Method for detecting a target substance in a sample

Abstract

To provide a new measurement system that makes it possible to perform high-sensitivity measurement without requiring a dedicated facility, environment, knowledge and technology.SOLUTION: Provided is a method for detecting a detection substance in a sample, the method including a step for measuring, with an image sensor, a change of fluorescence intensity of the conjugate of a detection substance locally concentrated in the sample by electrophoresis or dielectriophorsis and a detection substance recognition material, the detection substance recognition material being labeled with a fluorescent substance that emits fluorescence in a specific wavelength and specifically recognizing and binding to the detection substance, the sample being present in a detection cell with an electrode provided on the bottom inside of the detection cell. Said method includes: (1) providing a transparent insulating film on the image sensor and measuring fluorescence; (2) providing the image sensor so as to be in contact with the bottom outside of the detection cell and measuring fluorescence; (3) providing a slit having anisotropy similar to the electrode on the image sensor and measuring fluorescence, with the electrode having anisotropy in a direction X and a direction Y; or (4) irradiating excitation light from the bottom of the detection cell, providing the image sensor on a side surface of the detection cell, and measuring fluorescence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting an analyte in a sample, and more particularly to a method for detecting an analyte in a sample, which is highly sensitive and capable of being detected at high speed. [Background technology]

[0002] Biological substances such as enzymes, nucleic acids, and antibodies have a common characteristic of binding with target substances with high specificity. Biosensing technology, which applies the properties of biological substances, has been evolving at an accelerating pace, as disclosed in Non-Patent Documents 1 and 2, and is now so widespread that it is familiar to everyone. As disclosed in Non-Patent Document 1, various measurement methods using antibodies by immunochromatography have been developed. Immunochromatography does not require any knowledge, technique, equipment, or environment to be used, so for example, pregnancy testers using anti-human chorionic gonadotropin antibodies are widely used in ordinary households. Similarly, immunochromatography using antibodies that bind to influenza A and B viruses has greatly contributed to rapid influenza diagnosis in clinical settings.

[0003] Thus, although the immunochromatography method is easy to configure a system for easy testing, there is a technical barrier to improving the sensitivity. Therefore, as disclosed in Non-Patent Document 3, for example, attempts have been made to improve the sensitivity by replacing the method of labeling the antibody in the mobile phase in the immunochromatography method with a fluorescent substance or an enzyme that induces luminescence. Furthermore, as disclosed in Non-Patent Document 4, if the sensitivity of influenza viruses can be improved by 100 to 1000 times, it will be possible to test saliva instead of nasal secretions, which is expected to reduce the burden on medical professionals and patients.

[0004] As disclosed in Non-Patent Document 5, there have been many attempts in recent years to artificially create combinations of compounds called aptamers, etc., that can specifically bind to specific molecules as targets.

[0005] Non-Patent Document 6 discloses an example in which bacteria in water are electrically concentrated and the concentration of the bacteria is measured from the change in impedance using the electrode system. Furthermore, Non-Patent Document 7 discloses that bacteria are concentrated near an electrode, then an antibody specific to the bacteria is added to cause agglutination, and the bacteria are then washed to determine whether or not they have agglutinated due to the antibody.

[0006] Here, Patent Document 1 discloses a microbial count / concentration measuring device for measuring the number of microorganisms in a solution. Specifically, the device targets Legionella bacteria, fluorescently labels antibodies that bind to the bacteria, and collects the Legionella bacteria near an electrode by positive dielectrophoresis. The change in the fluorescence in the gap between the electrodes is observed using an optical fiber that is about the same size as the gap between the electrodes. Patent Document 2 also discloses a method for separating a complex substance from molecules other than a specific molecule contained in a sample. Specifically, AFP is used as a detection target substance, an anti-AFP antibody is bound to latex particles in advance, and an antigen-antibody reaction is carried out with a fluorescently labeled Fab of antibody WA1 that binds to a different epitope of AFP. Dielectrophoresis is then performed, and the state of dielectrophoresis with and without AFP is evaluated using the sandwich-reacted fluorescently labeled Fab. That is, in the technology disclosed in Patent Document 2, dielectrophoresis is performed to separate the fluorescently labeled Fab bound to latex particles via AFP from the fluorescently labeled Fab that exists in a free state, and therefore the presence or absence of an antigen is distinguished by the presence or absence of dielectrophoresis. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2002-174636 A [Patent Document 2] JP 2001-165906 A [Non-patent literature]

[0008] [Non-Patent Document 1] Nishijima, SCANS NEWS 2005 II [Non-Patent Document 2] C. Sebastian et al, Indo American Journal of Pharmaceutical Research.2016:6(07). [Non-Patent Document 3] CK Lee et al, Journal of Clinical Virology 55 (2012) 239- 243 [Non-Patent Document 4] A. Sueki et al, Clinica Chimica Acta 453, 71-74(2016) [Non-Patent Document 5] Taiichi Sakamoto, Journal of the Crystallographic Society of Japan 60, 129-134(2018) [Non-Patent Document 6] J. Suehiro et al 1999 J. Phys. D: Appl. Phys. 32 2814 [Non-Patent Document 7] J. Suehiro et al. J. Electrostatics 58 229(2003) Summary of the Invention [Problem to be solved by the invention]

[0009] However, the method described in Non-Patent Document 6 is unable to identify bacteria or distinguish between bacteria and airborne particles. In addition, the method described in Non-Patent Document 7 makes it possible to identify bacteria by the specificity of antibodies, but the reaction cell must be large enough to ensure a sufficient flow rate, and the device must be large because it needs to switch between three types of liquid.

[0010] As mentioned above, concentrating the target substance in the sample while maintaining the ease of use typified by immunochromatography requires complex and large-sized equipment. Also, the biggest problem with immunochromatography is the need for so-called B / F separation, which involves washing before and after the antigen-antibody reaction. Therefore, an object of the present invention is to provide a new measurement system that enables highly sensitive measurement to be performed without requiring dedicated equipment, environment, knowledge, and technology. [Means for solving the problem]

[0011] Through extensive research, the inventors have concluded that one of the most rational methods for increasing the sensitivity of the entire measurement system is not only to increase the sensitivity of the detection part, but also to concentrate the target substance in the sample. Furthermore, while based on a homogeneous detection system that does not require B / F separation, they have succeeded in incorporating a technology for electrically concentrating the target substance, and in the present invention, when measuring the change in the fluorescence intensity of the fluorescence caused by the binding of the target substance with the target substance recognition material using an image sensor, they have reached the point where they can provide a measurement system different from conventional methods by using a specified method, thereby creating new value that combines sensitivity and simplicity.

[0012] The present invention is as follows. [1] A method for detecting a target substance in a sample, comprising: measuring, by an image sensor, a change in the fluorescence intensity of a complex between a detection target substance and a detection target substance recognition material, the complex being locally concentrated in a sample by electrophoresis or dielectrophoresis; The detection target substance recognition material is labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and specifically recognizes and binds to the detection target substance, The sample is present in a detection cell having electrodes disposed inside the bottom of the detection cell; (1) Place a transparent insulating film on the image sensor to measure the fluorescence, or (2) Fluorescence is measured by placing an image sensor on the outside of the bottom of the detection cell, or (3) The electrodes have anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrodes is provided on the image sensor to measure the fluorescence, or (4) A method in which excitation light is irradiated from the bottom of a detection cell and fluorescence is measured by providing an image sensor on the side of the detection cell. The method for detecting a target substance in a sample is as follows: a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; Optionally, a step of binding a substance to be detected and a material for recognizing the substance to be detected in a sample; measuring a change in fluorescence intensity of a complex between the detection target substance and the detection target substance recognition material by an image sensor; confirming the presence of the target substance in the sample based on the measured fluorescence intensity; The method may include: [2] The method according to [1], in which the substance to be detected and the detection substance recognition material are bound to each other by mixing the substance to be detected in a sample with a detection substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength and a detection substance recognition material labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or with a quenching substance that absorbs fluorescence of the specific wavelength. [2-1] A method for detecting a target substance in a sample, comprising: a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; Optionally, a step of binding a substance to be detected and a material for recognizing the substance to be detected in a sample; measuring the fluorescence intensity of a complex between the target substance and a target substance recognition material; confirming the presence of the target substance in the sample based on the measured fluorescence intensity; Including, In order to measure the fluorescence intensity, a substance to be detected in a sample is mixed with a substance to be detected recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a substance to be detected recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or with a quenching substance that absorbs fluorescence at the specific wavelength, thereby binding the substance to be detected and the substance to be detected recognition material; A method in which the detection target substance recognition material is labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and specifically recognizes and binds to the detection target substance. [3] The method according to [1] or [2], in which the detectable substance in the sample is bound to the detectable substance recognition material by mixing the detectable substance in the sample with a detectable substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and the detectable substance labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or with a quenching substance that absorbs fluorescence at the specific wavelength, or a substance that is not the detectable substance but is recognized at the same site on the detectable substance recognition material as the detectable substance. [3-1] A method for detecting a target substance in a sample, comprising: a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; Optionally, a step of binding a substance to be detected and a material for recognizing the substance to be detected in a sample; measuring the fluorescence intensity of a complex between the target substance and a target substance recognition material; confirming the presence of the target substance in the sample based on the measured fluorescence intensity; Including, In order to measure the fluorescence intensity, the target substance in the sample is bound to the target substance recognition material by mixing the target substance in the sample, the target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and the target substance that is labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength, or a substance that is not the target substance but is recognized at the same site in the target substance recognition material as the target substance; A method in which the detection target substance recognition material is labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and specifically recognizes and binds to the detection target substance. [4] The method according to any one of [1] to [3], in which a substance to be detected that is labeled with a fluorescent substance that emits a specific wavelength in a sample and a substance to be detected that is labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or with a quenching substance that absorbs fluorescence of the specific wavelength are simultaneously bound to the substance to be detected. Regarding [2] or [3], [2] may be [2-1] and [3] may be [3-1]. [5] The method according to any of [1] to [4], wherein a fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that is excited with wavelength 1 and emits wavelength 2, and a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength is a fluorescent substance that absorbs wavelength 2 and emits wavelength 3, and the fluorescence intensity of wavelength 3 is measured by exciting with wavelength 1. Here, the measurement of the fluorescence intensity at wavelength 3 may be the measurement of wavelength 3 alone, or both wavelengths 2 and 3. [6] The method according to any of [1] to [5], wherein a fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that is excited at wavelength 1 and emits wavelength 2, a quenching substance that absorbs the fluorescence at the specific wavelength is a quenching substance that absorbs wavelength 2 but does not emit fluorescence at least in a measurement wavelength range, and the fluorescence intensity of wavelength 2 is measured by exciting at wavelength 1. [7] The method according to any of [1] to [6], wherein any one selected from the group consisting of a target substance recognition material, a target substance, and a substance that is not a target substance and is recognized at the same site on the target substance recognition material as the target substance is physically or chemically bound to a carrier particle. [8] a detection target substance recognition material labeled with a fluorescent substance that emits a specific wavelength in a sample, and a detection target substance recognition material labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence of the specific wavelength, are simultaneously bound to the detection target substance; The method according to any of [1] to [7], further comprising a analyte recognition material that is physically or chemically bound to a carrier particle, wherein the analyte and the analyte recognition material form a bond in the sample. [9] The method according to any one of [1] to [8], wherein the carrier particles are metal microparticles, metal oxide microparticles, non-metallic microparticles, metal-coated resin microparticles, or non-infectious spherical biological microparticles.

[10] The method according to any one of [1] to [9], which comprises locally concentrating the substance by positive dielectrophoresis.

[11] The method according to any one of [1] to

[10] , wherein the analyte recognition material is an antibody or an antibody fragment.

[12] The method according to any one of [1] to

[11] , wherein the substance to be detected is a bacterium, a virus, a nucleic acid, a protein or a peptide.

[13] The method according to any one of [1] to

[12] , wherein the substance to be detected is an influenza virus-derived substance.

[14] The method according to any one of [1] to

[13] , wherein at least one of the fluorescent substances is a quantum dot.

[15] The method according to any one of [1] to

[14] , wherein the analyte is a nucleic acid, and the analyte recognition material is a nucleic acid complementary to at least a portion of the nucleic acid of the analyte.

