding of detection antibody biotin and Streptavidin.
POD changes colorless dye precursors (eg, non-fluorescent 10-acetyl-3, 7-dihydroxyphenoxazine) into colored or fluorescent dyes (eg, fluorescent resorufin) in the presence of hydrogen peroxide. .
Therefore, if a dye precursor and hydrogen peroxide are dropped into a well, a colored or fluorescent reaction occurs, and a target substance (in this case, TNF-α) in the sample solution can be detected by measuring absorption or fluorescence.
As long as the dye precursor is present, the reaction of absorption or fluorescence proceeds by the enzyme reaction, so that the antigen can be detected with high sensitivity. Such a technique for enhancing the sensitivity using an antigen-antibody reaction (immune reaction) and an enzyme is referred to as an enzyme-linked immunosorbent assay (ELISA).

This technique is a method that enables a very small amount of detection without loss by performing this ELISA method in a capillary tube and performing sampling and detection with the same capillary tube.
Flow charts of an ELISA method using a capillary tube of the present technology (capillary ELISA method) (for example, see FIG. 12) are shown in FIGS. As an example, the tip of the sensitive capillary portion 11 of this capillary-ELISA method is used by inserting it into the gingival sulcus.

[1.1. Preparation of antibody-immobilized capillary tube (sensor / probe / chip)]
1 mL of phosphate buffered saline was added to the Accubine Human TNF-α ELISA Coating antibody vial and dissolved to prepare a 720 μg / mL solution of Coating antibody. 100 μL was dispensed into a microtube and heated at 25 ° C. for 20 minutes using a heat block. 3.6 μL of 1% immobilizing solution R (manufactured by Hirasol Bio) containing a diazide compound and a water-soluble polymer chain was added thereto, mixed, and heated again at 25 ° C. for 5 minutes.
5 μL of the coating antibody solution was taken and dropped into a weighing boat. One side end face of a Delrin capillary (inner product 0.8 μL, 20 mm length) was immersed in the dropped solution for 10 seconds to absorb the solution into the capillary. Absorption was confirmed with a stereomicroscope. After drying for 20 minutes by reducing the pressure to a vacuum, the coating antibody was immobilized on the inner wall of the capillary by UV exposure.
A syringe pump capable of suction was prepared, and a syringe was set. After connecting the set syringe and capillary tube with a tube, the tip of the capillary tube was immersed in pure water, and 1 mL was sucked to wash the inside of the capillary tube. After washing, the pressure was again reduced to vacuum and dried overnight. This produced a capillary tube in which the Coating antibody was immobilized on the inner wall (see FIG. 12).

[1.2. (Preparation of sample solution)
The 720 ng / mL TNF-α Standard of the kit of 1.1. Was dispensed into a 14 μL microtube, and 36 μL of ultrapure water was added to dilute to 200 pg / μL. From this, sample solutions of 0 (ultra pure water, control), 0.2, 20, 200 pg / μL were prepared.

[1.3. Preparation of detection antibody)
1 mL of phosphate buffered saline was injected into the Detection antibody vial of the kit of 1.1 and dissolved to obtain a 13.5 μg / mL solution. After heating in a heat block at 25 ° C. for 15 minutes, it was diluted 10-fold to prepare a 1.35 μg / mL Detection antibody solution.

[1.4. Preparation of Streptavidin-POD (Peroxidase) Solution]
The Streptavidin-HRP (horseradish peroxidase) solution in the kit was diluted 10-fold with phosphate buffered saline to prepare a Streptavidin-POD solution.

[1.5. Preparation of substrate solution)
0.4 mg / μL ADHP (AnaSpec) dissolved in dimethyl sulfoxide (DMSO) is dispensed into a 2.5 μL microtube, 42.5 μL of ultrapure water and 50 μL of 30% hydrogen peroxide are added, and the substrate solution is added. Prepared.

