JP2009103455A - Element, apparatus, and method for detection of target substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and highly sensitive target substance detection method, a detection element, and a detection apparatus. <P>SOLUTION: In an antigen-antibody reaction inspection by a sandwich technique for quantifying target substances by measuring particulates through the use of secondary antibodies modified with the particulates, the number of particulates immobilizes to a substrate is quantitatively evaluated through the use of the orientation state of liquid crystals. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a detection material, a detection element, a detection device, a detection method, and a manufacturing method thereof for detecting a target substance in a specimen. In particular, the present invention can be suitably applied to so-called biosensors that utilize the specific molecular recognition ability of biologically derived substances or similar substances.

  A biosensor is a measuring device that utilizes the excellent molecular recognition ability of living organisms and biomolecules. In the living body, there are, for example, enzyme-substrate, antigen-antibody, DNA-DNA and the like as combinations of substances having affinity for each other. Biosensors use the principle that one of these combinations can be fixed or supported on a substrate and used to selectively measure the other substance. In recent years, biosensors are expected to have a wide range of applications not only in the medical field, but also in the environment and foodstuffs. In order to widen their use, biosensors are small, lightweight, and highly sensitive that can be installed or carried anywhere. A sensor is desired.

  One of the high-sensitivity sensing methods is a method using a labeling substance in which an antibody is supported around fine particles. This is a sensor that uses a method in which a labeling substance reacts with a target substance in a sample and reacts with the target substance and an antibody applied to a substrate, and the target substance is quantitatively measured by measuring the labeling substance. It is an evaluation method that can be evaluated. Since it has a structure in which a target substance is sandwiched between two antibodies, it is generally referred to as a sandwich method, and research is actively conducted. In this method, the presence or absence of the target substance and its concentration can be easily detected by measurement of the labeling substance, and therefore, application as a simple and highly sensitive biosensor is expected. One of the active researches is a magnetic biosensor using a magnetic substance as a substrate and a labeling substance made of magnetic fine particles containing a magnetic substance in the fine particles. The operation principle is as follows.

  An element for detecting the presence and concentration of a target substance in a specimen by the sandwich method can be specifically sensed by the following operation. First, antibodies that specifically bind to two different regions existing in the target substance are employed. Here, a first target substance capture molecule (primary antibody in the case of an antigen-antibody reaction) that can specifically bind to one region of the target substance (referred to as an epitope in the case of an antigen-antibody reaction) on the substrate surface Fixed). Next, an antigen-antibody reaction is performed between the surface of the substrate and the target substance in the specimen by bringing the primary antibody into contact with the specimen containing the target substance. Thereafter, by bringing the other region of the target substance into contact with a second target substance capturing molecule (referred to as a secondary antibody in the case of an antigen-antibody reaction), a state in which the antigen is sandwiched between two antibodies can be realized. . Here, when the antigen-antibody reaction is performed using the magnetic fine particles having the secondary antibody supported on the surface, the magnetic label is immobilized on the surface of the sensor element. By measuring the number of magnetic labels by some method, it is possible to determine the number and concentration of the target substance.

  Although the order is different from the above reaction, a second target substance capture molecule is added in advance to a sample containing the target substance to bring the target substance and the second target substance capture molecule into contact with each other. A “target substance capture molecule” complex is formed. Then, by bringing the complex into contact with the first target substance-capturing molecule immobilized on the sensor element, it is possible to adopt a method in which the magnetic label is immobilized on the surface of the sensor element as a result.

  The following method has been proposed as a target substance detection element using such a magnetic detection method.

(Method 1)
An immunoassay method is disclosed in which a magnetic substance as a label is bound to a specimen by an antigen-antibody reaction, the label is magnetized, and then the label is detected by a SQUID (superconducting quantum interferometer) as a magnetic sensor (for example, Patent Document 1).

(Method 2)
A biosensor characterized in that a detection element for detecting a magnetic field formed by bound magnetic molecules includes a semiconductor Hall element, and an object to be measured is analyzed based on the amount of the specified magnetic molecules Is disclosed (for example, Patent Document 2).

(Method 3)
A method of detecting a magnetic signal of a magnetic fine particle labeled with a first capture molecule on a sensor element and a second capture molecule via a target molecule using a magnetoresistive element is disclosed (for example, patent document). 3)).

(Method 4)
On the other hand, Method 4 using liquid crystal is known as a biosensor technique different from Methods 1 to 3. Details are described in Non-Patent Documents 1 and 2 and Patent Document 4. These are methods using only the first target substance-capturing molecule (referred to as a primary antibody in the case of an antigen-antibody reaction) provided on the substrate surface among the antigen-antibody reactions described above. Compared with the above-mentioned method, this method is the same procedure as described above until the primary antibody is brought into contact with the specimen containing the target substance and the surface of the substrate and the target substance in the specimen are subjected to the antigen-antibody reaction. is there.

In Method 4 using liquid crystal, after the above procedure, the liquid crystal material is aligned on the substrate on which the primary antibody is present. Here, it is known that the bulk alignment of the liquid crystal material is governed by the alignment state of the substrate interface. That is, in this case, the state of the interface between the substrate and the liquid crystal is different between the interface state where only the primary antibody exists and the interface state where the target substance is captured on the primary antibody. Therefore, the difference between the state in which the target substance is present in the sample and the target substance is captured on the primary antibody and the state in which the target substance is not present in the sample is the optical alignment state of the liquid crystal molecules. It becomes possible to detect easily by observing automatically. That is, in Method 4, it can be said that the liquid crystal material itself plays the role of the secondary antibody and the labeling substance in the sandwich method. This method is sometimes referred to as a liquid crystal assay.
JP 2001-033455 A International Publication WO 03-067258 US Pat. No. 5,981,297 JP-T-2006-501860 Science 279, 2077 (1998) Materials Science and Engineering C 24 (2004) 237-240

  Each of these conventional methods has advantages and disadvantages. The method using magnetic fine particles of methods 1 to 3 is useful as a method capable of detecting the presence / absence and concentration of an actual target substance with high sensitivity using a magnetic label. However, since the method 1 uses superconductivity, the apparatus scale and the apparatus cost are increased. For this reason, it can only be installed in large hospitals and other institutions with sufficient budget and space. In addition, the diagnosis cost increases for the same reason.