[16] The method according to any one of [1] to

[15] , wherein the substance to be detected is single-stranded RNA. Effect of the Invention

[0013] The present invention makes it possible to perform highly sensitive measurements without requiring dedicated equipment, environment, knowledge, and technology. In particular, the present invention makes it possible to provide a measurement system different from conventional methods by measuring, with an image sensor, the change in the fluorescence intensity of the fluorescence caused by the binding of the target substance with the target substance recognition material. [Brief description of the drawings]

[0014] [Figure 1] The fluorescence spectrum of Ab1Qd565 is shown. [Diagram 2] The absorption spectrum of NPQSY9 is shown. [Diagram 3] 1 shows the change in the fluorescence spectrum of Ab1Qd565 upon addition of NPQSY9. [Figure 4] A reference diagram of a detection cell is shown. [Diagram 5] A reference diagram of a detection device is shown. [Figure 6] The fluorescence spectrum of Ab1Qd655 is shown. [Figure 7] 1 shows the absorption spectrum of BSAQSY21. [Figure 8] The graph shows the time course of the fluorescence intensity of Ab1Qd655 upon addition of BSAQSY21. [Figure 9] The figure shows the inhibition of quenching by the addition of BSA. This is the result of the enhancement of the fluorescence of Ab1Qd655 that was quenched by BSAQSY21. [Figure 10] A comparison of the fluorescence spectra (excitation 370 nm) at each stage is shown. [Figure 11] FIG. 1 shows a block diagram of a dielectrophoretic cell. [Figure 12] A configuration diagram of the measurement unit is shown. [Figure 13] The absorption spectrum (dashed line) and fluorescence spectrum (solid line, 390 nm excitation) of Ab2AT390 are shown. [Figure 14] The absorption spectrum (dashed line) and fluorescence spectrum (solid line, 470 nm excitation) of Ab3DY485 are shown. [Figure 15] FIG. 2 shows a configuration diagram of a detection cell. [Figure 16] The diagram of the test container is shown. By lifting the swab 164 in the direction of the arrow, saliva is squeezed out and mixed into the reaction liquid 162. [Figure 17] 1 shows a configuration diagram of an embodiment in which a transparent insulating film is provided on an image sensor to measure fluorescence. [Figure 18]FIG. 1 shows a cross-sectional view of a configuration of an embodiment in which a transparent insulating film is provided on an image sensor to measure fluorescence. [Figure 19] FIG. 13 shows a configuration diagram of an embodiment in which an image sensor is provided so as to be in contact with the outside of the bottom of a detection cell to measure fluorescence. [Figure 20] The electrodes have anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrodes is provided on the image sensor to measure fluorescence. [Figure 21] The electrodes have anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrodes is provided on the image sensor to measure the fluorescence. This embodiment shows one aspect of the determination method. [Figure 22] FIG. 1 shows a configuration diagram of an embodiment in which excitation light is irradiated from the bottom of a detection cell and an image sensor is provided on the side of the detection cell to measure fluorescence. [Figure 23] The absorption and fluorescence spectra of IgG1Qd585 are shown. [Figure 24] The absorption and fluorescence spectra of IgG2AF594 are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, the embodiment of the present invention will be described in detail. Note that the present invention is not limited to the following embodiment, and various modifications can be made within the scope of the present invention.

[0016] The detection method of the present invention comprises: A method for detecting a target substance in a sample, comprising: The method includes a step of measuring a change in the fluorescence intensity of a bond between a detection target substance and a detection target substance recognition material that has been locally concentrated in a sample by electrophoresis or dielectrophoresis. In the detection method of the present invention, the substance to be detected is labeled with a fluorescent substance that emits fluorescence at a specific wavelength and specifically recognizes and binds to the substance to be detected. The sample is placed in a detection cell in which an electrode is provided inside the bottom of the detection cell, and the change in fluorescence intensity is measured by an image sensor. In the detection method of the present invention, the measurement is carried out by any one of the following methods (1) to (4), thereby measuring the change in fluorescence intensity and detecting the analyte in the sample. (1) A transparent insulating film is placed on the image sensor to measure fluorescence; (2) An image sensor is placed in contact with the outside of the bottom of the detection cell to measure the fluorescence; (3) The electrode has anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrode is provided on the image sensor to measure the fluorescence; (4) Excitation light is irradiated from the bottom of the detection cell, and fluorescence is measured by an image sensor provided on the side of the detection cell.

[0017] As a method for detecting a target substance in a sample, a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; Optionally, a step of binding a substance to be detected and a material for recognizing the substance to be detected in a sample; measuring a change in fluorescence intensity of a complex between the detection target substance and the detection target substance recognition material by an image sensor; confirming the presence of the target substance in the sample based on the measured fluorescence intensity; The method may include: In this case, the method for detecting the target substance in the sample is as follows: a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; Optionally, a step of binding a substance to be detected and a material for recognizing the substance to be detected in a sample; measuring a change in fluorescence intensity of a complex between the detection target substance and the detection target substance recognition material by an image sensor; confirming the presence of the target substance in the sample based on the measured fluorescence intensity; In the step of measuring the change in fluorescence intensity with an image sensor, (1) A transparent insulating film is placed on the image sensor to measure fluorescence; (2) An image sensor is placed in contact with the outside of the bottom of the detection cell to measure the fluorescence; (3) The electrode has anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrode is provided on the image sensor to measure the fluorescence; (4) Excitation light is irradiated from the bottom of the detection cell, and fluorescence is measured by an image sensor provided on the side of the detection cell.

[0018] The detection method of the present invention can detect a target substance by measuring changes in fluorescence spectrum, etc., using a fluorescent substance bound to a target substance recognition material. In addition, by detecting changes in fluorescence spectrum, etc., a method can be provided in which electrophoresis or dielectrophoresis can be used for a purpose other than the measurement of changes in fluorescence spectrum, etc., that is, independently for direct or indirect concentration of a target substance. Thus, the present invention provides a detection method that can detect with higher sensitivity and speed than conventional methods using immunochromatography. In addition, in the detection method of the present invention, the detection substance may be detected by concentrating the conjugate of the detection substance and the detection substance recognition material that is specifically bound to the detection substance and labeled with a fluorescent substance, and measuring the change in the fluorescence spectrum, etc., using the fluorescent substance bound to the detection substance recognition material. In this case, the detection method of the present invention provides a detection method that can detect the detection substance with higher sensitivity and speed than the conventional method using immunochromatography.

[0019] In the present invention, the concentration, dissociation constant, binding curve, etc. of the binding between the target substance and the target substance recognition material were considered, and it was thought that in order to detect the target substance in a sample, it would be a fundamental measure to increase the sensitivity by bringing the concentration of the target substance closer to the inflection point in the binding curve to increase the sensitivity of the system. Therefore, it was thought that concentrating the sample would lead to an increase in sensitivity, and electrophoresis or dielectrophoresis was adopted as a concentration method, and it was thought that dielectrophoresis is preferable. In addition, when considering a case where a virus or the like is used as the target substance, it is possible to use a virus as the target substance and an antibody that binds to the surface antigen of the virus as the target substance recognition material, but since the virus surface is subject to large mutations over time, nucleoprotein may also be used as the target substance. Generally, nucleoprotein can be extracted using a surfactant, but since single-stranded RNA is extracted under the same conditions as nucleoprotein for influenza viruses and the new coronavirus (SARS-CoV-2) that caused the pandemic in 2020, RNA can also be used as the target substance.

[0020] In the detection method of the present invention, a detection substance recognition material that specifically binds to the detection substance is used, and preferably a detection substance recognition material that can specifically bind to the detection substance in water is used. At this time, the detection substance recognition material is in a free form. As described later, the detection substance or the detection substance recognition material that specifically binds to the detection substance may be physically or chemically bound to the carrier particle, but even in this case, the detection substance recognition material is bound to the carrier particle but is not fixed to a substrate or the like and is in a free state. That is, the detection method of the present invention is carried out in a solution, and the detection substance recognition material moves in the solution. That a material for recognizing a detectable substance specifically binds to a detectable substance means that the material for recognizing and binding to a detectable substance has specificity for the detectable substance, and in the present invention, "specificity" means that the material for recognizing a detectable substance binds to a specific detectable substance.

[0021] The sample to be measured in the detection method of the present invention is not particularly limited, but examples thereof include samples derived from living organisms, food, and samples collected from within buildings such as factories, schools, and hospitals. In hospitals, nosocomial infections can be a problem, and by applying the detection method of the present invention, bacteria causing nosocomial infections can be detected with high sensitivity and high speed. The living organism of the biological sample is not particularly limited, but examples thereof include mammals, specifically, humans and livestock, and biological samples such as saliva and blood may be used as the sample. In the present invention, the above-mentioned sample may be used as the detection target as it is, or the sample may be used after being diluted, suspended or dissolved in a solvent such as water or alcohol. Furthermore, in order to detect the presence of a substance to be detected in a sample, it is preferable to use the sample as is for detection, but before detection, the sample may be subjected to ultrasonic treatment or the like to pulverize the sample and elute the substance to be detected.

[0022] The substance to be detected is not particularly limited, but examples thereof include bacteria, viruses, nucleic acids, proteins, and peptides. By confirming the presence of bacteria or viruses, it is possible to confirm the presence of harmful bacteria or viruses, and by confirming the presence of biological substances such as nucleic acids, proteins, and peptides, it is possible to detect the presence of harmful substances or useful substances in a sample. When a substance present in a living body is to be detected, it is preferable to subject the sample to ultrasonic treatment or the like in advance to dissolve or precipitate the substance to be detected in the sample. Bacteria to be detected are not particularly limited, but examples include Escherichia coli (preferably pathogenic Escherichia coli), Streptococcus pneumoniae, Trichophyton, Chlamydia, Staphylococcus aureus, Salmonella, Campylobacter, and Mycobacterium tuberculosis. Viruses to be detected include, but are not limited to, influenza virus, norovirus, coronavirus (including the new coronavirus), rotavirus, hepatitis virus, varicella-zoster virus, human immunodeficiency virus, and human papillomavirus. As bacteria and viruses, those having pathogenicity are suitable as detection target substances. Examples of those having pathogenicity include those causing infectious diseases and food poisoning, and may be those useful for testing from the viewpoint of hygiene management in the food manufacturing process. For example, non-pathogenic Escherichia coli, which is required not to be contaminated in food, may be mentioned. The nucleic acids, proteins and peptides to be detected include those derived from bacteria or viruses, and those other than those derived from bacteria or viruses include poisons such as snake venom, abnormal prions and various tumor markers. They may also be substances that exist in a living body and are the subject of tests such as blood tests carried out to measure the state of the living body.

[0023] Influenza virus will be described as an example of the substance to be detected below, but the substance may be derived from influenza virus, such as influenza virus nuclear protein or influenza virus particles, and examples of materials that can be used to recognize the substance to be detected include antibodies that bind to surface antigens of influenza virus and antibodies that bind to nuclear protein extracted from influenza virus.

[0024] In the present invention, the analyte recognition material that recognizes and binds to the analyte in the sample is preferably an antibody or an antibody fragment, or may be a nucleic acid fragment (aptamer) that recognizes a virus such as influenza virus. The antibody or antibody fragment that can be used may be an antibody or antibody fragment that is known to recognize the analyte whose presence is to be detected in the sample. The antibody or antibody fragment serving as the analyte recognition material can be produced by a conventionally known method. The antibody or antibody fragment is preferably a molecule to which a sugar chain is bound, and the antibody fragment is not particularly limited as long as it can be labeled with a fluorescent substance or a quenching substance and can recognize the substance to be detected. Examples of the antibody fragment include, but are not limited to, Fv, Fab, and F(ab')2. When the substance to be detected is a nucleic acid, the substance to be detected recognition material may be a nucleic acid having a base sequence complementary to a part of the base sequence of the nucleic acid of the substance to be detected. The nucleic acid can be produced by a conventionally known method.

[0025] In the present invention, it is preferable to use an antibody or an antibody fragment as the analyte recognition material, and it is preferable to use the entire antibody molecule including Fc as it is, and it is preferable that the antibody contains a sugar chain. It is possible to maintain affinity by using an antibody having a glycan and fluorescently labeling it via the glycan in the Fc region. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. Monoclonal antibodies can be produced by conventional methods known in the art. In the present invention, antibodies produced by known methods may be used, or commercially available antibodies may also be used. In addition, the origin of the antibody is not particularly limited, and thus, examples of the antibody include mammals, and may be laboratory animals, specifically, antibodies derived from mice, rats, rabbits, camels, etc. The antibody may be a human antibody, and chimeric antibodies, humanized antibodies, etc. may also be used. Antibody classes include IgG, IgA, IgM, IgD, and IgE, and IgG or IgM may be preferably used, but is not particularly limited thereto. For example, even when IgG is used, the subclasses, such as IgG1 to IgG4, are not particularly limited. The antibody fragment may be a fragment of these antibodies as explained above.

[0026] The present invention has an advantage in that the target substance can be detected without B / F separation. In particular, when an antibody or an antibody fragment is used as the target substance recognition material, the present invention has an advantage in that the target substance can be detected without B / F separation. Here, the B / F separation in the present invention refers to the separation of the target substance (F) present in water in a free state from the target substance (B) bound to the target substance recognition material. As a most common example, the ELISA method includes the steps of 1) immobilizing an antibody on the surface of a container and washing, 2) adding a blocking agent to suppress non-specific adsorption and washing, 3) adding a sample and discarding it after a certain period of time and washing, 4) adding an antibody (enzyme label) that binds to the antigen and washing, and 5) adding some kind of substrate to detect the enzyme reaction, but each step requires a washing operation, which makes each step complicated, complicates the equipment, and requires the person performing the test to be familiar with the technique to a certain level.

[0027] In the present invention, the object of measurement is a complex formed by binding a substance to be detected with a material for recognizing the substance to be detected. Since the binding between the target substance and the target substance recognition material occurs in a sample containing the target substance, the sample is preferably a liquid sample. The binding between the analyte and the analyte recognition material is achieved by mixing a sample to be detected in order to confirm the presence of the analyte with the analyte recognition material. When a sample containing no target substance is used, no binding occurs between the target substance and the target substance recognition material. However, in the detection method of the present invention, a step is carried out to confirm the presence of the target substance in the sample based on a change in the measured fluorescence intensity, thereby confirming that no binding has occurred between the target substance and the target substance recognition material. Therefore, in the detection method of the present invention, in the step of binding the target substance to the target substance recognition material in a sample, binding occurs in a sample in which the target substance is present, but does not occur in a sample in which the target substance is not present. That is, the binding step in one embodiment of the detection method of the present invention is understood to be essentially a step of mixing the sample with the detection target substance recognition material. The bond between the target substance and the target substance recognition material may be a bond that results in a reversible equilibrium reaction, and may be a bond in a manner similar to the bond between a ligand and a receptor in the body or the bond between an antigen and an antibody.