[1.6. (Operation of antigen-antibody reaction in antibody-immobilized capillary tube)
Said 1.2. 5 μL of the sample solution prepared in (1) is taken into a weighing boat, and 1.1. When the tip of the antibody-immobilized capillary tube prepared in step 1 was dipped and visually confirmed with a stereomicroscope whether the sample solution was absorbed, it was found to be absorbed.
Said 1.2. 5 μL of the sample solution having a series of concentrations prepared in (1) was taken and dropped onto a weighing boat, and the tip of the antibody-immobilized capillary was immersed for 5 seconds to be absorbed. The above-described syringe pump was prepared, and a syringe was set. The inside of the capillary was washed by connecting the antibody-immobilized capillary and the syringe with a tube, immersing the tip of the capillary in ultrapure water, and aspirating 1 mL.
Next, 1.3. 2 μL of the detection antibody solution was dropped onto a weighing boat and sucked with a syringe pump to be absorbed into the capillary tube and left for 5 minutes. The capillary tube was washed by immersing in ultrapure water and aspirating 1 mL with a syringe pump.
Next, in 1.4. 2 μL of the Sterptavidin-POD solution was dropped onto a weighing boat and sucked with a syringe pump to be absorbed into the capillary tube and left for 5 minutes. After immersing in ultrapure water and sucking 1 mL with a syringe pump, the inside of the capillary was washed and then dried.

[1.7. (Fluorescence observation with substrate solution)
ADHP is converted to resorufin by the action of POD in the presence of hydrogen peroxide. Although ADHP is non-fluorescent, resorufin is a fluorescent substance and emits orange fluorescence when excited with green.
Therefore, the above 1.6. It was confirmed whether or not an antigen-antibody reaction was performed.
1.5. 5 μL of the substrate solution was dispensed into a weighing boat, and 1.6. As a result of immersing the tip of the antibody-immobilized capillary tube subjected to the above operation and performing fluorescence observation, the fluorescence in the capillary tube was visually confirmed.

[1.8. (Fluorescence measurement by absorption of substrate solution by capillary force)
The tip of the optical fiber of the optical fiber type fluorescence measurement system (Nippon Sheet Glass) is 4.6. It was held on the side of the antibody-immobilized capillary tube where the above operation was performed. Thereby, the capillary can be excited and the generated fluorescence can be measured. If the substrate solution is absorbed by capillary force and an increase in fluorescence is measured, the target substance is measured.
5 μL of the substrate solution was dropped on a weighing boat, the tip of the capillary tube was immersed for 10 seconds, and the fluorescence of the capillary tube was measured. The change of fluorescence intensity was shown with respect to the density | concentration of TNF- (alpha) (FIG. 13). The increase in fluorescence at 0 pg / μL (ultra pure water) was assumed to be that ADHP in the substrate solution was changed to resorufin. Although the variation was observed at a low concentration and the fluorescence intensity tended to be saturated at a concentration of 20 pg / μL or more, an increase in fluorescence corresponding to an increase in TNF-α could be confirmed.

  Capillary ELISA showed that TNF-α can be measured with a minute amount (capillary inner product) of about 800 nL. If the sandwich structure of Coating antibody, TNF-α (target substance), and Detection antibody can be formed on the inner wall of the capillary, the target substance can be measured, so that it is not necessary to fill the inner product of the capillary. What is necessary is just to be able to measure the change due to absorption or fluorescence of the dye precursor in the substrate solution due to the action of an enzyme (POD) labeled with a streptavidin-biotin bond. If the region where fluorescence measurement is performed with a capillary tube is 0.5 mm long, it is sufficient that each solution is collected at about 20 nL at the tip of the capillary tube.

  If the inner product of the capillary is reduced, it is clear that measurement can be performed with a smaller sample, and measurement with a nanoliter amount can also be realized. For example, a glass capillary having an inner diameter of 37 μm has been put into practical use. If this is used, the inner product is about 0.5 nL with a length of 0.5 mm, and measurement by sampling a sub-nanoliter amount is also possible. In the measurement with a nanoliter amount or less, there may be a case where the fluorescence of the dye cannot be detected significantly due to external noise such as stray light even if the fluorescence is generally said to be highly sensitive. In that case, it becomes possible by using a chemiluminescence method such as a luciferin-luciferase reaction system.

[1.9. (Fluorescence measurement by flow method)
By attaching a tube to the end of the capillary tube and piping it to a syringe set in a syringe / pump, the substrate solution and the washing solution can be flowed by suction. Therefore, a sample solution of 200 pg / μL of TNF-α was used, and 1.6. Using the antibody-immobilized capillary tube, the substrate solution was aspirated by 5 μL, and 1 mL of ultrapure water was aspirated for washing. The results are shown in FIG.
It can be seen that after the fluorescence due to the formation of resorufin is observed, the fluorescence disappears by washing. Absorption by capillary force is also possible by suction using a pump, so 1.6. It was shown that all the steps of the capillary ELISA including the step can be performed by the flow method. That is, the entire process can be realized by a suction method using a differential pressure such as a pump.