  Method 2 and method 3 are methods using a Hall element or a magnetoresistive (MR) element as a substrate. Since the substrate used here is formed through many film forming steps and patterning steps by a photolithography process as in the case of a semiconductor memory or the like, the manufacturing cost of the member to be used increases. In general, a device for strictly detecting a substance in a living body should use a new member each time in order to avoid contamination with other patient test substances, like syringes and injection needles in blood tests. desired. However, in these two methods, it is necessary to use an expensive member having a cost equivalent to that of a semiconductor memory as a so-called disposable member. Therefore, compared with the method 1, although the scale and cost of the whole apparatus are small, the member cost required for the assay per time becomes high.

  In Method 4, since the base material can be a simple structure similar to a general segment type liquid crystal element used for a wristwatch, a calculator, or the like, the cost of a member used for the assay is extremely low. In addition, since an optical evaluation method using polarized light can be adopted for detection of the target substance, the apparatus scale is also compact and inexpensive. For this reason, it is possible to easily realize a low-cost assay.

However, in view of Non-Patent Document 1, experiments were conducted at a target substance detection concentration of about 0.5 μM (= 5 × 10 ⁻⁷ mol / liter). This detection concentration is described in Patent Document 2 and the like. Higher than the concentration of magnetic biosensor. For this reason, in order to influence the bulk alignment of the liquid crystal, the state of the interface must be changed significantly, and for that purpose, it is presumed that it is necessary to act a relatively high concentration analyte. In addition, when the concentration is low, a region where the orientation is slightly changed may be observed. However, it is considered difficult to clearly distinguish these slight differences from alignment defects that occur in a general liquid crystal process. That is, there is a concern that the accuracy of diagnosis decreases in a region where the concentration is low.

  Accordingly, an object of the present invention is to provide a simple diagnostic method that solves the above-described problems, and to provide an element and a device used therefor.

  That is, the present invention supports a labeling substance on a substrate by a reaction between an antibody (primary antibody) supported on the substrate, a target substance, and an antibody (secondary antibody) modified with the labeling substance. Is a target substance detection element in which a liquid crystal is disposed around.

  The present invention also includes a labeling substance supported on a substrate by a reaction between an antibody (primary antibody) supported on the substrate, a target substance, and an antibody modified with a labeling substance (secondary antibody). A target substance detection device that detects the presence or absence and the number of labeling substances by detecting the alignment state of liquid crystal disposed around the substrate, thereby detecting the presence or absence and concentration of the target substance in the specimen, A target substance detection apparatus having a light source, a polarization control element, and an imaging element for optically detecting the alignment state of liquid crystal.

  The present invention also includes a labeling substance supported on a substrate by a reaction between an antibody (primary antibody) supported on the substrate, a target substance, and an antibody modified with a labeling substance (secondary antibody). This is a target substance detection method in which the presence / absence and number of labeling substances are detected by optically detecting the alignment state of the liquid crystal disposed around the substrate, thereby detecting the presence / absence and concentration of the target substance in the specimen.

  According to the present invention, a highly sensitive and simple diagnostic method can be provided, and a target substance detection material, a detection element, a detection device, and a detection method can be provided.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

  As described above, in a biosensor, an antibody is fixed to or supported on a substrate. As described in Method 4 above, a conventionally reported liquid crystal assay method has been proposed in which a target substance is supported on this antibody and then a liquid crystal material is used as a labeling substance. On the other hand, the biosensor of the present invention employs a sandwich method using fine particles as a labeling substance as in methods 1 to 3, and further uses a liquid crystal material.

  That is, in the present invention, a minute substance in which an antigen-antibody reaction has occurred is detected by the following process.

  First, a first antigen-antibody reaction is performed by immersing a specimen containing a target substance in a site where a primary antibody is disposed on a substrate. Next, the substance that has not contributed to the antigen-antibody reaction is washed (first antigen-antibody reaction process).

  Next, a secondary antibody labeled with fine particles is brought into contact with the substrate, and a second antigen-antibody reaction is performed. As a result, a structure in which the target substance is sandwiched between the antibodies is formed. Next, the secondary antibody that has not reacted is washed, and then water is dried (second antigen-antibody reaction process).

  Subsequently, another liquid crystal material is applied onto the substrate, dropped, or another substrate is disposed so as to provide a predetermined gap from the substrate, and the liquid crystal material is injected between the two substrates. In the element thus obtained, the presence or absence of the target substance can be easily measured by optically observing the alignment state of the liquid crystal (liquid crystal element production and optical evaluation process).

  In addition to that, by applying an evaluation method to be described later, it becomes possible to perform measurement with higher sensitivity (high sensitivity optical evaluation process).

  Hereinafter, each of the above processes will be described in detail.

[First antigen-antibody reaction process and second antigen-antibody reaction process]
As the primary antibody used in the present invention, a substance including a molecular structure having specific affinity and binding property to a target substance such as an immunoglobulin G molecule is used. In addition, any other material having similar properties can be applied to the present invention.

  Further, by using a gold thin film as the functional film located on the surface of the sensor element, the protein disclosed in International Application WO2005 / 095461 can be used as the first capture molecule. Furthermore, by using at least a part of the surface of the fine particle as the labeling substance as gold, it can be used as the second capture molecule.

This gold-binding protein is
(1) a first domain having a binding site for gold and comprising at least a part of an immunoglobulin G light chain variable region (VL) or an immunoglobulin G heavy chain variable region (VH);
(2) a second domain having a binding site for a target substance and comprising at least a part of an immunoglobulin G heavy chain variable region (VH) or an immunoglobulin G light chain variable region (VL);
And may be referred to as gold-binding diabody.

  In this case, the above (1) serves as an anchor when the sensor element or the labeling substance is fixed to the fine particles, and can be simply fixed in a state where the capturing ability of the capturing molecule is maintained. An example of this case is shown in FIG. 1 as a schematic diagram. On the sensor element 1 whose surface is covered with the gold thin film 10, the gold-binding protein 12 having the characteristics (1) and (2) above is immobilized as the first capture molecule, and the target substance 4 is bound to the protein. Has been. Similarly, the labeled fine particle 6 is coated with a gold thin film 11, and the gold-binding protein 13 having the characteristics (1) and (2) as a second capture molecule is coated with the gold thin film on the fine particle. It is fixed to the part. Then, the second capture molecule is immobilized in such a manner that it binds to the other region of the target substance immobilized on the first capture molecule.