[0028] The binding step is not particularly limited, but examples thereof include the following methods. (1) Mixing a target substance in a sample with a target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a target substance recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or with a quenching substance that absorbs fluorescence at the specific wavelength, thereby binding the target substance in the sample with the target substance recognition material; (2) Mixing a target substance in a sample with a target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a target substance labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength, or a substance that is not a target substance but is recognized at the same site as the target substance in the target substance recognition material, thereby binding the target substance in the sample with the target substance recognition material; and (3) The substance to be detected in the sample is mixed with a material for recognizing the substance to be detected that is labeled with a fluorescent substance that emits fluorescence at a specific wavelength, thereby binding the substance to the material for recognizing the substance to be detected in the sample.

[0029] When the binding step is the above (1), it is preferable that the recognition sites for the analyte in the analyte recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength and the analyte recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength are different. By having different recognition sites, the two types of analyte recognition materials can bind to the analyte simultaneously, forming a so-called sandwich structure. In this case, the order of mixing the detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, the detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength, and the detection target substance in the sample is not particularly limited, but the sample to be confirmed whether the detection target substance is contained is added to the space where the detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength and the detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength are present, and vice versa is also possible, but it is preferable to mix the detection target substance recognition material and the detection target substance to bind them. The detection target substance in the sample and one of the detection target substance recognition materials may be mixed and bound first, and then the other detection target substance recognition material may be mixed. In this case, the other detection target substance recognition material may be mixed after the conjugate is concentrated. In addition, the detection target substance or the detection target substance recognition material may be concentrated, and then the conjugate may be formed, or the concentration and the formation of the conjugate may be performed at the same time. When the binding step is the above-mentioned (1), the substance to be detected is bound to a substance to be detected recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a substance to be detected recognition material labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence of the specific wavelength. Thus, typically, the substance to be detected is sandwiched between two different substance to be detected recognition materials.

[0030] When the binding step is the above (2), the detection substance in the detection substance labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength is selected as the detection substance to be confirmed to be present in the sample. In addition, a substance that is not a detection substance and is recognized at the same site as the detection substance in the detection substance recognition material, which is labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength, may be used, and the non-detection substance is labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength, and is recognized at the same site as the detection substance in the detection substance recognition material, so it behaves in the same way as the detection substance. Hereinafter, in this specification, the explanation of the detection substance labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength also applies to a non-detection substance that is recognized at the same site as the detection substance in the detection substance recognition material. A substance other than the analyte substance that is recognized at the same site as the analyte substance in the analyte substance recognition material may be understood to be a pseudo analyte substance. In this case, one of the target substances and the target substance recognition material may be mixed and bound first, and then the other target substance may be mixed. In this case, the other target substance may be mixed after concentrating the conjugate. In addition, the target substance or the target substance recognition material may be concentrated, and then the conjugate may be formed, or the concentration and the formation of the conjugate may be performed at the same time. When the binding step is the above-mentioned (2), the analyte in the sample and the analyte labeled with a fluorescent substance or a quencher competitively bind to the analyte recognition material. It is preferable to combine a detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength with a detection target substance labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength, and then mix the detection target substance in the sample. It is also possible to combine a detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength with a detection target substance in the sample, and then mix the detection target substance labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs the fluorescence of the specific wavelength.

[0031] The fluorescent substance or quenching substance must be bound at a site that does not inhibit the binding between the target substance and the target substance recognition material, or does not inhibit the binding as much as possible. In addition, when two types of fluorescent substances are used, or when a fluorescent substance and a quenching substance are used, it is preferable to select the binding site with the target substance or the target substance recognition material so that they are present in the vicinity of each other in the bound body, specifically, so that they can transfer optical excitation energy.

[0032] In the present invention, the fluorescent substance that labels the detection target substance or the detection target substance recognition material may be appropriately selected. As the fluorescent substance, a substance that satisfies some or all of the following requirements is preferably used. The excitation wavelength is long, preferably 350 nm or longer, more preferably 400 nm or longer. The fluorescence wavelength is short, preferably 1500 nm or less. - High extinction coefficient, preferably a molar extinction coefficient of 10,000 or more. High quantum yield, preferably 0.1 or higher. When multiple fluorescent substances are used, the wavelength that excites one fluorescent substance does not excite the other fluorescent substance, and the fluorescence emitted by one fluorescent substance does not overlap when the fluorescence of the other fluorescent substance is measured. In this case, it is preferable that the peaks of the fluorescence spectrum of one fluorescent substance and the excitation spectrum of the other fluorescent substance are sufficiently close to each other so that the fluorescence of one fluorescent substance can efficiently excite the other fluorescent substance. Furthermore, it is preferable that the fluorescence spectrum of one fluorescent substance is sufficiently narrow and the excitation spectrum of the other fluorescent substance is sufficiently wide, and it is even more preferable that the difference between the positions of the peak of the excitation spectrum and the peak of the fluorescence spectrum (Stokes shift) is sufficiently large. In addition, it is preferable that the fluorescent material satisfy some or all of the following requirements. -Do not inhibit the water solubility of the substance to be labeled. After labeling, the specific binding between the target substance and the target substance recognition material must not be inhibited. The label does not aggregate unlabeled material and / or enhance light scattering. In the present invention, the quenching substance that labels the detection target substance or the detection target substance recognition material may be appropriately selected. In addition to the requirements described above for fluorescent substances, the quenching substance must satisfy the following requirements. -Energy transfer does not produce a second fluorescence, or it emits light at a wavelength farther away than the measurement range.

[0033] In the detection method of the present invention, it is preferable that at least one of the fluorescent substances is a quantum dot. As a fluorescent label, semiconductor fluorescent particles with a particle size of several nm, commonly called quantum dots, have an extinction coefficient and quantum yield several tens of times that of organic dyes, and because they have strong fluorescence and a narrow half-width, it is easy to obtain high performance as a system. Due to the narrow half-width, during energy transfer called the FRET phenomenon, the excitation energy can be received by the partner dye (acceptor) without leaking within the emission wavelength range. Another major advantage is that by binding quantum dots to the glycans of antibodies, it is possible to completely avoid the reduction in affinity caused by chemical manipulation of the Fv region, which sometimes occurs. In the present invention, it is not necessary for the energy transfer to be FRET-like in the strict sense, but it is sufficient if energy transfer occurs that results in a change that can be distinguished as significant.

[0034] In the present invention, the detectable substance, the detectable substance and a substance other than the detectable substance that is recognized at the same site as the detectable substance in the detectable substance recognition material, or the detectable substance recognition material may be physically or chemically bound to the carrier particle. The decision as to whether or not to use a detection target substance recognition material bound to carrier particles may be made depending on the properties of the detection target substance. In one embodiment of the detection method of the present invention, the target substance, the target substance recognition material, or the combination of the target substance and the target substance recognition material is locally concentrated by electrophoresis or dielectrophoresis.Therefore, the formation of the combination of the target substance and the target substance recognition material may be a step before or after the concentration step, or the concentration and the combination may be performed simultaneously.For example, if electrophoresis or dielectrophoresis is performed when the target substance and the target substance recognition material are mixed, it can be said that the concentration step and the combination step are performed simultaneously. Although not particularly limited, for example, as long as it is a combination of a target substance and a target substance recognition material that can be locally concentrated by electrophoresis or dielectrophoresis, a target substance recognition material that is not bound to carrier particles can be used. Note that even if it is a combination of a target substance and a target substance recognition material that can be locally concentrated by electrophoresis or dielectrophoresis, the target substance recognition material may be bound to carrier particles to perform electrophoresis or dielectrophoresis more efficiently. Therefore, before performing the detection method of the present invention, it may be possible to confirm whether the target substance itself, the target substance recognition material itself, or a combination of the target substance and the target substance recognition material can be locally concentrated by electrophoresis or dielectrophoresis, and then decide whether to physically or chemically bind carrier particles to the target substance recognition material. When the target substance itself, the target substance recognition material itself, or the combination of the target substance and the target substance recognition material cannot be locally concentrated by electrophoresis or dielectrophoresis, carrier particles may be physically or chemically bound to either the target substance itself, the target substance recognition material itself, or the combination of the target substance and the target substance recognition material. The selection of carrier particles may be determined based on the efficiency of local concentration by electrophoresis or dielectrophoresis. The method for physically or chemically binding the analyte recognition material to the carrier particles is not particularly limited, and the binding may be carried out by a conventionally known method or according to the manual attached to the carrier particles.

[0035] In the present invention, the carrier particles may contain a material for recognizing a target substance that is physically or chemically bound to the carrier particles. The target substance recognition material may or may not be labeled with a fluorescent substance or a quenching substance, but may be an unlabeled target substance recognition material. In this case, for example, a detection target substance recognition material labeled with a fluorescent substance that emits a specific wavelength in a sample, and a detection target substance recognition material labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence of the specific wavelength, are simultaneously bound to the detection target substance; The method further comprises the step of physically or chemically binding a detection target substance recognition material to the carrier particles, and the detection target substance and the detection target substance recognition material form a conjugate in the sample. or, The following may be used in a sample: a substance to be detected in the detection target recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength; a substance to be detected labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength; or a substance that is not the substance to be detected and is recognized at the same site on the detection target recognition material as the substance to be detected; and a detection target recognition material that is physically or chemically bound to a carrier particle. Instead of using a target substance recognition material that is not labeled but is physically or chemically bound to a carrier particle, a labeled target substance recognition material may also be physically or chemically bound to a carrier particle.

[0036] The carrier particles used in the detection method of the present invention are not particularly limited as long as they can locally concentrate the complex with the target substance when physically or chemically bound to the target substance recognition material, but any conventionally known carrier particles may be used. The carrier particles used in the examples are an example of the carrier particles that can be suitably used in the present invention. Specific examples of carrier particles include, but are not limited to, metal microparticles, metal oxide microparticles, non-metallic microparticles, metal-coated resin microparticles, and non-infectious spherical biological microparticles. Specific examples of metal particles include gold, silver, platinum, titanium, palladium, iron, and aluminum. Specific examples of metal oxide fine particles include titanium dioxide, aluminum oxide, magnesium oxide, ITO (indium tin oxide), ATO (antimony tin oxide), and the like. Specific examples of non-metallic fine particles include magnetic particles, and fine particles made of graphite, polystyrene, and conductive resin. The non-infectious spherical biological particles may be bacteria, oomycetes, myxomycetes or fungi belonging to the fungus family, specific examples of which include lactococci and yeast. Fine particles whose surfaces are plated with a conductive metal, such as those sold under the trade names Micropearl and Micropearl Au by Sekisui Chemical Co., Ltd., may also be used.

[0037] The particle size of the carrier particles is not particularly limited and is appropriately selected depending on the carrier particles used. The particle shape is not particularly limited, but for example, it is preferable that gold colloidal particles are in the range of 30 to 100 nm, preferably 40 to 60 nm, polystyrene particles are in the range of 100 nm to 3 μm, and lactobacillus or yeast are in the range of 1 to 5 μm. As the metal-coated resin fine particles, fine particles having a particle size of 5 μm or less can be suitably used.

[0038] In the present invention, the substance to be detected and the material to be detected recognition are bound in a sample to form a complex of the substance to be detected and the material to be detected recognition, and the complex may then be locally concentrated by electrophoresis or dielectrophoresis while measuring the change in fluorescence intensity. Here, the target substance recognition material may be bound to carrier particles. Electrophoresis or dielectrophoresis of the bound substance can be carried out by methods known in the art. In the present invention, dielectrophoresis is preferably used, and it is possible to concentrate the bond between the target substance and the target substance recognition material by performing dielectrophoresis, and detect the desired target substance. Note that, even when the bond is concentrated by electrophoresis, it is possible to detect the desired target substance in the same way as in the case of dielectrophoresis.

[0039] One method of applying an electric field to the binder for electrophoresis or dielectrophoresis is to apply a direct current. Usually, fine particles in water are charged with either a positive or negative surface charge. For example, E. coli is negatively charged. When a direct current voltage is applied to a sample containing suspended E. coli using a gel electrode, the E. coli aggregate as if forming a solid near the gel at the positive electrode. The inventors have confirmed that if the voltage is cut off at this time, this solidified state will loosen, and if the positive and negative polarities are reversed, the bacteria will move in the opposite direction. Measuring E. coli in this state increases the sensitivity of the detection system. Applying a direct current (electrophoresis) can induce electrolysis of water, and by the same principle, the electrode material may be ionized and dissolved, so applying an alternating current (dielectrophoresis) is preferable. When applying an alternating current, electrolysis does not occur even when several volts are applied to a metal electrode, and it is known that particles move between regions of different electric field densities due to a phenomenon called dielectrophoresis. It is known that the particles are related not to their surface charge, but to the dielectric and electrical conductivity of the particles and solvent, and the frequency of the electric field (R. Pethig, BIOMICROFLUIDICS, 4, 022811(2010)). The voltage to be applied is not particularly limited, but may be, for example, 0.1 to 10 V, and may be, for example, 1 to 5 V, 1 to 4 V, or 2 to 4 V. The frequency is not particularly limited, but may be, for example, 100 Hz to 200 MHz, and is preferably on the order of MHz, and may be around 2 MHz.