  Above 1.8. And 1.9. Further, since TNF-α in a very small amount of sample liquid was produced, it was shown that the capillary ELISA method can be applied to a sample such as a body fluid that exudes only a small amount such as a gingival crevicular fluid in a healthy gingiva. Inflammatory mediators other than TNF are considered to be detectable as well.

[2. Systematization)
In-situ measurement is possible by holding the capillary as the measurement probe and the optical fiber of the fluorescence measurement system by the holder. The washing step is provided with a suction mechanism such as a pump at the end of the capillary tube, so that all operations necessary for ELISA can be performed even when a capillary tube is used. Hand-held miniaturization can be easily realized. An assumed basic configuration diagram is shown in FIG.

As per normal gingival crevicular fluid, only 50 nL / min or less per tooth was exuded [Reference 2: edited by Fujita, Otsuka et al., “Standard Biochemistry / Oral Biochemistry” p. 294 (2003, Gakken Shoin), a detection method (ELISA method) using an antigen-antibody reaction (immune reaction) can be realized using a sample that can be collected only in a very small amount.
In addition, the measurement system for the ELISA method can be reduced in size.
In addition, since the reaction system is provided inside the capillary that is the sample collection tool, the sample collection amount can be reduced to the absorption amount of the capillary tube, the time for sample collection etc. can be reduced, and the burden on the subject is reduced. Can be reduced.
In addition, since measurement can be performed immediately after sample collection without transferring the sample to another container, sample alteration can be reduced. Only the amount can be collected and the loss can be reduced.

In addition, since the amount of sample handled is very small, the risk of exposure to pathogens via the sample can be drastically reduced.
Further, since the amount of sample collected is small, the body fluid such as the affected part to be collected can be divided into fine regions and the pathological condition can be examined for each micro region of the tissue. For example, if it is a gingival crevicular fluid, the pathological condition in each part of the gingiva showing a pathological condition can be examined.
In addition, since the reaction is performed on the spot after the collection, the result can be obtained in a short time, so that the treatment is performed simultaneously with the examination and contributes to the QOL of the patient.

In addition, this technique can also take the following structures.
[1] In a sensitive capillary equipped with a sampling field for collecting a target substance and a reaction field for immobilizing a trapping substance for trapping the target substance and performing an interaction reaction between the substances, On the other hand, a target substance detection method for detecting emitted light or radioactive information by interacting substances with an immobilized trap substance.
[2] The method for detecting a target substance according to [1], wherein the immobilized trap substance is a substance in which a trap substance that traps the target substance is immobilized by absorbing a solution containing the trap substance in a capillary tube. .
[3] The sample liquid containing the target substance is absorbed in the capillary, the substance is allowed to interact with the target substance using the immobilized trap substance, and then the inside of the capillary is washed.
Absorbing a solution containing a detection substance labeled with light or radioactive in the capillary to cause the labeled detection substance to interact between the target substance and the substance;
The method for detecting a target substance according to the above [1] or [2], comprising detecting light or radioactive information emitted from the bound labeled detection substance.
[4] After the reaction of the interaction between the substances with the immobilized trap substance, the capillary is made to absorb the solution containing the detection substance labeled with the enzyme, and the inside of the capillary is washed. ,
Absorbing a solution containing the enzyme substrate into the capillary;
The method for detecting a target substance according to any one of [1] to [3], further comprising: detecting optical information generated by changing the enzyme substrate.
[5] The target substance detection method according to [4], wherein the enzyme substrate is a dye precursor.
[6] Any one of [1] to [6], wherein the immobilized trap substance and / or the labeled detection substance is obtained by biotin-avidin crosslinking or biotin-streptavidin crosslinking. The target substance detection method as described.
[7] The target substance detection method according to any one of [1] to [6], wherein the sample liquid containing the target substance is a body fluid or blood.

[8] A collection part capable of collecting the target substance with a capillary tube;
A probe for detecting a target substance, comprising: a sensitive capillary section provided with a sensitive part for immobilizing a trapping substance that binds to the target substance and causing an interaction between the substances.
[9] The target substance detection target according to [8], wherein the immobilized trapping substance is a substance in which a trapping substance that traps a target substance is immobilized by absorbing a solution containing the trapping substance in a capillary tube. probe.
[10] The probe for detecting a target substance according to [8] or [9], wherein the labeled detection substance is a detection substance labeled with an enzyme.
[11] The probe for detecting a target substance according to any one of [8] to [10], wherein the enzyme substrate is a dye precursor.
[12] Any one of [8] to [11], wherein the immobilized trapping substance and / or the labeled detection substance is obtained by biotin-avidin crosslinking or biotin-streptavidin crosslinking. The probe for target substance detection as described.