  The size of the labeled fine particles used in the present invention can be selected depending on the shape, size or application of the element. A particle having a diameter of approximately 10 nanometers to several tens of micrometers is preferable from the viewpoint that the size of fine particles that affect the alignment state of liquid crystal molecules is necessary. Moreover, the substance which comprises the microparticles | fine-particles can be selected suitably according to the high sensitivity evaluation method mentioned later. For example, magnetic fine particles can be used in the same manner as the labeling substance used in the methods described in the above-mentioned methods 1 to 3, and normally used paramagnetic and superparamagnetic magnetic fine particles and magnetic beads can be used. As an example, a mixture or coating of iron oxide particles such as ferrite and magnetite and polymers such as styrene, dextran, and acrylamide are used. Further, for example, charged fine particles that can be used as an electrophoretic display element described in US Pat. No. 3,612,758 are also possible. Alternatively, it is preferable to select the characteristics of the fine particles according to the evaluation method described later, such as dielectric fine particles having a dielectric constant different from that of the liquid crystal material used in the present invention and fine particles having different absorptance, and use as a labeling substance.

  The target substance that can be detected and bound by the primary antibody immobilized on the substrate or the secondary antibody labeled with microparticles should be any substance that can perform the antigen-antibody reaction described above. Is possible.

  Biological substances that are usually applied as target substances include biological substances selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof. Specifically, it comprises a biomolecule selected from nucleic acids, proteins, sugar chains, and lipids. Specifically, it includes a substance selected from any of DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide, and sphingolipid. Any material can be applied. Furthermore, bacteria and cells themselves that produce the above-mentioned “biological substances” can also be targeted substances as “biological substances” targeted by the present invention.

  Among them, in particular, so-called disease markers can be mentioned as specific examples of proteins (lipid proteins, glycoproteins, protein complexes, protein multimers).

  Examples include acidic glycoproteins that are produced by hepatocytes in the fetal period and are present in fetal blood, and are used as markers for hepatocellular carcinoma (primary liver cancer), hepatoblastoma, metastatic liver cancer, and Yorksack tumor. Phetoprotein (AFP), an abnormal prothrombin that appears at the time of liver parenchymal disorder, PIVKA-II that is confirmed to appear specifically in hepatocellular carcinoma, glycoprotein that is immunohistochemically a breast cancer-specific antigen, primary BCA225, a marker of advanced breast cancer, recurrent / metastatic breast cancer, basic fetal protein found in human fetal serum, intestinal and brain tissue extracts, ovarian cancer, testicular tumor, prostate cancer, pancreatic cancer, biliary tract cancer, liver CA15 which is a sugar chain antigen serving as a marker for cell cancer, kidney cancer, lung cancer, stomach cancer, bladder cancer, basic fetoprotein (BFP) which is a marker for colon cancer, advanced breast cancer, recurrent breast cancer, primary breast cancer, ovarian cancer 3. CA19-9 which is a sugar chain antigen serving as a marker for pancreatic cancer, biliary tract cancer, gastric cancer, liver cancer, colon cancer and ovarian cancer, glycan antigen serving as a marker for ovarian cancer, breast cancer, colorectal cancer, gastric cancer and pancreatic cancer CA72-4, ovarian cancer (especially serous cystadenocarcinoma), endometrial adenocarcinoma, fallopian tube cancer, cervical adenocarcinoma, pancreatic cancer, lung cancer, colon cancer CA125, which is a marker for cancer, epithelium CA130, a glycoprotein that is a marker for primary ovarian cancer, fallopian tube cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer (especially serous cystadenocarcinoma), endometrial adenocarcinoma, cervical adenocarcinoma CA602 which is a core protein antigen. CA54 / 61 (CA546), a nucleoside sugar chain-related antigen that serves as a marker for ovarian cancer (particularly mucinous cystadenocarcinoma), cervical adenocarcinoma, and endometrial adenocarcinoma, colon cancer, stomach cancer, rectal cancer, biliary tract cancer Currently, carcinoembryonic antigen (CEA), pancreatic cancer, biliary tract cancer, hepatocytes, which are most widely used to assist cancer diagnosis as tumor-related marker antigens such as pancreatic cancer, lung cancer, breast cancer, uterine cancer, urinary tract cancer, etc. DUPAN-2, a sugar chain antigen that serves as a marker for cancer, stomach cancer, ovarian cancer, and colon cancer, is present in the pancreas and specifically hydrolyzes elastic fiber elastin (which constitutes arterial walls and tendons) in the connective tissue Pancreatic exocrine proteolytic enzyme, elastase 1 as a marker for pancreatic cancer, pancreatic sac cancer, biliary tract cancer, glycoprotein present in high concentration in ascites and serum of human cancer patients, lung cancer, leukemia, esophageal cancer, pancreatic cancer, Ovarian cancer, kidney cancer, bile duct cancer, stomach cancer, bladder cancer, colon cancer, NCC-ST-, a sugar chain antigen that is a marker for immunosuppressive acidic protein (IAP), a marker for thyroid adenocarcinoma, malignant lymphoma, pancreatic cancer, biliary tract cancer, breast cancer, colon cancer, hepatocellular carcinoma, lung adenocarcinoma, gastric cancer 439, γ-seminoprotein (γ-Sm), a glycoprotein that is a marker for prostate cancer, is a glycoprotein extracted from human prostate tissue and is therefore only present in prostate tissue, and is therefore a marker for prostate cancer Prostate-specific antigen (PSA), an enzyme that hydrolyzes phosphate esters under acidic pH secreted from the prostate, specific for prostate acid phosphatase (PAP), neural tissue and neuroendocrine cells used as a tumor marker for prostate cancer Is a glycolytic enzyme that is a marker of lung cancer (especially small cell lung cancer), neuroblastoma, nervous system tumor, pancreatic islet cancer, esophageal small cell carcinoma, gastric cancer, kidney cancer, breast cancer Heteroenolase (NSE), a protein extracted and purified from liver metastases of cervical squamous cell carcinoma, markers for uterine cancer (cervical squamous cell carcinoma), lung cancer, esophageal cancer, head and neck cancer, skin cancer Sialyl LeX-i antigen (SLX) which is a sugar chain antigen serving as a marker for squamous cell carcinoma-related antigen (SCC antigen), lung adenocarcinoma, esophageal cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, uterine cancer , Pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, sugar chain antigen SPan-1, which is a marker for colon cancer, esophageal cancer, stomach cancer, rectal / colon cancer, breast cancer, hepatocellular carcinoma, biliary tract cancer, pancreatic cancer, lung cancer, uterine cancer Tissue polypeptide antigen (TPA), ovarian cancer, metastatic ovarian cancer, which is a single-chain polypeptide that is useful for prediction of recurrence and treatment progress, especially in combination with other tumor markers , Stomach cancer, colon cancer, biliary tract cancer, pancreatic cancer, lung Sialyl Tn antigen (STN) which is a mother nucleus sugar chain antigen serving as a cancer marker, Cytokeratin (CYFRA) which is an effective tumor marker for detecting non-small cell lung cancer, particularly squamous cell carcinoma of the lung, in gastric juice Inactive precursors of two types of pepsin (PG I and PG II), secreted protein digestive enzymes, gastric ulcer (especially lower gastric ulcer), duodenal ulcer (especially relapsed or intractable), Brunnell adenoma, Solinger Pepsinogen (PG), a marker of Ellison syndrome, acute gastritis, an acute phase reaction protein that changes in plasma due to tissue damage and infection, and C-reaction that shows high values when necrosis occurs in the myocardium due to acute myocardial infarction, etc. Protein (CRP), serum amyloid A protein (SAA), which is an acute phase reaction protein that changes in plasma due to tissue damage and infection, and molecular weight mainly present in cardiac muscle and skeletal muscle It is a heme protein of about 17500, and is present mainly in the soluble fraction of myoglobin, skeletal muscle, and myocardium that are markers for acute myocardial infarction, muscular dystrophy, polymyositis, and dermatomyositis, and is released into the blood by cell damage Creatine kinase (CK), an enzyme that is a marker of acute myocardial infarction, hypothyroidism, progressive muscular dystrophy, polymyositis (CK-MM type derived from skeletal muscle, CK-BB derived from brain, smooth muscle Type, myocardial CK-MB type isozyme, mitochondrial isozyme and immunoglobulin-binding CK (macro CK)), and troponin complex together with troponins I and C on thin filaments of striated muscle It is a 39000 molecular weight protein that is involved in the regulation of muscle contraction and is a marker of rhabdomyolysis, myocarditis, myocardial infarction, and renal failure. Troponin T, a protein contained in both skeletal muscle and myocardial cells, and an increase in measurement results indicates skeletal muscle, myocardial damage or necrosis, and is a marker for acute myocardial infarction, muscular dystrophy, and renal failure Examples include ventricular myosin light chain I, and chromogranin A, thioredoxin, and 8-OhdG, which have recently been attracting attention as stress markers.