[0040] When using AC, the governing factors of dielectrophoresis change depending on the dielectric constant and conductivity of the target particles and solvent, and the applied voltage and frequency (in the case of a sine wave). We have explained positive dielectrophoresis, that is, particles gathering at places with high electric field density such as the corners of the electrodes, but depending on the combination of frequency band, dielectric constant, and conductivity, negative dielectrophoresis may occur and particles may move away from the electrodes. In the present invention, either positive or negative dielectrophoresis may be used, but positive dielectrophoresis is preferred.

[0041] In the present invention, the change in fluorescence intensity may be measured while the conjugate is locally concentrated. In this case, it is possible to confirm the presence of the target substance in the sample without performing B / F separation. In the present invention, measuring the change in fluorescence intensity while concentrating means that B / F separation is not performed. If B / F separation is not performed, the fluorescence intensity may be measured after locally concentrating the bound substance. In one embodiment of the present invention, a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; Optionally, a step of binding a substance to be detected and a material for recognizing the substance to be detected in a sample; measuring a change in fluorescence intensity of a complex between the detection target substance and the detection target substance recognition material by an image sensor; However, concentration and fluorescence intensity measurement may be performed simultaneously, concentration and binding may be performed simultaneously, binding and fluorescence intensity measurement may be performed simultaneously, or concentration, binding and fluorescence intensity measurement may be performed simultaneously. In one embodiment of the present invention, in order to increase design freedom, it is preferable to separate concentration and fluorescence intensity measurement, and to perform a step of detecting from a change in fluorescence using a system in which the intensity of fluorescence changes, and a step of concentrating the reaction system by electrophoresis or dielectrophoresis to improve the sensitivity of the entire system, independently of the step of detecting from a change in fluorescence. In addition, as a preferred embodiment, a detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength in a sample and a detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength are mixed together to bind the detection target substance and the detection target substance recognition material; or, a detection target substance in a sample, a detection target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a detection target substance labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength, or a substance that is not a detection target substance but is recognized at the same site in the detection target substance recognition material as the detection target substance, thereby binding the detection target substance in the sample to the detection target substance recognition material; or, a detection target substance recognition material labeled with a fluorescent substance that emits a specific wavelength in a sample, and a detection target substance recognition material labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence of the specific wavelength, are simultaneously bound to the detection target substance; The detection device further comprises a analyte recognition material that is physically or chemically bound to the carrier particles, and the analyte and the analyte recognition material form a bond in the sample. In addition, another preferred embodiment is The target substance recognition material, which is physically or chemically bound to the carrier particle, is locally concentrated by electrophoresis or dielectrophoresis, with or without being bound to the target substance, and optionally the target substance and the target substance recognition material are bound in the sample, and the change in fluorescence intensity of the target substance is measured by any of the methods described above as a preferred embodiment.

[0042] The measurement of the fluorescence intensity can be appropriately set depending on the fluorescent substance. The conditions for measuring the fluorescence intensity can also be set appropriately, and the excitation light generally has a wavelength of 300 to 600 nm, preferably 350 nm or more, and more preferably 400 nm or more. Fluorescence intensity can be measured using a filter and optical sensor (photodiode, phototransistor), or the glowing state can be captured as an image using a smartphone image sensor and compared with the known electrode shape for image recognition, making it possible to extract only the glowing parts, which enhances contrast and enables more accurate detection.

[0043] The step of measuring the change in fluorescence intensity in the present invention is not particularly limited, but examples thereof include the following method. When the binding step is the above (1) or (2) and two kinds of fluorescent substances are used, the fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that is excited with wavelength 1 and emits wavelength 2, and the fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength is a fluorescent substance that absorbs wavelength 2 and emits wavelength 3, and the fluorescence intensity of wavelength 3 or the fluorescence intensities of wavelengths 2 and 3 are measured by exciting with wavelength 1. When the binding step is the above (1) or (2) and a quencher is used, the fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that is excited at wavelength 1 and emits wavelength 2, the quencher that absorbs the fluorescence at the specific wavelength is a quencher that absorbs wavelength 2 but does not emit fluorescence at least in the measurement wavelength range, and the fluorescence intensity of wavelength 2 is measured by exciting at wavelength 1. When the binding step is the above (3), the fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that emits fluorescence at wavelength 2 upon excitation at wavelength 1, and the fluorescence intensity of wavelength 2 is measured upon excitation at wavelength 1. In measuring the change in fluorescence intensity, a step of measuring a change in the fluorescence intensity of a complex between a detection target substance and a detection target substance recognition material by measuring a change in the wavelength of the fluorescence or a change in the magnitude of the fluorescence may be used.

[0044] When the binding step is the above (1) and two kinds of fluorescent substances are used, it is a method for measuring fluorescence intensity utilizing energy transfer caused by the two kinds of fluorescent substances being present in the vicinity of the substance to be detected via a material for recognizing the substance to be detected, and high sensitivity can be achieved by observing the energy transfer between the two kinds of fluorescent substances. Among them, it is preferable to measure the fluorescence emitted by a fluorescent substance that fluoresces at a wavelength different from the specific wavelength that was not observed before mixing.

[0045] When the binding step is the above (1) and a fluorescent substance and a quencher are used, the method is a fluorescence intensity measurement method utilizing the energy transfer caused by the fluorescent substance and the quencher being present in the vicinity of the substance to be detected via a material for recognizing the substance to be detected, and high sensitivity can be achieved by observing the energy transfer between the fluorescent substance and the quencher. Among them, it is preferable to measure the fluorescence emitted by a fluorescent substance that fluoresces at a specific wavelength and is reduced by mixing.

[0046] When the binding step is (1) above and the material is supported on a carrier particle, either the target substance recognition material labeled with a fluorescent substance that fluoresces at a specific wavelength, or the target substance recognition material labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quenching substance may be supported on the carrier particle, but it is preferable that the target substance recognition material labeled with a fluorescent substance that fluoresces at a specific wavelength be supported on the carrier particle.

[0047] When the binding step is the above (2) and two kinds of fluorescent substances are used, it is a method for measuring fluorescence intensity utilizing energy transfer due to the presence of two kinds of fluorescent substances in close proximity, and high sensitivity can be achieved by observing the energy transfer between the two kinds of fluorescent substances. In particular, it is preferable to measure the fluorescence emitted by a fluorescent substance that fluoresces at a wavelength different from the specific wavelength that is reduced by mixing. In this case, it is preferable that the measurement utilizes the phenomenon caused by the release of the detected substance labeled with the fluorescent substance, since the detected substance in the sample is replaced by the detected substance labeled with the fluorescent substance and binds to the detected substance recognition material.

[0048] When the binding step is the above (2) and a fluorescent substance and a quencher are used, the method is a fluorescence intensity measurement method that utilizes the energy transfer caused by the fluorescent substance and the quencher being in close proximity, and high sensitivity can be achieved by observing the energy transfer between the fluorescent substance and the quencher. In particular, it is preferable to measure the fluorescence emitted by a fluorescent substance that fluoresces at a specific wavelength that increases upon mixing. In this case, it is preferable to use a measurement that utilizes the phenomenon caused by the release of the quencher-labeled analyte in the sample, since the analyte in the sample replaces the analyte labeled with the fluorescent substance and binds to the analyte recognition material.

[0049] When the binding step is the above-mentioned (2), a substance to be detected that is labeled with a fluorescent substance that fluoresces at a specific wavelength and a substance to be detected that is labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quencher are used; however, the substance to be detected may be labeled with a fluorescent substance that fluoresces at a specific wavelength, and the substance to be detected may be labeled with a fluorescent substance that fluoresces at a wavelength different from the specific wavelength or a quencher.

[0050] When the binding step is the above (3), the detection target substance recognition material is present throughout the sample, and fluorescence due to the fluorescent substance labeled on the detection target substance recognition material is observed as background. When the binding step is the above (1) or (2), the detection target substance recognition material labeled with a fluorescent substance that fluoresces at a specific wavelength may be supported on carrier particles, but when the binding step is the above (3), it is preferable that the detection target substance itself is concentrated by electrophoresis or dielectrophoresis.

[0051] In any of the above-mentioned cases where the binding step is (1) to (3), in each of the above-mentioned embodiments described as the fluorescence intensity measuring step in the present invention, a detection target substance recognition material that is physically or chemically bound to the carrier particle may be further included, and the detection target substance and the detection target substance recognition material may form a conjugate in the sample. In this case, the detection target substance is locally concentrated through the binding with the detection target substance recognition material by the detection target substance recognition material that is physically or chemically bound to the carrier particle. In addition, the detection target substance recognition material is physically or chemically bound to the carrier particle, but is not labeled with a fluorescent substance or a quenching substance.

[0052] In the present specification, when carrier particles are physically or chemically bound to a target substance or a target substance recognition material, the carrier particles are preferably particles that promote dielectrophoresis.

[0053] Any of the fluorescent substance that fluoresces at a specific wavelength, the fluorescent substance that fluoresces at a wavelength different from the specific wavelength, or the quenching substance used in the present invention may be a carrier particle.

[0054] The detection method of the present invention makes it possible to perform highly sensitive measurements without requiring dedicated equipment, environment, knowledge and / or technology, and therefore makes it possible to test for infectious diseases using samples that were previously impossible, to test in-situ where testing was necessary but not previously possible, and to test for items that, in principle, were impossible to test using genetic testing. More specifically, when detecting influenza viruses, it will become possible to use saliva instead of nasal secretions as a sample, and it will become possible to carry out testing at border points such as customs, and it will become possible to directly test food processing plants and distribution processes that previously required several days of incubation, and obtain results right in front of the customer. It will also become possible to test for pathogenic factors that do not contain genes, such as abnormal prions.

[0055] Examples of detectors for detecting a substance to be detected in a sample for carrying out the detection method of the present invention are given below or in the Examples, but the present invention is not limited thereto. The detection device is equipped with a detection cell having a pair of microelectrodes in a liquid reservoir into which an aqueous sample can be introduced and held, the microelectrodes being electrically connected to an external or internal voltage generator, and a means for concentrating the detectable substance in the sample by electrophoresis or dielectrophoresis by applying a direct or alternating current between the electrodes while in contact with the sample, and a detection means for measuring the fluorescence intensity of the label, which has at least a detectable substance recognition material that is capable of specifically binding to the detectable substance and has been fluorescently labeled in advance, and is capable of measuring the presence of the detectable substance in the sample by qualitatively or quantitatively measuring the state of binding between the detectable substance and the detectable substance recognition material by evaluating the fluorescence intensity, without moving the aqueous sample and performing B / F separation of the bond (B) between the detectable substance and the detectable substance recognition material and the free detectable substance recognition material (F).

[0056] FIG. 4 shows a reference diagram of the detection cell, and FIG. 5 shows a reference diagram of the detection device. The detection device includes a light source 63 and a lens 65 as detection means, and excitation light 64 having a wavelength that excites the fluorescent substance is irradiated from the light source 63 through the lens 65 to the detection cell 61. In addition, the device includes an optical sensor 68 and an optical filter 67 for measuring the fluorescence intensity to confirm the presence of the substance to be detected, and the fluorescence 66 of the detection wavelength or the fluorescence 66 other than the detection wavelength is collected by the optical filter 67, and the fluorescence intensity is measured by the optical sensor 68. It is preferable that the optical filter 67 cuts off the fluorescence of wavelengths other than the detection wavelength. The detection cell includes a means for concentrating the substance to be detected in the sample by electrophoresis or dielectrophoresis, and a microelectrode 42 is printed on the substrate 41. The pair of microelectrodes is shown as electrode 1 47 and electrode 2 48. The liquid reservoir in the detection device is formed by the substrate 41, the spacer 43, and the cover 44, and is shown as the capillary 45. The cover 44 is provided with an air hole 46. In FIG. 4, the direction of introduction of the sample is shown as 4A. As a concentrating means, a terminal 69 is connected to the electrode 1 47 and the electrode 2 48. When performing dielectrophoresis, for example, a high frequency with an optimal voltage and frequency is applied from a function generator (not shown) to the terminal 69.

[0057] The detection device may be provided with a means for forcibly stirring the cell by vibrating it after the sample is introduced into the detection cell. For example, when the substance to be detected is a nucleoprotein of a virus, the means is used to destroy the envelope of the virus and expose the nucleoprotein during detection. In FIG. 5, the means is exemplified as a vibrator 62.

[0058] Furthermore, when measuring the fluorescence intensity in the detection means, the optical sensor 68 has an electronic means for capturing an image of the fluorescence with an image sensor via an optical filter 67 that does not pass excitation light, and extracting the image by referring to the shape of the microelectrode. The portion not extracted is treated as background noise, and information on changes in the fluorescence intensity is corrected and contrast is emphasized, enabling more sensitive measurements.

[0059] In addition, a grinder may be provided that can disperse a sample containing the substance to be detected in water and process it into a slurry that is close to liquid so that it can be introduced into a detection cell as a sample in the detection method. In this case, solids containing the substance to be detected can also be introduced as a sample.