[13] A target substance detection apparatus comprising at least the detachable target substance detection probe according to any one of [6] to [12].
[14] Detachable, equipped with a sensitive capillary part that provides a collecting part capable of collecting a target substance with a capillary, and a sensitive part for immobilizing a trapping substance for trapping the target substance and interacting with the substance A target substance detection probe;
A light irradiating unit for irradiating light to the sensitive unit of the target substance detection probe;
A target substance detection device comprising at least a light detection unit that detects light information from the sensitive unit by light irradiation by the light irradiation unit.

According to the present technology, it is possible to measure a target substance in real time, with high accuracy, and noninvasively. By using this technology, medical field (pathology, tumor immunology, transplantation, genetics, regenerative medicine, chemotherapy, etc.), drug discovery field, clinical laboratory field, food field, agricultural field, engineering field, forensic field , Can contribute to the improvement of analysis and analysis technology in various fields such as criminal forensics.
Moreover, this technique can reduce a test subject's burden and can also reduce the risk of exposure of a pathogen etc. via a sample drastically. The present technology can examine the pathological condition for each minute region of the tissue, and can be put into treatment at the same time as the examination to contribute to the QOL of the patient. Therefore, the present technology can contribute to a wide range of fields, but can particularly contribute to the development of the medical field and the like.
In addition, the present technology can reduce the size of the measurement system for the ELISA method.

  DESCRIPTION OF SYMBOLS 1 Target substance detection probe; 10 Target substance detection apparatus; 11 Sensitive capillary part; 12 Sensitive part; 13 Sampling part; 14 Support part; 15 Optical measuring holder; 16 Optical measuring system; 17 Optical waveguide; 2 Fixed object; Trapping substance; TA trapping substance; X target substance; YA detection substance; M label; C cross-linked product

Claims (9)

In a sensitive capillary comprising a collection field for collecting a target substance and a reaction field for immobilizing a trapping substance for trapping the target substance and performing an interaction between the substances,
A target substance detection method for detecting an emitted light or radioactive information by performing an interaction between substances with an immobilized trap substance on a collected target substance.   2. The target substance detection method according to claim 1, wherein the immobilized trap substance is obtained by immobilizing a trap substance for trapping a target substance by absorbing a solution containing the trap substance in a capillary tube. A sample liquid containing a target substance is absorbed in the capillary, and the substance is allowed to interact with the target substance with the immobilized trap substance, and then the inside of the capillary is washed;
Absorbing a solution containing a detection substance labeled with light or radioactive in the capillary to cause the labeled detection substance to interact between the target substance and the substance;
The target substance detection method according to claim 1, comprising detecting light or radioactive information emitted by the bound labeled detection substance. After the reaction of the interaction between the substances with the immobilized trap substance, the capillary is made to absorb a solution containing the detection substance labeled with an enzyme, and the inside of the capillary is washed;
Absorbing a solution containing the enzyme substrate into the capillary;
The method for detecting a target substance according to claim 3, comprising: detecting optical information generated by changing the enzyme substrate.   The target substance detection method according to claim 4, wherein the enzyme substrate is a dye precursor.   The target substance detection method according to claim 4, wherein the immobilized trap substance and / or the labeled detection substance is obtained by biotin-avidin crosslinking or biotin-streptavidin crosslinking.   The target substance detection method according to claim 4, wherein the sample liquid containing the target substance is a body fluid or blood. A collecting part capable of collecting the target substance with a capillary;
A probe for detecting a target substance, comprising: a sensitive capillary section provided with a sensitive part for immobilizing a trapping substance that binds to the target substance and causing an interaction between the substances. Removable target substance detection comprising a sensitive capillary part that provides a collecting part capable of collecting a target substance with a capillary and a sensitive part for immobilizing a trapping substance that traps the target substance and interacting with the substance Probe for,
A light irradiating unit for irradiating light to the sensitive unit of the target substance detection probe;
A target substance detection device comprising at least a light detection unit that detects light information from the sensitive unit by light irradiation by the light irradiation unit.