  After the target substance is bound to the primary antibody by the first antigen-antibody reaction, the secondary antibody labeled with the microparticles and the second antigen-antibody reaction are performed.

  Next, after the second antigen-antibody reaction, fine particles that have not contributed to the reaction are washed with physiological saline or the like. That is, in general, biological substances are water-soluble, and thus proteins such as target substances or antibodies can be washed with an aqueous solvent.

  Dry water after washing. This is because after the second antigen-antibody reaction, the liquid crystal material is brought into contact with the substrate for inspection. In other words, since a general liquid crystal material is an organic material and is not compatible with water, it is preferable to keep the vicinity of the substrate interface free from moisture in an assay using liquid crystal.

  Furthermore, when magnetic fine particles are used as a labeling substance in the second antigen-antibody reaction, a known magnetic manipulation technique can be used. As a result, by applying a magnetic field from the outside, the secondary antibodies modified with the magnetic fine particles are collected in the vicinity of the substrate interface, and the reaction can be promoted. Furthermore, the magnetic fine particles that have not contributed to the reaction can be moved to a position away from the substrate by using a magnetic field during the above-described washing, thereby increasing the efficiency of washing the secondary antibody.

  Similarly, when charged fine particles are used as the labeling substance in the second antigen-antibody reaction, a known electrophoresis technique can be used. As a result, the same accumulation and cleaning effects as in the magnetic manipulation technique can be enhanced.

  These antigen-antibody reactions may be performed in a reaction well having a predetermined size or may be performed in a microchannel. That is, any method can be used as long as the binding state in which the target substance is sandwiched between the antibodies as described above can be realized.

  In the present invention, it is preferable to align the liquid crystal material in a predetermined direction in order to obtain a highly accurate measurement result. For this reason, it is preferable to use a substrate provided with a liquid crystal alignment film on the substrate on which antigen-antibody reaction is performed. As described above, since the primary antibody has the property of being immobilized on gold, for example, by allowing the presence of gold to coexist in a part of the liquid crystal alignment film region, the alignment property and the antibody Since immobilization can be made compatible, it is preferable. As this manufacturing method, for example, after applying a liquid crystal alignment film, the film may be formed by mask vapor deposition or the like so that gold is formed in a sea-island shape.

  The alignment film used here may be a polyimide alignment film used in a general liquid crystal display, or an alignment film made of an inorganic material such as an oblique deposition film of silicon oxide. In addition, any material can be used as long as it has a property capable of defining the alignment state of the liquid crystal.

  For the gold film formation and patterning, a photolithography process and a lift-off process can be used in addition to the above-described mask deposition.

  In general, since the orientation of liquid crystal is not defined on the gold surface, it has a random orientation. Therefore, a fine uneven shape may be provided on the gold surface to define the alignment direction of the liquid crystal. Alternatively, if a method using circularly polarized light, which will be described later, is adopted, highly accurate measurement can be performed even if the liquid crystal on the substrate side subjected to antigen-antibody reaction is in a random orientation. Since there is no need for patterning as described above, it is advantageous in terms of manufacturing cost of the substrate.

  In the liquid crystal assay, when an evaluation is performed with a liquid crystal sandwiched between two substrates, it is preferable to use an alignment film for the other substrate different from the substrate that undergoes the antigen-antibody reaction. In this case, since the gold patterning as described above is unnecessary, the same alignment film forming process as that of a normal liquid crystal display can be employed.

  For example, in order to detect the alignment state while applying a voltage to the liquid crystal, it is necessary to form the above-described configuration on the substrate having the electrode, depending on whether or not a technique for high sensitivity measurement described later is adopted. is there. Here, in order to observe the alignment state of the liquid crystal in a transmissive state, a predetermined transmittance can be obtained by forming the gold on the substrate with a transparent electrode so thin that light can be transmitted. On the other hand, in order to observe the alignment state of the liquid crystal in the reflection state, the thickness of the gold and the transmittance of the electrode to be used are not required.

[Liquid crystal device fabrication and optical evaluation process]
By the above process, a substrate after the antigen-antibody reaction in which the target substance is sandwiched between the primary antibody and the secondary antibody labeled with the microparticles is obtained. As shown in FIG. 2, the liquid crystal is aligned on this substrate, and the presence or absence of fine particles is detected from the alignment state.