[0060] The detection cell provided in the detection device of the present invention may be in a state where an appropriate amount of a detection substance recognition material labeled with a fluorescent substance or a quenching substance, a detection substance, a pH adjustment buffer optimal for the reaction, a surfactant, etc. may be attached to the detection cell wall by adding these in advance as a solution and freeze-drying. In the present invention, the reaction solution used for fluorescence detection is preferably a solution capable of carrying out the binding reaction, electrophoresis or dielectrophoresis between the substance to be detected and the substance to be detected recognition material. For example, a sample containing the substance to be detected may be mixed with the solution to detect the substance to be detected. The reaction solution may suitably be, for example, the solution described in the Examples. Desirably, a solvent that minimizes the electrical conductivity within a range that does not inhibit the reaction between the substance to be detected and the substance to be detected recognition material, such as non-ionized water or a mixture of this with a sugar alcohol, may be used. In addition, in consideration of the fact that the substance to be detected and the substance to be detected recognition material can be bound and concentrated in the same solvent, for example, a phosphate buffer solution may be used, and PBS with a concentration of 1 mM or less may be used. The pH adjusting buffer used here is not particularly limited, and may be appropriately selected taking into consideration the analyte to be detected and the binding reaction between the analyte and the analyte recognition material. The surfactant is used, for example, to decompose the virus and extract the nuclear protein therein, and also to guide the sample into the cell by capillary action. One type of surfactant may be used, or two or more types may be used depending on the required function. For example, TritonX-100 can be suitably used to extract the nuclear protein from the influenza virus.

[0061] In the present invention, a small tube is prepared in addition to the detection cell, and the components used for detection may be mixed in the tube and then introduced into the detection cell. In this case, the tube may contain a surfactant and other components in advance. The method of introduction into the tube is not particularly limited, but the surfactant and other components may be present in the tube by freeze-drying. Alternatively, the surfactant and other components may be contained in a separate container and mixed with the sample in the tube before being introduced into the cell. In the present invention, calibration may be performed by measuring with and without a sample, detection may be performed using a standard solution, detection may be performed using a standard reference value for each lot, or detection may be performed without using a reference value by implementing strict quality control.

[0062] In the detection method of the present invention, a sample is present in a detection cell having an electrode provided inside the bottom of the detection cell, and when a change in the fluorescence intensity of a bond between a detection target substance that has been locally concentrated in the sample by electrophoresis or dielectrophoresis and a detection target substance recognition material is measured by an image sensor, (1) Place a transparent insulating film on the image sensor to measure the fluorescence, or (2) Fluorescence is measured by placing an image sensor on the outside of the bottom of the detection cell, (3) The electrodes have anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrodes is provided on the image sensor to measure the fluorescence, or (4) Excitation light is applied from the bottom of the detection cell, and the fluorescence is measured by an image sensor provided on the side of the detection cell. Note that the bottom can also be read as the bottom surface. By performing the measurement by any one of the above methods (1) to (4), the change in fluorescence intensity is measured to detect the target substance in the sample. In one embodiment of the present invention, the presence or absence of the target substance in the sample is confirmed by measuring the change in fluorescence intensity. By carrying out the measurement using any one of the above methods (1) to (4), it is possible to more clearly distinguish between the concentrated complex of the analyte substance and the analyte substance recognition material and the entire sample.

[0063] In the detection method of the present invention, it is convenient to use an optical sensor as a method for measuring the fluorescence intensity of the entire sample in bulk, but in the present invention, an image sensor is used to measure the change in fluorescence intensity. According to the study by the present inventors, since the electric field strength is proportional to the voltage and inversely proportional to the distance, a voltage between the electrodes of several V and a distance between the electrodes of several μm are suitable in terms of design and specifications for detecting the target substance. Furthermore, since the detection method of the present invention aims to locally concentrate the bond between the target substance and the target substance recognition material to increase the detection sensitivity, it is necessary to detect the vicinity of the electrodes and the entire sample with a resolution of several μm. Therefore, in the present invention, by measuring the fluorescence intensity using an image sensor, it becomes possible to detect the target substance with higher resolution.

[0064] In the step of measuring the change in fluorescence intensity, when the method (1) is used, a transparent insulating film is provided on the image sensor. In the case where an optical filter is provided, the transparent insulating film may be provided in the order of optical filter, transparent insulating film, and image sensor in the fluorescent light path, or in the order of transparent insulating film, optical filter, and image sensor. In addition, since existing image sensors usually have an optical filter for each pixel, it is possible to use the optical filter of the existing image sensor without providing a separate optical filter, or, if the conditions are suitable, to distribute the output of the three primary color pixels by software to imitate the effect of a color filter. Furthermore, since the surface of an image sensor is usually insulated by a color filter and a microlens, these may be provided as a transparent insulating film. Also, the image sensor and the transparent insulating film may be provided to form the bottom of the detection cell. In this case, an electrode may be provided on the image sensor via a transparent insulating film, and the transparent insulating film forms the bottom of the detection cell. The electrode present inside the bottom of the detection cell, the bottom of the detection cell (transparent insulating film), and the image sensor present outside the bottom of the detection cell are arranged in this order. In this case, an optical filter does not need to be present. Also, a part or all of the electrode present inside the bottom of the detection cell, the bottom of the detection cell (transparent insulating film), and the image sensor present outside the bottom of the detection cell may be integrated. In method (1), the fluorescence from the conjugates concentrated on the electrode is imaged on an image sensor. When the fluorescence is imaged as light spots, the number of light spots before and after application of an electric field can be counted to determine the number of conjugates concentrated locally.

[0065] A configuration diagram for method (1) is shown in Fig. 17, and its cross-sectional view is shown in Fig. 18. In Fig. 17 and Fig. 18, microelectrodes (201, 211), transparent insulating films (202, 212), and image sensors (203, 213) are shown. The image sensor is not particularly limited, but may be, for example, an image sensor of 5 mm x 5 mm and 5000 x 5000 elements (pitch 1 μm), and the most common image sensor is a back-illuminated CMOS. As shown in Figure 18, an image sensor is composed of a circuit, a sensor, a color filter, and a microlens from the bottom. The microlens and color filter are composed independently of the three primary colors. In other words, the three elements of the microlens, color filter, and sensor come together to form one pixel. At present, the smallest pixel in the widespread market is less than 1 μm in size. A transparent insulating substrate (insulating film) with microelectrodes printed on it is placed on top of the microlens of the image sensor, and the detection liquid is stored on top of that. The width and spacing of the microelectrodes are preferably 10 μm or less, and may be 5 to 10 μm wide and 2 to 5 μm apart, but are not particularly limited thereto. For example, the electrode width is 10 μm, the electrode spacing is 5 μm, and the length is 2 mm. With this configuration, the fluorescence emitted from the conjugates gathered at the ends of the microelectrodes can be directly captured as an image by the image sensor, and the change in fluorescence intensity can be measured by comparing the output of the image sensor before and after voltage application. Preferably, for ease of use, the excitation light (320 nm or 450 nm) emitted by the LED is irradiated from above through a light guide plate. Also, a color filter built into the image sensor may be used to separate the excitation light by blocking blue information that reacts to the excitation light among the signals emitted by the image sensor. The transparent insulating film is not particularly limited, but may be, for example, a thin glass film, a synthetic resin film, etc. The thickness of the insulating film is preferably 1 μm or less. The explanations given regarding the image sensor and electrodes (including microelectrodes) in the method (1) are also applicable to the methods (2) to (4).

[0066] In the step of measuring the change in fluorescence intensity, when the method (2) is used, an image sensor is provided so as to be in contact with the outside of the bottom of the detection cell. In the method (2), compared to the method (1), the bottom of the detection cell is not a transparent insulating film, and the method (2) can be carried out in the same manner as described in the method (1). The method (2) has the simplest configuration among (1) to (4), and its configuration diagram is shown in FIG. The method (2) differs from the method (1) in that the thickness of the bottom of the detection cell is preferably 100 μm or less, more preferably 10 μm or less. The material of the detection cell is not particularly limited as long as it transmits fluorescence. By placing an image sensor very close to the electrode, it is possible to receive the fluorescence from the conjugate near the electrode. Also, by measuring the strength of the signal received by the image sensor before and after application of an electric field, it is possible to count the number of particles near the electrode, and the change in fluorescence intensity is measured. If the thickness of the bottom of the detection cell is 50 μm or more, the detection cell can be made of plastic or glass, making it easy to manufacture the detection cell, but this creates a distance between the image sensor and the fluorescent light spot, which may make it difficult to form an image on the image sensor.In this case, it is also possible to measure the change in fluorescence intensity by evaluating it as an area sensor that compares the brightness before and after voltage application.

[0067] In the step of measuring the change in fluorescence intensity, when the method (3) is used, the electrodes have anisotropy in the X and Y directions, and slits having the same anisotropy as the electrodes are provided on the image sensor. In the methods (2) and (3), the detection cell may be used as a disposable cell. Method (3) is an example of (2) that is devised when the thickness of the detection cell is about 0.5 to 1.0 mm, and a configuration diagram of method (3) is shown in FIG. 20. Explaining with reference to FIG. 20 as an example, for example, in the XY plane (bottom surface of the detection cell), comb-shaped electrodes extending in the Y direction face each other in the X direction. Note that the detection cell is not shown in FIG. 20. Between the microelectrode 231 and the image sensor 233, a slit 233 having anisotropy corresponding to the anisotropy of the electrode in the X and Y directions is provided. The width of the slit 233 does not necessarily have to be the same as that of the microelectrode 231, but it is preferable that 0.1 to 5 electrodes are inserted into one slit. The width and spacing of the microelectrodes are preferably 10 μm or less, and may be 5 to 10 μm wide and 2 to 5 μm apart. Although not particularly limited, for example, the electrode width is 10 μm, the electrode spacing is 5 μm, and the length is 2 mm. The width of each slit and the slit interval are preferably 0.2 to 1.0 μm. The thickness of the slit 233 is preferably 1 to 5 μm in order to capture the fluorescence from the light spot with good sensitivity. The fluorescence from the conjugate diffuses in all directions as a point light source, but the diffused light can be controlled by the slits.

[0068] In the method (3), it is preferable to compare the integral value of the image sensor output in the X direction and the integral value of the image sensor output in the Y direction before and after the application of a voltage (an overview of this is shown in FIG. 21). In this method, the sensitivity can be increased by increasing the exposure time. The change in fluorescence can be understood by integrating the brightness in each of the X and Y directions. Therefore, the background fluorescence from the conjugates and the like located away from the electrodes is dispersed in the same manner in the X and Y directions, and therefore the desired change in fluorescence can be measured.

[0069] In the step of measuring the change in fluorescence intensity, when the method (4) is used, an image sensor is provided on the side of the detection cell. In the method (4), excitation light is irradiated from the bottom of the detection cell. A configuration diagram of the method (4) is shown in FIG. 22. In the method (4), if the bottom of the detection cell is the XY plane, the side of the detection cell is the YZ plane, and since the image sensor is provided on the YZ plane, the change in the fluorescence intensity is measured by comparing the change in the depth direction (Z direction) before and after the application of a voltage. Also, by measuring the strength of the signal received by the image sensor before and after the application of an electric field, it is possible to measure the number of particles near the electrode, and the change in the fluorescence intensity is measured. By providing the image sensor on the side of the detection cell, the image sensor can detect differences in fluorescence intensity between locations near the electrode and locations farther away from the electrode, in particular, compared to when the image sensor is provided on the bottom of the detection cell. In the method (4), the excitation light may be, for example, light emitted from an LED that is emitted through a light guide plate and then emitted from the surface (the same principle as the backlight of a liquid crystal display). The detection cells and image sensors can be used by referring to the descriptions regarding the above methods (1) to (3). EXAMPLES

[0070] The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples.

[0071] <Example 1> Antibody labeling FIA3298 (Bio Matrix Laboratories) was used as an antibody that binds to the nucleoprotein of influenza A virus. Quantum dots (Qdot565, Thermo Fisher) were chemically labeled to the glycan of the Fc region of the antibody according to the method of the Qdot Antibody Conjugation Kit (Bio Matrix Laboratories). The fluorescence spectrum of the quantum dot-labeled antibody Ab1Qd565 measured using a fluorescence spectrophotometer (F2500, Hitachi High-tec) had a fluorescence peak at 561 nm (Figure 1). As an example, a quantum dot-labeled antibody may be represented by the formula: [ka]

[0072] Labeling of nucleoprotein (NP) The recombinant influenza A (H1N1) nucleoprotein (NP) used was AAM75159.1 (Sino Biological), and the quencher was the NHS ester of QSY9 (Thermo Fisher). The NHS ester of QSY9 is represented by the following formula: [ka] The NHS ester of QSY9 was dissolved in dimethyl sulfoxide (DMSO). The DMSO solution of the NHS ester of QSY9 was added to an aqueous solution of NP dissolved in 10 mM sodium bicarbonate buffer (pH 8.0) so that the number of molecules of QSY9 was 30 times the number of molecules of NP, and the mixture was left at room temperature overnight. The mixture was purified into the nuclear protein NPQSY9 labeled with a quencher using a Biorad Micro Bio Spin Column 30. The purification conditions were as per the protocol provided by Biorad, except that the developing solution was changed to PBS (pH 7.2). The absorption spectrum of the fraction obtained by purification, measured using a UV / VIS spectrophotometer (UV2500PC, Shimadzu Corporation), is shown in Figure 2. A comparison of Figures 1 and 2 shows that the fluorescence peak of the quantum dot-chemically labeled antibody Ab1Qd565 and the absorption peak of the quencher of the quencher-labeled nuclear protein NPQSY9 overlapped almost ideally.

[0073] Quenching confirmation experiment Ab1Qd565 was diluted with PBS to a concentration of 10 nM. 200 μL of the diluted solution was used to measure the fluorescence spectrum using a microcell and a fluorescence spectrophotometer (F2500, Hitachi). NPQSY9, whose protein concentration was adjusted to 10 μM, was mixed in 2 μL portions into this solution, and the change in fluorescence intensity at 561 nm was observed (Figure 3). The results in FIG. 3 confirmed that the fluorescence having a peak at 561 nm was quenched by binding between NPQSY9 and Ab1Qd565 in solution.

[0074] Dielectrophoresis of polystyrene microparticles Polystyrene with a particle size of 3 μm slight A particle water dispersion (Polysciences) was diluted 100,000 times with deionized water. The conductivity was measured using a conductivity meter (EC-33, Horiba, Ltd.) and found to be 13 μS / cm. Next, the dielectrophoresis of the polystyrene fine particles was observed under the following conditions. Cell: Based on a BAS interdigitated microelectrode (Au, 5 μm interelectrode distance), with a cover glass attached to the microelectrode via a 12 μm spacer. High frequency generator: Tektronix AFG3022 function generator Oscilloscope: Hewlett-Packard 54602B Microscope: Tektronix VHX900 with 1000x lens Approximately 10 μL of the diluted solution of polystyrene microparticles prepared as described above was placed on the microelectrode, and the voltage and frequency were swept while observing under a microscope. It was confirmed that the polystyrene microparticles aligned along the contour of the electrode under the condition of 2 V / 1 kHz. That is, positive dielectrophoresis was observed in polystyrene microparticles. The time required for aggregation to reach a steady state was about 1 to 2 seconds. Similarly, positive dielectrophoresis was observed when yeast was used, and the yeast was concentrated along the electrode.

[0075] Antibody loading Ab1Qd565 was supported on the surface of polystyrene microparticles using the method specified by the latex particle manufacturer. After that, blocking treatment was performed with BSA to prevent unnecessary non-specific adsorption. NPQSY9 was mixed with this so that the number of antibody / NP molecules was the same, and a pre-quenched state was created. Using a Leica Microsystems DFC360FX fluorescence microscope and an ebq100 light source, dielectrophoresis was performed using the above cell, and it was confirmed that the fluorescence from Ab1Qd565 gathered along the contour of the electrode at 2V / 1kHz. A mixture of Ab1Qd565-supported polyethylene microparticles and NPQSY9 under the same conditions as above was mixed with unlabeled NP (same product as above) in an amount equal to the protein concentration of NPQSY9, and incubated for 5 minutes. While observing this with a fluorescence microscope, it was confirmed that the particles lined up along the contour of the electrode at 2V / 1kHz. Fluorescence microscope images taken during dielectrophoresis with and without unlabeled NPs were electronically stored as still images on a connected computer. Comparing the two, it was confirmed that the fluorescence intensity was higher in the case with NPs. These electronic images were loaded into Adobe Photoshop, and it was confirmed that the difference between the two companies could be made clearer by cutting out the image near the microelectrode, eliminating background noise, and enhancing the contrast.

[0076] Based on the above results, it is believed that by creating dedicated software, these tasks can be carried out consistently and quantitative judgments can be made. In addition, when an inactivated virus (81N73, Hy Test) was passed through an immunochromatography column (anti-NP antibodies FIA2121 and FIA3298, both manufactured by Sino Biological) using a developing solution containing TritonX-100 (1%), the virus was destroyed during the development, and NP appeared, causing a sandwich reaction between the two antibodies, which was confirmed to be detectable by immunochromatography. Considering all the above, it is believed that this example makes it possible to combine dielectrophoresis and fluorescence observation, thereby demonstrating the possibility of high-sensitivity, high-speed detection.

[0077] <Example 2> Antibody labeling Anti-BSA mouse monoclonal antibody (Funakoshi) was used as the antibody that binds to BSA (Sigma). TM Qdot TMThe antibody was concentrated to 100 μL (1 mg / mL) using the antibody concentrator included in the 655 Antibody Labeling Kit (Invitrogen). The glycans in the Fc region of the antibody were selectively chemically labeled with quantum dots (Qdot655, Thermo Fisher) according to the method in the kit. The quantum dot-chemically labeled antibody Ab1Qd655 was purified using the gel filtration device included in the kit. The fluorescence spectrum (excitation light 370 nm) of Ab1Qd655 in PBS solution measured using a fluorescence spectrophotometer (F7100, Hitachi High-tec) had the peaks shown in Figure 6. The faint peak visible at 720 nm is due to scattering of light with twice the wavelength of the excitation light.

[0078] Antigen labeling As a quencher, QSY21 succinate (QSY21NHS, Thermo Fisher) was used. QSY21NHS is represented by the following formula. QSY21 has a large absorption around 650 nm, but it is known to be a quencher that does not emit fluorescence at least in the visible light region. [ka] QSY21NHS was dissolved in anhydrous DMSO at a concentration of 10 mg / mL (12.3 mM). A DMSO solution of QSY21NHS was added to a 10 mM PBS solution of BSA (5 mg / mL) so that the number of molecules of BSA:QSY21NHS was 30 times the number of molecules of BSA, and the mixture was stirred by vortexing. Furthermore, an aqueous NaHCO3 solution was added to a final concentration of 50 mM, and the mixture was left at room temperature for 3 hours. The mixture was purified into BSA labeled with a quencher using a spin column (BioRad P6). The absorption spectrum of the purified product, measured using a UV / VIS spectrophotometer (UV2500PC, Shimadzu Corporation) for a solution diluted 5 times with PBS, is shown in Figure 7. The number of bonds was calculated to be 1.5 molecules of QSY21 per BSA molecule.

[0079] FRET quenching experiment Ab1Qd655 in PBS for 10 -8The diluted solution was placed in a fluorescence microcell (excitation side optical path length 10 mm, fluorescence side optical path 2 mm, Hellma) and irradiated with 370 nm excitation light using a fluorescence spectrophotometer (F-7100, Hitachi High-Tech Science), and the fluorescence spectrum was measured from 350 nm to 800 nm. The wavelength range of the fluorescence spectrum in the following quenching confirmation experiments was all the same. 10 -8 M Ab1Qd655 1.0E -6 BSAQSY21 at a final concentration of 10 -8 The mixture was mixed to give a final concentration of M, and then stirred for 5 seconds using a vortex mixer. After stirring, the mixture was quickly placed in a fluorescence microcell, and the fluorescence spectrum was measured using a fluorescence spectrophotometer. After that, the fluorescence spectrum was measured every 5 minutes until 30 minutes, and then every 10 minutes from 30 minutes to 150 minutes. The results are shown in Figure 8. Immediately after the start of the measurement, 20% quenching occurred, and then, although the rate was slower, a final decrease in fluorescence of 72% was confirmed. In other words, the decrease in fluorescence was confirmed to be due to intermolecular energy transfer (FRET) based on the antigen-antibody reaction.

[0080] Quenching inhibition confirmation experiment 10 for the reaction mixture for the quenching confirmation experiment -5 M unlabeled BSA final concentration 10 -7 M. After addition, the mixture was quickly stirred by pipetting, and the fluorescence spectrum was measured from 350 nm to 800 nm using a fluorescence spectrophotometer (F-7100, Hitachi High-tec) with 370 nm excitation light, as in the quenching confirmation experiment. The wavelength range of the fluorescence spectrum in the following inhibition confirmation experiments was the same. The fluorescence spectrum was then measured after 3 minutes, and thereafter was measured 12 times at 5 minute intervals. The results are shown in Figure 9. The 72% quenched fluorescence increased by 37% when unlabeled BSA was added. It was confirmed that Ab1Qd655 and BSAQSY21 bound to each other through an antigen-antibody reaction, displacing the unlabeled BSA from the quenched state, and the fluorescence of Ab1Qd655 was enhanced. By observing the increase in fluorescence, it was confirmed that BSA can be detected without using B / F separation. Figure 10 shows the fluorescence spectra at each stage for comparison. In addition, when modified with Qd655, the solution of Ab1Qd655 has the property of easily agglutinating and precipitating during storage at 4°C for several days. This phenomenon can be suppressed by, for example, arginine (10 mM-500 mM, preferably 50 mM-200 mM).

[0081] Dielectrophoresis Figure 11 shows a dielectrophoresis cell using a micro interdigital electrode manufactured by BAS. In Figure 11, 111 is a microelectrode, in which gold electrodes with an electrode length of 2 mm, an electrode width of 5 μm, and an electrode interval of 5 μm are printed on a substrate 112, and each electrode is electrically connected to terminals 113 and 113'. A cover glass 114 is attached to the top of this via a 130 μm spacer 115 to form a space (sample part). A Tektronix AFG3022 was used to generate high frequency waves. One of the output terminals of the AFG3022 was connected to terminal 113 via a 0.001 μF capacitor to prevent direct current. An oscilloscope (Hewlett-Packard 54602B) was connected in parallel to terminals 113 and 113' to monitor the waveform. When a sample of yeast dispersed in non-ionized water was introduced into the sample section of the dielectrophoresis cell and a voltage of 2 MHz and 2 V was applied between terminals 3 and 3' while recording images with a digital microscope (VHX900, 1000x, Keyence), it was observed that the dispersed yeast instantly aligned on the end surface of the electrode. The same phenomenon was observed at frequencies of 5 MHz, 1 MHz, 300 kHz, and 100 kHz. When dielectrophoresis was performed with the following related particles, ITO (indium tin oxide), ATO (antimony tin oxide), and Sekisui Chemical's polybeads (polystyrene particles with gold-plated surfaces), type A (MB-A) and type B (MB-B), all of which have a particle size of 2 μm, dielectrophoresis was not observed. However, when these were immersed in a PBS solution of 0.1% BSA, centrifuged at 5000g for 20 minutes after 8 hours, and the supernatant was discarded twice to coat the surface with BSA, ITO, ATO, MB-A, and MB-B all instantly aligned at 2 MHz 5 V in 1 mM PBS. In other words, they were concentrated at the electrode edge. With graphite particles (Ito Graphite Industries), regardless of whether they were coated with BSA, very fast dielectrophoresis was observed at 2 MHz, 5 MHz, and 300 kHz in 1 mM PBS. Dielectrophoresis was confirmed even when the PBS concentration was increased to 10 mM, although a slight decrease in speed was observed. Note that the dielectrophoresis described above was positive, with the particles gathering at the ends of the electrodes (more precisely, at the corners of the printed electrodes). For reference, the conductivity of PBS of various concentrations was measured using a Horiba conductivity meter (Laqua Twin EC-33) and the results were 10mM: 13,000μS / cm, 2mM: 3,360μS / cm, and 1mM: 2,110μS / cm. According to the reference (Yen-Pei Lu et. al, Scientific Reports | (2019) 9:14771), the conductivity of saliva is listed as 2,000-8,000μS / cm, and the actual measured value using the actual sample was 5,780μS / cm.

[0082] Dedicated measuring device The basic configuration of the measurement unit is shown in Figure 11. The detection cell 121 is a four-sided transparent fluorescence measurement cell with an optical path length of 10 mm, and the color filter 123 (sharp cut filter Y50, HOYA) completely blocks light with wavelengths shorter than 500 nm. The LED 122 is an OSV2YL5111A (manufactured by Opto Supply) that emits light at 370 nm. An excitation wavelength of around 370 nm was used. The sensor 124 is an S7183 (Hamamatsu Photonics) that has its center of sensitivity to light around 650 nm. The AD converter was made with ArduinoUno and the program was made with ArduinoIDE. The PC and Arduino were connected via USB and the program on the PC was made with Processing. The operation steps are as follows: The surface of the detection cell is blocked in advance with a protein other than BSA to prevent non-specific adsorption. Ab1Qd655 and BSAQSY21 in PBS (10 -8 M) and incubate for 24 hours at room temperature. The resulting mixture is placed in the detection cell and the application on the PC is launched. Instructions are sent from the software on the PC, and the AD converter measures the output voltage of the sensor, performs AD conversion, and sends a signal to the PC via the USB interface. This is done every 0.5 seconds for a total of five times, and the average value is saved as the initial value. -Display the message "Ready" on the PC monitor. The tester adds the sample to the detection cell (at this time, the volume change is negligible). A vibrator (not shown) in contact with the detection cell vibrates and agitates the liquid for 3 minutes. The sensor signal is read and sent to the PC. This is repeated five times, and the PC calculates the average value and saves it as the measurement value. Stop the vibrator stirring. The PC compares the initial value with the measured value, and if there is an increase in fluorescence intensity of more than a predetermined threshold (e.g. 5%), it displays the result as "positive." At this time, the percentage of increase is displayed as a reference value, and in addition to the two levels of "positive" and "negative," a semi-quantitative value on a scale of 5-10 may be displayed. By simply mixing the sample, it is possible to qualitatively or quantitatively detect the target substance in a homogenous solution without performing a development process like the lateral flow method, a washing process like ELISA, or other B / F separation.