  When evaluating the orientation of the liquid crystal, a liquid crystal material may be coated on the substrate after the antigen-antibody reaction, and analysis using polarized light may be performed immediately after that. Further, as in a general liquid crystal display, a method in which a cell in which a liquid crystal material is sandwiched between two upper and lower substrates is manufactured and measured using polarized light may be used.

  In the former method, the liquid crystal is just on the substrate, one interface of the liquid crystal is the interface between the substrate and the liquid crystal (solid-liquid interface), and the other interface is the air-liquid crystal interface ( Gas-liquid interface). Although there are few reports on the alignment state of the liquid crystal at the gas-liquid interface, the liquid crystal is aligned at a certain angle according to the specific properties of the liquid crystal material, such as an optical film using a discotic liquid crystal described in Japanese Patent No. 0258398. It is known. For this reason, the alignment state in the vicinity of the gas-liquid interface becomes substantially uniform. At the remaining solid-liquid interface, the alignment state of the liquid crystal varies depending on the presence or absence of fine particles. In other words, since one gas-liquid interface is uniform and the remaining solid-liquid interface changes its orientation depending on the presence or absence of fine particles, the presence of fine particles, that is, the presence of the target substance can be detected by the method of applying liquid crystal. Is possible.

  The coating method used at this time is difficult to perform correct optical measurement if the thickness of the liquid crystal is uneven, such as a method of spin coating after hanging a liquid crystal material on a substrate, screen printing, transfer printing method, ink jet method, etc. A general liquid film forming method can be employed. The application is preferably controlled to a constant thickness.

  On the other hand, in the method of manufacturing and measuring the same cell configuration as the latter liquid crystal display, A method of injecting liquid crystal after producing an empty cell; Any of the methods of superposing the upper substrate after dropping the liquid crystal material on the substrate after the antigen-antibody reaction may be used.

  I. In order to fabricate the empty cell, a process is required in which two substrates are maintained with a predetermined gap therebetween. Therefore, a cell manufacturing process similar to that of a general liquid crystal display can be used. For example, spacer beads having a predetermined diameter are scattered on one substrate, and the remaining one substrate is overlaid. Alternatively, a spacer material having a predetermined thickness such as a Mylar film is disposed on a part of the outer periphery of one substrate, and then the remaining one substrate is overlaid. Alternatively, it is preferable in terms of simplicity if a step is formed so that a gap is created when the two substrates are preliminarily overlapped. These are all the same as the manufacturing process of a normal liquid crystal display.

  Alternatively, when the antigen-antibody reaction is performed in a microchannel, the microchannel itself can be evaluated as a liquid crystal cell.

  A liquid crystal material is injected into the empty cell thus obtained. This injection is the same as the manufacturing process of a normal liquid crystal display, and adopts a method in which the empty cell and the liquid crystal are brought into contact under vacuum and the atmosphere is opened to the atmosphere to pressurize by pressure difference. Can do. Or in order to carry out more simply, the injection | pouring using a capillary phenomenon may be performed in air | atmosphere, without employ | adopting such a vacuum process.

  B. This method is also a so-called dropping injection method used as a method for manufacturing a liquid crystal display. This method is a process of making a liquid crystal element by dropping a liquid crystal material on one substrate and then overlaying the other substrate, and is generally adopted as a manufacturing process for large liquid crystal panels that take time to inject liquid crystal. Has been.

  Also in this case, a spacer material is appropriately selected and used in order to set a predetermined distance between the two substrates. This method is a. This is advantageous in terms of time required for evaluation and convenience.

  A liquid crystal element (hereinafter simply referred to as a liquid crystal element) using the substrate obtained after the antigen-antibody reaction thus obtained is optically evaluated. Since this optical evaluation method is the same as the display principle of a general liquid crystal display, it can be easily performed.

  As described above, a transmissive liquid crystal element and a reflective liquid crystal element can be manufactured.

  In the case of the transmission type, evaluation is performed using two polarizing plates so as to sandwich the element. Light is irradiated from the lower side of the element, the first polarizing plate out of the two polarizing plates is linearly polarized light, and when the light passes through the liquid crystal element, it becomes elliptically polarized light due to the birefringence effect, and after passing through the element, The second polarizing plate can produce a difference in transmittance or coloring state depending on the state of elliptically polarized light.

  At this time, when the labeling substance, that is, the fine particles are fixed to the substrate by the antigen-antibody reaction, the alignment state of the liquid crystal around the portion where the fine particles are present is different from the other portions. As a result, the state of elliptically polarized light passing through the periphery of the fine particle is different from the state of elliptically polarized light passing through the portion where the fine particle does not exist, so that only the periphery of the fine particle is observed with a different transmittance or colored state. The That is, the presence of the fine particles can be detected optically.

  On the other hand, in the case of the reflective type, a gold substrate for immobilizing the primary antibody can be used as it is as a reflective layer. In order to observe in more detail, it is preferable to form a reflective layer also in parts other than gold. The evaluation of the alignment state is the same as the measurement principle of the transmission type, and the evaluation can be performed by observing the difference in the polarization state between the periphery of the fine particles and the other. At this time, for example, a change in polarization can be observed by arranging a linearly polarizing plate on the upper part of the device, or a circularly polarizing plate may be used. Alternatively, polarization measurement under crossed Nicols using PBS (polarization beam splitter) is advantageous in terms of contrast.

  As the substrate used for the liquid crystal element, it is preferable to provide an alignment control film as described above on the substrate on which antigen-antibody reaction is performed, but it goes without saying that an alignment control film is also preferably provided on the opposite substrate. . At this time, the counter substrate side may be subjected to uniaxial alignment treatment such as rubbing using an alignment film that is aligned substantially parallel to the substrate, or using a vertical alignment film that is aligned perpendicular to the substrate. Also good.

  In particular, when it is difficult to give alignment control ability to the substrate on which antigen-antibody reaction is performed, and the liquid crystal is aligned in parallel and randomly with respect to the substrate, the counter substrate is subjected to vertical alignment treatment, It is preferable to employ the configuration used. That is, in this configuration, one counter substrate side remains vertical and the other one is in parallel orientation, so that the retardation value in the cell thickness direction is substantially uniform in the cell. Although the orientation azimuth is random, it is possible to perform measurement independent of the orientation azimuth by adopting circularly polarized light as the incident polarized light.

  Such a method makes it possible to evaluate the presence or absence of fine particles. The difference in orientation at this time may be observed using a microscope, or may be evaluated by enlarging an image using an optical system similar to a liquid crystal projector. Although this evaluation can be performed visually, it is also possible to adopt an evaluation based on automatic calculation using an image processing apparatus including a digital image sensor and a computer.

[High-sensitivity optical evaluation process]
The evaluation described above described a method of observing a state in which liquid crystal is simply injected into a cell using a substrate after an antigen-antibody reaction. Here, we will show the evaluation process that takes advantage of the characteristics of liquid crystals to achieve higher sensitivity.

Since the role of the fine particles used in the present invention is to influence the alignment of the liquid crystal, there are no particular restrictions on the material and properties of the fine particles. On the other hand, high-sensitivity measurement can be realized by adopting the following measurement method using fine particles of materials (1) Method using magnetic fine particles (2) Method using charged fine particles (3) Surrounding substances and Is a method using fine particles with different absorption coefficients.

Hereinafter, the measurement method will be described in order. (1) Method using magnetic fine particles In this method, magnetic fine particles used in the magnetic biosensor described in the methods 1 to 3 described at the beginning can be used. That is, similarly to these, by reacting the target substance on the substrate having the primary antibody and then reacting with the secondary antibody labeled with the magnetic microparticles, the magnetic microparticles can be immobilized on the substrate. In the above methods 1 to 3, the magnetic fine particles are detected by a magnetic sensor, whereas in the present invention, highly sensitive detection is performed using a liquid crystal.

  Since two antibodies and a target substance exist between the substrate and the magnetic fine particles, the magnetic fine particles are immobilized on the substrate, but the target substance and the two antibodies are between them. There are gaps for the length of which are arranged substantially linearly. For example, since the molecular length of a known immunoglobulin G used as an antibody is approximately 15 nm, there is a gap of 30 to 50 nm between the substrate and the magnetic fine particles, depending on the size of the target substance. To do. In addition, the antibody and the target substance are not immobilized in a state where they do not move completely. For example, it is considered that the molecule rotates relatively freely, such as a carbon single bond portion of each molecule. Alternatively, the binding state between the antibody and the target substance is assumed to be due to an equilibrium reaction, and the binding state is not necessarily strong. Therefore, the movement of the magnetic fine particles immobilized on the substrate can be controlled by an external field.

  Evaluation using the liquid crystal element is performed using this property. That is, for example, an alternating magnetic field is applied as an external field to a liquid crystal element in which magnetic fine particles are present on the substrate interface. As a result, the magnetic fine particles vibrate in response to the alternating magnetic field. That is, a part of the interface of the liquid crystal element vibrates due to the external field. Since the bulk alignment state of the liquid crystal element is known to be governed by the interface alignment angle (pretilt angle), the liquid crystal bulk alignment state can be modulated by the alternating magnetic field.

  If the magnetic fine particles to be used are sufficiently large, the magnetic fine particles can be directly counted with an optical microscope without using liquid crystal, but it is difficult to count small fine particles of 2 microns or less with an optical microscope. In particular, it is impossible to measure fine particles having a size equal to or smaller than the wavelength of visible light with an optical microscope. On the other hand, since the method of the present invention is a technique for detecting the presence of fine particles by expanding the liquid crystal, it can be detected regardless of the size of the fine particles.

  At this time, more accurate measurement is possible by comparing the alignment state depending on the presence or absence of an external field. For example, the state of orientation in the absence of an external field is stored as an image, and then the state of orientation in a state where an external field is applied is stored as an image. If these two stored images are compared and calculated by the image processing apparatus, only the part affected by the external field can be extracted.

  If a response such as the liquid crystal molecules themselves vibrate or change orientation due to an external magnetic field, the evaluation results will be affected, so a liquid crystal material with zero or sufficiently low magnetic anisotropy should be used. Use. When the magnetic anisotropy is zero, it is more preferable because the liquid crystal does not respond at all to an external magnetic field. When magnetic anisotropy exists, it is preferable to perform the evaluation within a range below the threshold of the Fredericks transition so that the liquid crystal does not respond by the presence of an external magnetic field. At this time, it is preferable to use a liquid crystal material having sufficiently small magnetic susceptibility anisotropy in order to apply an external field sufficient to vibrate the magnetic fine particles.

  In the evaluation, even if an image that looks like a fine particle is detected in an image measured without applying an external field, it can be distinguished whether it is a true fine particle that modifies the secondary antibody or dust generated in the process. There may not be. On the other hand, in the present invention, since the presence / absence of external field application is compared and extracted, it is possible to eliminate erroneous determination due to dust and to quantify only the magnetic fine particles.

  For example, when a defect inspection device for a liquid crystal panel is used, when a full high-definition liquid crystal panel is inspected, it is possible to instantaneously measure the defect of one pixel out of 6 million pixels. That is, foreign matter inspection with a dynamic range of approximately 8 digits can be performed at high speed. Therefore, when such an optical inspection apparatus is used also in the present invention, high-sensitivity measurement can be easily realized in the fine particle measurement of the present invention. That is, according to the example of the defect inspection apparatus, detection can be performed even if only one fine particle exists on the substrate, so that ultra-sensitive measurement is possible.

  The method of the present invention is also advantageous in terms of quantitativeness. In the magnetic biosensor for detecting the magnetic field from the fine particles described in the methods 1 to 3, since the distance between the substrate and the magnetic fine particles varies depending on the length of the antibody and the binding state, there is a concern that the signal intensity may vary. In addition, there is a concern that the signal intensity changes depending on where on the magnetic biosensor element the magnetic fine particles adhere. On the other hand, in the method of the present invention, since the number of fine particles on the substrate can be optically measured by the inspection apparatus, quantitative evaluation can be performed easily and accurately.

  The direction of the magnetic field applied during this evaluation may be applied in a specific direction. However, when the alignment of the liquid crystal molecules on the substrate side that has reacted with the antigen-antibody is not fixed, especially in the case of a random alignment state, measurement is performed from a plurality of magnetic field application azimuth angles, and the results are comprehensively judged to determine the fine particles. You may count. The change in the magnetic field orientation at this time may be caused by rotating the magnetic field application device or rotating the liquid crystal element while the magnetic field application device is fixed. When the liquid crystal element is rotated, it is preferable to simultaneously rotate the image pickup element for image acquisition in order to correctly compare the image information.

(2) Method Using Charged Fine Particles In the above (1), magnetic fine particles are used. However, charged fine particles can be used based on the same concept.