[0083] <Example 3> In a so-called sandwich-type antigen-antibody reaction in which two types of antibodies (Ab2, Ab3) simultaneously bind to one antigen, Ab2 and Ab3 are labeled with different fluorescent dyes (F2, F3), and F3 is excited by the fluorescence (wavelength λF2) emitted by F2 and emits light of wavelength λF3, but is not excited by the excitation light for F2 (wavelength λE2). By detecting the fluorescence intensity of wavelength λF3 by excitation with λE2, it is possible to monitor the progress of the sandwich reaction. Below, we describe an example in which two types of antibodies were prepared against the nucleoprotein (NP) of influenza A virus. At the same time, antibodies FIA2121 and FIA3298 (both anti-NP mouse monoclonal antibodies from Biomatrix Research Institute) were used as antibodies that reacted with NP in a sandwich manner to form antibody-antigen complexes of Ab2·NP·Ab3. Using antibody FIA2121 as Ab2, 10 μL of PBS solution (1 mg / mL) was used as the starting point and the labeling reaction was carried out according to the protocol disclosed by ATTO390 NHS ester (ATTO-Tec). The reaction solution was purified using Nanosep molecular weight cutoff 30k. The number of dye bonds per antibody molecule calculated from the absorption spectrum was 23. The absorption spectrum (dashed line) and the fluorescence spectrum (solid line) at 390 nm excitation of the obtained labeled antibody (Ab2AT390) are shown in Figure 13. Antibody FIA3298 was used as Ab3, and 100 μL of PBS solution (2 mg / mL) was prepared, 20 μL of 1M NaHCO3 aqueous solution was added, and 25 μL of DMSO solution (2 mM) of dye DY485XLNHS ester (Dyomic) (37.5 times the number of molecules of the antibody) was immediately added and mixed well. After 5 minutes, the reaction solution was purified using Nanosep molecular weight cutoff 30k (Nihon Genetics). The number of dye bonds per antibody molecule calculated from the absorption spectrum was 37. The absorption spectrum (dashed line) and the fluorescence spectrum (solid line) at 470 nm excitation of the obtained labeled antibody (Ab3DY485) are shown in Figure 14. From the results of Figures 13 and 14, it can be understood that when the reaction solution containing two kinds of labeled antibodies is irradiated with light at 390 nm, only Ab2AT390 is excited and emits fluorescence around 470 nm, but Ab3DY485 is not excited because the Ab2AT390 and Ab3DY485 molecules are sufficiently far apart. In addition, if NP is present in the solution, Ab2AT390 and Ab3DY485 molecules bind to NP via NP at a short distance of estimated 5-10 nm, and energy transfer occurs by FRET, that is, the excitation energy equivalent to the fluorescence around 470 nm emitted by Ab2AT390 is transferred, and Ab3DY485 is excited and emits fluorescence at 560 nm. In other words, the presence of NP can be determined by exciting the reaction solution with light at 390 nm and observing the fluorescence at 560 nm. From the results of Examples 1 to 3, the present invention can be practiced by the following embodiments. Note that the embodiments shown below are shown as examples and do not limit the scope of the present invention, and the present invention can be practiced by various modifications.

[0084] <Embodiment 1> The monoclonal antibodies mAb11, mAb12, and mAb13 are used, which have epitopes at three different sites on the nucleoprotein (NP) of influenza A virus. Disperse 2 μL of ITO with an average particle size at a concentration of 0.2 mg / mL in 1 mL of a 10 μg / mL solution of mAb11. Incubate for 2 h with sufficient stirring, centrifuge at 5000 g / 5 min, and discard the supernatant. Disperse the precipitate in a 1 mg / mL BSA solution (containing 0.05% sodium azide and 1% TritonX-100) to prepare Reagent 11. On the other hand, mAb12 and mAb13 were labeled with ATTO390 and DY485XL, respectively, to obtain labeled antibodies Ab12AT390 and Ab13DY485. -7 Prepare 1 mM PBS (containing 0.05% sodium azide) containing M and use this as reagent 12. As a sample, 100 μL of saliva containing inactivated influenza A virus is mixed with 400 μL of reagent 11 and left for 5 minutes. At this time, the viral envelope is destroyed by TritonX-100, and NPs are released (confirmed by evaluation using immunochromatography developed in-house). The released NPs bind to mAb 11 adsorbed on the surface of ITO. It is then mixed with 500 μL of reagent 12. At this point, the saliva has been diluted 10 times, and the conductivity has fallen to below 1000 μS / cm, which is sufficient for dielectrophoresis to occur. The microelectrode is introduced into the detection cell at the bottom. Detection is performed in the following steps. 1) An AC voltage of 2 MHz, 5 V is applied to the microelectrode. 2) Illuminate the area near the microelectrode with a 390 nm light source and observe the fluorescence intensity at around 560 nm. 3) Fluorescence observation is performed 10 times in succession and the average value is sent to the PC. 4) This is repeated every 5 seconds for a total of 2 minutes, and the final intensity is calculated from the trend of increase in fluorescence intensity. 5) Based on the final intensity, a negative / positive determination is made along with quantitative data. In the first embodiment, NPs derived from a specimen are bound to ITO via mAb 11, and the NP concentration is locally increased near the microelectrode by dielectrophoresis. Concentration on the electrode surface (XY surface) is expected to be about 100 times, but in the depth direction (Z axis), by efficiently convecting in the depth direction of the cell using an oscillator, the frequency of forced contact with the electrode surface can be increased, and it becomes possible to concentrate the specimen by more than 1000 times in total. In the first embodiment, for example, the fluorescent dye is a dye with a high molecular extinction coefficient and a high quantum yield, and a combination of dyes that efficiently cause the FRET phenomenon can be applied. Although the reaction is prepared as different reagents to make it easier to understand, the reagents 11 and 12 may be mixed in advance, and it is self-evident that the number of steps during testing can be further reduced by preparing reagents 11 and 12 attached to a detection cell and freeze-dried. Note that the same can be selected appropriately and applied in the second and third embodiments.

[0085] <Embodiment 2> We use monoclonal antibodies mAb21 and mAb22 that target two different epitopes on the nucleoprotein (NP) of influenza A virus. We also use a fragment of NP (NP fragment) that binds to mAb22. Since NP can be synthesized by genetic recombination, mAb21 and mAb22 may be prepared by first determining the fragment of NP and then producing antibodies using the NP fragment as an immunogen. Confirm that the obtained antibody recognizes NP. In this case, the reaction rate constant (ka: binding rate constant, kd: dissociation rate constant) between mAb22 and the NP fragment can be determined by, for example, surface plasmon resonance, and is preferably ka>10 6 , kd<10 -3 By screening antibodies, high measurement speed can be achieved. mAb 21 is adsorbed onto the ITO surface, which is then blocked with BSA to prepare Reagent 21 containing 1% TritonX100. On the other hand, mAb22 was labeled with Qd655, and the NP fragment was labeled with QSY21. The labeled antibody Qd655Ab22 and the labeled antigen fragment QSY21NP fragment were both incubated for 10 min. -8 A 1 mM PBS solution (containing 0.05% sodium azide) containing 100 mM of erythritol is prepared as reagent 22. The test is carried out in the following steps. 1) 100 μL of the specimen (saliva containing influenza A virus) is mixed with 400 μL of Reagent 21 and left for 5 minutes. 2) The resulting mixture is mixed with 500 μL of Reagent 22. 3) The mixture is immediately introduced into a detection cell equipped with a microelectrode on the bottom. 4) As stirring by the vibrator begins, light with a wavelength of 370 nm or less is irradiated near the microelectrode, and the fluorescence emitted from near the microelectrode is measured by a sensor via a color filter that cuts off wavelengths of 500 nm or less. 5) The first measurement value is saved as the initial value, and the change in fluorescence intensity thereafter is measured and the final measurement value is calculated from the trend of that change. 6) In the state where Qd655Ab22 and ASY21NP have previously bound to each other and a certain degree of quenching has occurred, NP present near the electrode binds to Qd655Ab22, and the presence or absence of NP and the positive / negative status of infection are determined from the inhibition of quenching (i.e., increased fluorescence).

[0086] <Embodiment 3> Ab31 and Ab32 are used, which simultaneously bind to the surface antigen of influenza A virus. Ab31 and Ab32 are labeled with ATTO390 and DY485XL, respectively (hereafter referred to as Ab31AT390 and Ab32DY485). These are then mixed for 10 min. -7 Prepare 1 mM PBS (containing 0.05% sodium azide) containing 1 mM or more (Reagent 31). Perform the test using the following steps. 1) 100 μL of a specimen (saliva containing influenza A virus) is mixed with 900 μL of a reagent 31 and introduced into a detection cell having a microelectrode disposed on the bottom surface. 2) Immediately apply a high frequency voltage of 1 kHz-5 MHz, preferably 100 kHz, 5 V to the microelectrode and begin stirring with the vibrator. 3) Excite the area near the electrode with light at 390 nm, and measure the fluorescence intensity at around 560 nm. 4) The final measurement value is calculated from the change in fluorescence intensity over time. 5) The final measurement value is used to determine the virus concentration and infection status. In embodiment 3, the viruses themselves are concentrated near the electrodes by dielectrophoresis, so that surfactants and incubation time required for NP extraction are not required.

[0087] <Embodiment 4> A detection cell 151 is equipped with comb-shaped microelectrodes on the bottom surface, each with an electrode width and electrode interval of 5 μm and an electrode length of 5 mm, and an LED light source 153 and a sensor 155 are tilted toward the microelectrodes to form a detection device (Figure 15). The analog signal output by the sensor 155 is processed. BSAQSY21 to 10 -6ITO with a particle size of 2 μm was dispersed at 0.11 mg / mL in a PBS solution containing BSAQSY21, left overnight at room temperature, centrifuged at 5000 g for 20 minutes, and the supernatant was discarded. The ITO was then dispersed in skim milk at a concentration of 1 mg / mL, blocked, centrifuged, the supernatant was discarded, and the precipitate was recovered. The loss due to centrifugation was estimated to be 30%, and the final concentration of BSAQSY21 was 10. -8 Ab1Qd655 (final concentration 10 -8 Prepare a PBS dispersion solution with M) and leave it at room temperature overnight, then measure BSA using the following steps. The obtained mixed dispersion solution is placed in the detection cell and the application on the PC is launched. When the software on the PC sends an instruction, the LED irradiates 370 nm light, and at the same time, the AD converter measures the output voltage of the sensor, performs AD conversion, and sends a digital signal to the PC via the USB interface. This is done every 0.5 seconds for a total of five times, and the average value is saved as the initial value. -Display the message "Ready" on the PC monitor. The tester adds the sample to the detection cell (at this time, the volume change should be negligible). The vibrator (not shown) in contact with the detection cell vibrates to stir the liquid for 3 minutes. At the same time, a high frequency of 5 V and 2 MHz is applied between the microelectrodes. The sensor signal is read and sent to the PC. This is repeated five times, and the PC calculates the average value and saves it as the measurement value. Stop the vibrator stirring and stop applying high frequency. The PC compares the initial value with the measured value, and if there is an increase in fluorescence intensity above a predefined threshold (e.g. 5%), it displays the result as "positive." At this time, the percentage of increase is displayed as a reference value, and in addition to the two levels of "positive" and "negative," a pseudo-quantitative value may be displayed on a scale of 5-10. In the fourth embodiment, instead of the detection target, a substance equivalent to the detection target prepared in advance (pseudo antigen) is adsorbed on the carrier surface, and the pseudo antigen is concentrated near the electrode by dielectrophoresis. Similarly, it is possible to bind a labeled antibody to the carrier surface and concentrate it by dielectrophoresis. Since the concentration of the antigen-antibody complex in the antigen-antibody reaction is proportional to the free antibody concentration, the free antigen concentration, and the affinity of the antibody, in the case where the pseudo antigen and the antigen compete as in this case, there is a certain effect whether the antibody, the pseudo antigen, or the detection target is adsorbed on the carrier and concentrated by dielectrophoresis. It is desirable to concentrate the detection target substance in the sample because it is the most efficient way to improve the sensitivity of the system even if an antibody with the same affinity is used.

[0088] <Embodiment 5> As a specimen, 100 μL of saliva is collected. Any collection method can be used, but in order to obtain a certain degree of quantitativeness without using a dedicated tool, a cotton-tipped swab 164 is effective (FIG. 16). With reaction solution already filled up to 162 in a test container 161 equipped with a microelectrode 165, the swab 164 is hooked onto the recess of a squeeze plate 163 and squeezed, whereby the specimen is mixed into the reaction solution. The reaction solution is 1 mM PBS with 1% TritonX-100 dissolved therein, into which RNA1 and RNA2, each complementary to a different portion of the base sequence of influenza A RNA, have been mixed in advance as detection target substance recognition materials. Based on Murakami Akira et al., Chemistry and Biology, Vol. 41, No. 8, 2003, RNA1 is pyrene-modified 2'-O-methyl oligo RNA. RNA2 is physically adsorbed onto graphite with a particle size of 2 μm, and then blocked with 0.1% BSA. When the above reaction solution is loaded into the detector, the following process is carried out. 1) According to the control signal sent from the PC, the vibrator in contact with the test vessel starts, and mixing by convection begins. 2) By stirring at room temperature for 30 seconds, if the sample contains the influenza A virus, which is the substance to be detected, the viral envelope will be destroyed by the action of Triton X-100, exposing single-stranded RNA to the reaction solution, and RNA1 and RNA2, which serve as materials for recognizing the substance to be detected, will bind to this RNA (RNA binding product). 3) A signal sent from the PC applies a 2MHz, 5V high frequency to the microelectrode placed on the bottom of the detection vessel. This causes the RNA binding compounds to be concentrated in the XY directions on the bottom surface along the microelectrode. Furthermore, convective stirring causes the reaction solution to come into contact with the microelectrode evenly, resulting in concentration in the Z direction. 4) An LED (Stanley Electric, MNU1109EAE-275) irradiates the area near the microelectrode with 275 nm light. 5) A color filter cuts out light with wavelengths below 350 nm, and the 380 nm fluorescence that passes through the color filter is detected by a photosensor (HIOKI 9743). After AD conversion, the digital signal is sent to a PC. 6) The PC records the output fluctuations of the optical sensor for three minutes, evaluates the difference from the value detected in advance using a negative control (buffer adjusted to a conductivity of 8000 μS / cm), and displays the determination result for the presence or absence of influenza virus or semi-quantitative data. Although a carrier was used to ensure dielectrophoresis, the system could be made even simpler by directly concentrating RNA by dielectrophoresis without using a carrier, provided that the conductivity of the reaction solution is sufficiently low and an increase in the applied voltage and a decrease in the detection speed can be tolerated.