  The principle of evaluation is the same as above, comparing the difference in orientation depending on the presence or absence of an external field. At this time, the difference is that charged fine particles are used instead of magnetic fine particles, and that an electric field is applied instead of applying a magnetic field as an external field. At that time, in the above (1), the precautions of magnetic susceptibility anisotropy were described, but in this method, attention must be paid to the dielectric anisotropy.

  That is, as described above, if the liquid crystal responds due to an external field, the evaluation result is affected. Therefore, a liquid crystal material having a dielectric anisotropy of zero or a sufficiently small value is used. When the dielectric anisotropy is zero, it is more preferable because the liquid crystal does not respond at all to the external electric field. In the case where dielectric anisotropy exists, it is preferable to perform the evaluation within a range below the threshold of the Fredericks transition so that the liquid crystal does not respond even if an external electric field exists. At this time, it is preferable to use a liquid crystal material having a sufficiently small dielectric anisotropy in order to apply an external field sufficient to vibrate charged fine particles. When adjusting the dielectric anisotropy, a commercially available positive-type (positive dielectric anisotropy) liquid crystal material and negative-type (negative dielectric anisotropy) liquid crystal material may be mixed and used. Good.

  The process of high sensitivity evaluation is the same as [1] above. In this method, since an electric field is applied instead of a magnetic field, a coil or the like for applying a magnetic field is unnecessary, which is preferable in that the apparatus configuration is simplified.

(3) Method of using fine particles having a physical absorption coefficient different from that of the surrounding material Here, an evaluation method using heat will be described. When the physical absorption coefficient of light or the like is different from the surrounding members with respect to the fine particles to be used, the state of heating when light or the like is irradiated from the outside changes between the vicinity of the fine particles and the portion where no fine particles are present. That is, if the state of thermal change due to the presence or absence of fine particles is detected by liquid crystal, the fine particles can be quantitatively evaluated.

  The liquid crystal material has a refractive index anisotropy, and in the case of a nematic liquid crystal, the refractive index anisotropy generally has a temperature dependency. In the case of a smectic liquid crystal, there is almost no temperature dependence of the refractive index. Therefore, it is preferable to use a nematic liquid crystal in the present invention.

  Here, a nematic liquid crystal device using a nematic liquid crystal device and a substrate made by repelling an antigen-antibody with a secondary antibody modified with fine particles having a large light absorption coefficient and nematic liquid crystal is used to radiate light from the outside. The value of anisotropic refractive index of the liquid crystal decreases. Thereby, a difference arises in the polarization state around the fine particles depending on the presence or absence of light irradiation. Similar to the above (1) and (2), it is possible to quantitatively evaluate the fine particles by comparing these images with and without light irradiation.

  Apart from this, the evaluation can also be made by observing the distribution of the phase transition temperature. There is a temperature difference between the vicinity of the fine particles and other locations depending on the presence or absence of light irradiation. By utilizing this property, it is possible to detect the presence or absence of fine particles by observing the vicinity of the phase transition temperature of the liquid crystal.

  When light is not irradiated from the outside, phase transition occurs at almost the same timing in the liquid crystal element when the ambient temperature is changed. On the other hand, when light is irradiated from the outside, when the ambient temperature is changed, a local temperature difference occurs between the vicinity of the fine particles and other places, so that phase transition occurs in the liquid crystal element. The timing is observed differently. This makes it possible to quantitatively evaluate the presence or absence of fine particles.

  In the above example, fine particles with different physical absorption coefficients by light have been described. However, if they have the property of absorbing the external energy and raising the temperature in the vicinity of the fine particles, the same concept should be used for evaluation. Is possible. That is, as the physical absorption of the present invention, for example, absorption by electromagnetic waves of various wavelengths from gamma rays to microwaves may be used, or ultrasonic vibration may be used.

  In addition, said (1)-(3) can perform still more sensitive observation by performing the following evaluation. These use the response of fine particles to external perturbations to change the alignment state in the vicinity of the liquid crystal interface and at the same time observe it in a static state. This observation result is compared with the static orientation state without external perturbation.

  It is known that the electro-optical characteristics of a liquid crystal element vary greatly depending on the state of liquid crystal molecules at the interface. That is, only the pretilt angle of the liquid crystal and the surface alignment force of the alignment film are slightly different, and the electro-optical characteristics of the liquid crystal element are observed differently. Therefore, by using the method of the present invention and simultaneously measuring the electro-optical characteristics and comparing the electro-optical characteristics with or without external perturbation, the difference in interface orientation can be further expanded and evaluated. This is preferable from the viewpoint of measuring sensitivity.

[Target substance detection device]
Next, an apparatus for detecting a target substance of the present invention will be described. As described above, the labeling substance is supported on the substrate by the reaction between the antibody (primary antibody) supported on the substrate, the target substance, and the antibody (secondary antibody) modified with the labeling substance. This is a target substance detection device that detects the presence / absence and number of labeling substances by detecting the alignment state of the liquid crystal disposed around the labeling substance, thereby detecting the presence / absence and concentration of the target substance in the specimen. The apparatus of the present invention includes at least a light source for optically detecting the alignment state of the liquid crystal, a polarization control element, and an imaging element.

  Furthermore, the apparatus of the present invention aligns a liquid crystal on a substrate in which a target substance is sandwiched between a primary antibody and a secondary antibody labeled with microparticles, and detects the presence or absence of microparticles from the alignment state. Therefore, it has a function of changing the alignment state of the liquid crystal material around the labeling substance. Specific examples include the use of a substance having a surface energy different from that of the alignment film on the substrate and the surface energy of the labeling substance, and the use of a labeling substance whose size is adjusted according to the liquid crystal material used. it can.

  Further, in order to detect the presence / absence and concentration of the target substance in the specimen, the apparatus of the present invention further detects (1) the liquid crystal material from the alignment state of the liquid crystal disposed around the labeling substance. It can have a memory for storing the orientation state as image information and (2) a function of calculating to compare the orientation state with or without an external field. When the above (1) and (2) are provided, as already described, a part affected by an external field can be extracted, which is preferable in that higher-accuracy measurement is possible. Furthermore, in order to provide a highly sensitive and simple diagnostic method, it is possible to provide a device for evaluating electro-optical characteristics.

  In this embodiment, magnetic fine particles are used.