[0089] In the embodiment 5, the fluorescence of RNA1 is enhanced by the double strand, but in the embodiment 6, which is a modification of the embodiment 5, RNA11, which is complementary to a part of the base sequence of influenza A RNA and fixed to a carrier, and RNA12, which is complementary to RNA11, are labeled with a fluorescent material and a quencher, respectively, and usually RNA11 and RNA12 are bound to each other and quenched, but when influenza A RNA, which is a substance to be detected, is further mixed in, RNA11 binds to the substance to be detected and quenching is inhibited. In this case, if RNA11 is made sufficiently longer than RNA12, the affinity between RNA11 and the substance to be detected is high, so that the binding between RNA11 and the substance to be detected is prioritized over the binding between RNA11 and RNA12, which is preferable in terms of both reaction speed and sensitivity. Similarly, RNA21 that is complementary to a partial base sequence of influenza A RNA and is immobilized on a carrier is used as the detection target substance recognition material, and a molecular probe in which the 5' end of RNA21 is fluorescently labeled and the 3' end is labeled with a quenching probe is available as a molecular beacon from Merck or Primetech, so these may also be used. Although the above description has been given using RNA as the detection target substance recognition material, the detection target substance may be RNA, and the detection target substance recognition material may be DNA complementary to the detection target substance.

[0090] <Embodiment 7> Preparation of materials A typical combination is that the analyte is influenza A virus (InfV), and the analyte recognition materials 1 and 2 are both anti-hemagglutinin monoclonal antibodies (IgG1, IgG2), with IgG1 and IgG2 capable of simultaneously binding to InfV ​​in a sandwich-like manner. Such antibodies can be selected from Abnova's MAB5637-M40, MAB1268, MAB10133, MAB1269, MAB5637-M33, and MAB10128. Alternatively, any of the antibodies included in the kits from Sino Biological Company's SEK11684 and SEKA11684 may be used as a pair of antibodies previously confirmed to bind simultaneously. IgG1 was labeled using the Qdot 585 Antibody Conjugation Kit (Thermo Fisher) according to the protocol included in the kit. The absorption spectrum and fluorescence spectrum of the resulting labeled antibody IgG1Qd585 are shown in FIG. IgG2 is Alexa Fluor TM Labeling was performed using the 594 Antibody Labeling Kit (ThermoFisher) according to the protocol included in the kit. The absorption spectrum and fluorescence spectrum of the resulting labeled antibody IgG2AF594 are shown in FIG. As is clear from a comparison of Figures 23 and 24, for example, IgG1Qd585 has sufficient absorbance at around 320 nm or 450 nm, but at these wavelengths, IgG2AF594 is hardly excited. On the other hand, the fluorescence of IgG1Qd585 (fluorescence 1) peaks at 585 nm, and if the two molecules are close enough together and the FRET phenomenon occurs, IgG2AF594 is excited and emits fluorescence (fluorescence 2) with a peak at 620 nm. If fluorescence 2 is observed at around 650 nm, it can be separated from fluorescence 1. Furthermore, fluorescence 2 has a sufficient Stokes shift of 320 nm and 450 nm, which are the excitation light, and can be completely separated using an optical filter, etc.

[0091] Preparation of reaction mixture The prepared IgG1Qd585 and IgG2AF594 were desalted by dialysis against non-ionized water containing 0.02% sodium azide as a stabilizer, and a reaction solution containing both molecules was then prepared. Normally, a buffer such as PBS is used when carrying out an antigen-antibody reaction, but considering the subsequent dielectrophoresis, it is preferable for the buffer to have a low conductivity, and a suitable example of the buffer is non-ionized water containing 0.02% sodium azide. At this time, it is preferable that the concentrations of IgG1Qd585 and IgG2AF594 are high, as long as the background fluorescence 1 and fluorescence 2 are not too high. One suitable example is 10 -7 It's M.

[0092] Embodiment 7(1) Based on the configuration diagram of FIG. 17, detection is performed in the following steps. 1) Approximately 100 μL of saliva is collected as a sample, which is mixed with 500 μL of a reaction solution and stirred, preferably with a vortex. 2) The mixture obtained in 1) is introduced into the detection cell. 3) The image information obtained from the image sensor is stored as a control. 4) A voltage of 5 V and 2 MHz is applied to the microelectrode. At the same time, the vibration element in contact with the detection cell is operated to actively stir the reaction solution by convection, particularly in the depth direction. 5) With the voltage for dielectrophoresis and the transducer still operating, the image information obtained from the image sensor after two minutes is saved as measurement data. 6) Subtract the control from the measured data to obtain a difference image. 7) The obtained differential image data is processed by software to measure the amount of fluorescence collected along the microelectrode. At this time, the image may be cut out by software along the electrode shape and contrast may be enhanced.

[0093] Embodiment 7(2) Based on the configuration diagram of FIG. 19, detection is performed in the following steps. 1) Approximately 100 μL of saliva is collected as a sample, which is mixed with 500 μL of a reaction solution and stirred, preferably with a vortex. 2) The mixture obtained in 1) is introduced into the detection cell. 3) The image information obtained from the image sensor is stored as a control. 4) A voltage of 5 V and 2 MHz is applied to the microelectrode. At the same time, the vibration element in contact with the detection cell is operated to actively stir the reaction solution by convection, particularly in the depth direction. 5) With the voltage for dielectrophoresis and the transducer still operating, the image information obtained from the image sensor after two minutes is saved as measurement data. 6) Subtract the control from the measured data to obtain a difference image. 7) The obtained differential image data is processed using software to measure the amount of fluorescence collected along the microelectrode.

[0094] Embodiment 7(3) Based on the configuration diagram of FIG. 20, detection is performed in the following steps. 1) Approximately 100 μL of saliva is collected as a sample, which is mixed with 500 μL of a reaction solution and stirred, preferably with a vortex. 2) The mixture obtained in 1) is introduced into the detection cell. 3) The image information obtained from the image sensor is stored as a control. 4) A voltage of 5 V and 2 MHz is applied to the microelectrode. At the same time, the vibration element in contact with the detection cell is operated to actively stir the reaction solution by convection, particularly in the depth direction. 5) With the voltage for dielectrophoresis and the transducer still operating, the image information obtained from the image sensor after two minutes is saved as measurement data. 6) Subtract the control from the measured data to obtain a difference image. 7) The obtained differential image data is processed by software to measure the amount of fluorescence collected along the microelectrode. At this time, the image may be cut out by software along the electrode shape and contrast may be enhanced. In embodiment 7(3), light reaches only pixels in the vertical direction from the light point, so that an image is formed with a certain resolution. Although the luminous intensity is reduced by providing a slit, this can be compensated for by increasing the sensitivity of the image sensor or by lengthening the exposure time. As shown in FIG. 21, when scanning in the X direction at an arbitrary Y position, a peak is obtained along the electrode as shown. In addition, the value obtained by integrating this value in the X direction is Σx. When scanning in the Y direction at an arbitrary X position, there is a place in the Y direction where a high value continues as shown in the figure. If the value integrated in the Y direction at this place is Σy, then it is clear that Σy>Σx. This makes it possible to evaluate the fluorescence value more accurately. Note that the term "scanning" used in this embodiment does not refer to mechanical movement, but rather refers to the direction of software calculation in which the output of all pixels is actually treated as an image. [Industrial Applicability]

[0095] The detection method of the present invention is useful for testing for infectious diseases that were previously impossible to detect, for on-site testing where testing was necessary but not previously possible, and for testing for items that, in principle, were impossible to test by genetic testing. [Explanation of symbols]

[0096] 41 Substrate 42 Microelectrodes 43 Spacer 44 Cover 45 Capillary 46 Air Hole 47 Electrode 1 48 Electrode 2 4A Sample introduction direction 4B Microelectrode Enlarged View 61 Detection Cell 62 Transducer 63 Light source 64 Excitation Light 65 Lens 66 Fluorescence 67 Optical Filter 68 Light Sensor 69 Terminals 111 Microelectrodes 112 Substrate 113, 113' Terminal 114 Coverslip 115 Spacer 121 Detection Cell 122 LED 123 Color Filter 124 Sensors 151 Detection Cell 152 Microelectrodes 153 LED 154 Color Filter 155 Sensors 156 High Frequency 161 Inspection container 162 Reaction Solution 163 Aperture plate 164 Swab 165 Microelectrodes 201 Microelectrodes 202 Insulating film 203 Image Sensor 211 Microelectrodes 212 Insulating film 213 Image Sensor 214 Detection Cell 215 Samples 221 Microelectrodes 222 Image Sensor 223 Detection Cell 231 Microelectrodes 232 Slit 233 Image Sensor 241 Microelectrodes 242 Image Sensor 243 LED 244 light guide plate

Claims

1. A method for detecting a target substance in a sample, comprising: a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; a step of mixing a target substance in a sample, a target substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a target substance recognition material labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength, thereby binding the target substance to the target substance recognition material; measuring a change in fluorescence intensity of a complex between a detection target substance and a detection target substance recognition material in a sample using an image sensor; The target substance recognition material specifically recognizes and binds to the target substance, The step of measuring the change in the fluorescence intensity by the image sensor includes: a sample being present in a detection cell having an electrode provided inside a bottom portion of the detection cell; (1) Place a transparent insulating film on the image sensor to measure the fluorescence, or (2) Measure the fluorescence by placing an image sensor on the outside of the bottom of the detection cell, or (3) The electrodes have anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrodes is provided on the image sensor to measure the fluorescence, or (4) irradiating the bottom of the detection cell with excitation light and measuring the fluorescence by providing an image sensor on the side of the detection cell; A fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that is excited by light of wavelength 1 to emit fluorescence at wavelength 2, and a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength is a fluorescent substance that absorbs light of wavelength 2 to emit fluorescence at wavelength 3, and the fluorescence intensity of wavelength 3 is measured by exciting the fluorescent substance with light of wavelength 1; or A fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that emits fluorescence at wavelength 2 when excited by light of wavelength 1, a quenching substance that absorbs the fluorescence at the specific wavelength is a quenching substance that absorbs light of wavelength 2, and the fluorescence intensity of wavelength 2 is measured by exciting the fluorescent substance with light of wavelength 1; and

2. A method for detecting a target substance in a sample, comprising: a step of locally concentrating the target substance, the target substance recognition material, or a combination of the target substance and the target substance recognition material in the sample by electrophoresis or dielectrophoresis; mixing a substance to be detected in a sample, a detection substance recognition material labeled with a fluorescent substance that emits fluorescence at a specific wavelength, and a substance to be recognized by the detection substance recognition material, the substance being labeled with a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength or a quenching substance that absorbs fluorescence at the specific wavelength; measuring a change in fluorescence intensity of a complex between a detection target substance and a detection target substance recognition material in a sample using an image sensor; The target substance recognition material specifically recognizes and binds to the target substance, The step of measuring the change in the fluorescence intensity by the image sensor includes: a sample being present in a detection cell having an electrode provided inside a bottom portion of the detection cell; (1) Place a transparent insulating film on the image sensor to measure the fluorescence, or (2) Measure the fluorescence by placing an image sensor on the outside of the bottom of the detection cell, or (3) The electrodes have anisotropy in the X and Y directions, and a slit having the same anisotropy as the electrodes is provided on the image sensor to measure the fluorescence, or (4) irradiating the bottom of the detection cell with excitation light and measuring the fluorescence by providing an image sensor on the side of the detection cell; A fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that is excited by light of wavelength 1 to emit fluorescence at wavelength 2, and a fluorescent substance that emits fluorescence at a wavelength different from the specific wavelength is a fluorescent substance that absorbs light of wavelength 2 to emit fluorescence at wavelength 3, and the fluorescence intensity of wavelength 3 is measured by exciting the fluorescent substance with light of wavelength 1; or A fluorescent substance that emits fluorescence at a specific wavelength is a fluorescent substance that emits fluorescence at wavelength 2 when excited by light of wavelength 1, a quenching substance that absorbs the fluorescence at the specific wavelength is a quenching substance that absorbs light of wavelength 2, and the fluorescence intensity of wavelength 2 is measured by exciting the fluorescent substance with light of wavelength 1; and

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