After depositing gold on a 1 cm square glass substrate, the primary antibody is immobilized. On this substrate, the antigen-antibody reaction described in the specification is performed to fix magnetic fine particles having a diameter of 200 nm. This immobilization process is performed in the following order.
1. A sample containing a target substance, a diluted solution of the sample, and a labeling reagent containing magnetic fine particles are prepared.
2. The glass substrate is placed on the bottom of the reaction well.
3. A prescribed amount of the diluent is dispensed into the reaction well.
4). Next, a prescribed amount of sample is dispensed.
5). Stir the reaction.
6). Discard the reaction.
7). Wash.
8). Discard the cleaning solution.
9. Dispense labeling reagent.
10. Stir the labeling reagent.
11. Discard the labeling reagent.
12 Wash.
13. Discard the cleaning solution.
14 Dry and take out the glass substrate.

  By such an antigen-antibody reaction process, a glass substrate on which magnetic fine particles are immobilized according to the concentration of the target substance in the specimen is obtained.

  In these 10 processes, when a known magnetic manipulation technique is adopted and the magnetic fine particles are manipulated by an external field, the secondary antibody can be reacted more effectively.

  On the other hand, a 1 cm square glass substrate with ITO is prepared as a counter substrate. This substrate is subjected to a vertical alignment process, and a Mylar film having a thickness of 5 μm, a width of 0.5 mm, and a length of 1 cm is attached to two opposing sides.

  The substrate subjected to the antigen-antibody reaction and the counter substrate are overlapped, and MLC6608 (manufactured by Merck), which has a negative dielectric anisotropy as a liquid crystal material, is injected using a capillary phenomenon.

  The alignment state of the liquid crystal element thus obtained can be observed by attaching a broadband circularly polarizing plate to the counter substrate side. The image at this time is taken with a digital camera and stored. The apparatus used here is shown in FIG.

  On the other hand, an alternating magnetic field is applied to the liquid crystal element from the outside. An image is acquired and stored with a digital camera while applying this magnetic field. In this embodiment, the direction of the alternating magnetic field is 6 points in the direction of the azimuth angle with respect to the substrate surface in increments of 30 degrees (1.0 hour, 6 hour direction, 2.1 hour, 7 hour direction, 3.2 hour).・ Measure at 8 o'clock, 4.3 o'clock, 9 o'clock, 5.4 o'clock, 10 o'clock, 6.5 o'clock, 11 o'clock) and save each image.

  Thereafter, these six images are compared with images in a state where no external magnetic field is applied, and differences are extracted. At this time, the image difference can be extracted by acquiring an exclusive OR between the images with and without the external field.

  Next, by calculating the logical sum between the extracted six images, it is possible to accurately determine the presence of the fine particles.

  At this time, the measurement is performed while applying a voltage between the gold and ITO of the substrate as well as a method of obtaining without applying a voltage between the upper and lower substrates. By acquiring data in increments of 0.5 V from 0 V (no voltage applied) to 5 V, and analyzing these results, a more accurate number of fine particles can be obtained.

It is a schematic diagram explaining the sandwich method used for this invention. It is a schematic diagram explaining the target substance detection element using the liquid crystal of this invention. It is a schematic diagram showing the target substance detection apparatus used for the Example of this invention.

Explanation of symbols

1 Sensor element 4 Target substance 6 Labeled fine particle 10, 11 Gold thin film 12 Gold-binding protein (first capture molecule)
13 Gold-binding protein (second capture molecule)

Claims (18)

The labeling substance is supported on the substrate by the reaction between the antibody (primary antibody) supported on the substrate, the target substance, and the antibody modified with the labeling substance (secondary antibody),
A target substance detection element in which a liquid crystal is disposed around the labeling substance.   The target substance detection element according to claim 1, wherein the labeling substance is a magnetic fine particle.   The target substance detection element according to claim 1, wherein the labeling substance is a charged fine particle.   The target substance detection element according to claim 1, wherein a physical absorption coefficient of the labeling substance is higher than a physical absorption coefficient of a part where no labeling substance exists. A labeling substance is supported on the substrate by a reaction between the antibody (primary antibody) supported on the substrate, the target substance, and an antibody modified with the labeling substance (secondary antibody), and is disposed around the labeling substance. A target substance detection device that detects the presence or absence and number of labeling substances by detecting the alignment state of the liquid crystal thus detected, thereby detecting the presence or absence and concentration of the target substance in the specimen,
A target substance detection apparatus comprising at least a light source, a polarization control element, and an imaging element for optically detecting the alignment state of the liquid crystal.   The target substance detection apparatus according to claim 5, wherein the target substance detection device has a function of vibrating or heating the labeling substance by an external field to change the alignment state of the liquid crystal material around the labeling substance. The target substance detection device according to claim 6,
(1) a memory for storing the alignment state of the liquid crystal material as image information;
(2) a function for calculating the alignment state with or without an external field;
A target substance detection apparatus having   The target substance detection apparatus according to claim 5, wherein the labeling substance is a magnetic fine particle.   9. The target substance detection apparatus according to claim 8, which has a function of accelerating the reaction by operating the magnetic fine particles with an external magnetic field at least during the reaction between the target substance and the secondary antibody.   The target substance detection apparatus according to claim 5, wherein the labeling substance is a charged fine particle.   The target substance detection apparatus according to claim 5, wherein a physical absorption coefficient of the labeling substance is higher than a physical absorption coefficient of a portion where no labeling substance exists. The labeling substance is supported on the substrate by the reaction between the antibody (primary antibody) supported on the substrate, the target substance, and the antibody modified with the labeling substance (secondary antibody),
Target substance detection that detects the presence and number of the target substance in the specimen by detecting the presence and number of the labeling substance by optically detecting the alignment state of the liquid crystal disposed around the labeling substance Method.   The target substance detection method according to claim 12, wherein the labeling substance is detected by vibrating or heating the labeling substance according to an external field and changing an alignment state of a liquid crystal material around the labeling substance. The target substance detection method according to claim 12 or 13,
(1) measuring the alignment state of the liquid crystal when there is no external field;
(2) measuring the alignment state of the liquid crystal when an external field is present;
(3) a step of identifying the presence or absence of the labeling substance by comparing (1) and (2) and calculating the number,
A target substance detection method comprising:   The target substance detection method according to claim 12, wherein the labeling substance is a magnetic fine particle.   The target substance detection method according to claim 15, which has a function of accelerating the reaction by operating the magnetic fine particles with an external magnetic field at least during the reaction between the target substance and the secondary antibody.   The target substance detection method according to claim 12, wherein the labeling substance is a charged fine particle.   The target substance detection method according to claim 12, wherein a physical absorption coefficient of the labeling substance is higher than a physical absorption coefficient of a site where no labeling substance exists.

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