Fluorescence counting and quantitative device

Abstract

To provide a quantifying device that performs fluorescence counting of the amount of antigens or antibodies in a sample by using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS).SOLUTION: A measuring device executes a method for performing fluorescence-labeling of an antigen or an antibody that is a measurement object in a sample, capturing the antigen or antibody by an antigen-antibody reaction using a substrate solid-phased with plasmon metal complex quantum crystal, enhancing labeled fluorescence formed by excitation light by the surface plasmon enhancement effect of the plasmon metal complex quantum crystal, and performing fluorescence counting and quantify the labeled fluorescence.SELECTED DRAWING: Figure 19B

Description

Technical Field

[0001] The present invention relates to a novel fluorescence counting method in which solidification is carried out by a quantum crystal aggregation method, Using the resulting solid-phase substrate, a fluorescently labeled antigen or antibody, which is captured in an antigen-antibody reaction, is used. fluorescence is exhibited on a fluorescence screen by a surface plasmon excitation method, and quantification is carried out by fluorescence point counting. Quantitative device It relates to.

Background Art

[0002] Currently, gene testing by the PCR method is the mainstream for virus testing. It is a test that collects mucus and sputum from the nose and the back of the throat and examines proteins such as antigen viruses contained therein. This PCR method is a highly accurate method that collects a sample, amplifies the gene contained in the sample, and examines whether it matches a specific gene sequence. However, this method requires sophisticated pretreatment technology and sophisticated testing equipment, and the time required for testing is about 6 hours or more. Therefore, a simple and rapid gene amplification method is desired, and the LAMP (Loop-Mediated Isothermal Amplification) method has been proposed. However, since the PCR method only uses gene amplification, it is not suitable for on-site testing that requires rapidity. Moreover, the PCR method is a qualitative determination of positive or negative, and has a drawback in terms of quantification.

[0003] Therefore, as a supplement to the PCR method, simple and rapid serological diagnostic methods using immunochromatography or enzyme immunosorbent assay (ELISA) to detect virus-specific antibodies in serum have been proposed. In the case of typical acute viral infections, antibodies in the blood are induced about a week after the onset of symptoms. Therefore, in this type of serological diagnosis, it is necessary to measure the blood antibody titer during the acute and recovery phases of the disease and compare the changes in antibodies. Thus, it is relatively difficult to incorporate serum-specific antibody detection methods into diagnostic methods for acute viral infections, which require testing and diagnosis as soon as possible after the onset of symptoms. However, blood samples required for serological diagnosis are relatively easy to collect, and the risk of secondary infection to healthcare workers during sample collection is relatively low. Furthermore, virus-specific antibody detection methods using immunochromatography can be qualitatively analyzed by visual judgment, so they do not require special equipment and can be tested quickly and easily in outpatient settings or at the bedside, making their introduction into clinical practice as soon as possible desirable. However, in the case of COVID-19, it is currently difficult to detect virus-specific antibodies in the serum of COVID-19 patients up to 6 days after symptom onset. Furthermore, it has been revealed that the detection rate remains at only about 20% even in serum one week after symptom onset. Moreover, the antibody positivity rate increases over time, and after 13 days from symptom onset, while IgG antibodies in the serum are positive in most patients, the detection rate of IgM antibodies is low, and there are many cases where only IgG antibodies are positive. For this reason, it is considered necessary to evaluate paired serum samples—sera up to 6 days after symptom onset and serum from 13 days after symptom onset—for serological diagnosis of COVID-19 using this kit. Furthermore, non-gene amplification antibody testing methods may not be able to rule out non-specific reactions, and the interpretation of the results lacks reliability, requiring careful consideration based on a comprehensive assessment of multiple test results and clinical symptoms.

[0004] In the current situation, virus testing requires accuracy comparable to the PCR method, which amplifies genes, and rapid testing equivalent to immunochromatography. Therefore, the inventors have diligently conducted research to realize a method that can achieve both accuracy comparable to PCR and rapid testing equivalent to immunochromatography. There is a method called immunofluorescence that uses antibodies that specifically recognize antigens in tissues and cells to investigate the distribution of those antigens. By sequentially using primary and secondary antibodies, the distribution of primary antibodies in tissues and cells, that is, the distribution of the antigens they recognize, can be observed as the distribution of fluorescently labeled secondary antibodies. However, in order to use this method outside of tissue and cell systems, the virus in the sample collected from the patient must be immobilized outside of the tissue and cell system. Furthermore, even if the virus can be immobilized as a sample over time, it is easy for false samples to be present between the immobilized samples. This can trigger nonspecific reactions (a phenomenon in which some biological component other than the target of measurement causes an abnormal reaction with components such as the measurement reagent or additives in the blood collection tube, resulting in measurement values ​​that are far removed from the actual disease state), thus degrading the accuracy of the measurement. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent application No. 2020-74439 [Non-patent literature]

[0006] [Non-Patent Document 1] KONIKAMINORUTA Report 2012 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to realize a method and apparatus that possesses accuracy comparable to the PCR method described above, while simultaneously enabling a simple and rapid test equivalent to the immunochromatography method described above. To solve this problem, the inventors conducted extensive research. As a result, they found that by using a method of agglomerating quantum crystals of a plasmon metal complex that excites and enhances surface plasmons, the virus is simultaneously agglomerated during the aggregation of the quantum crystals, and the virus aggregates and disperses together with the quantum crystals to become a solid phase on a metal substrate. Due to the surface plasmon enhancement effect of the aggregated plasmon metal complex, the fluorescence of the labeled virus antibody appears as dots or granules in the fluorescence image, and the number of viruses can be counted as dot fluorescence counts (hereinafter referred to as the fluorescence counting method). Furthermore, they found that nonspecific reactions can be eliminated or reduced in this fluorescence counting method, and the accuracy is significantly improved. [Means for solving the problem]

[0008] The present invention is based on the above findings and is a novel fluorescence counting system for quantifying a sample (virus or antibodies produced by it in the immune function of a human or animal) in a fluorescence image using fluorescence dots or particles. The system involves aggregating an inactivated virus or its antibody onto a metal substrate with a plasmon metal complex using an electrode potential difference, and then immobilizing it on the metal substrate with a metal complex quantum crystal to form a solid phase. Solid-phase substrate Then, the immobilized virus or immobilized antibody is labeled using an antigen-antibody reaction. means The fluorescence points or particles in the fluorescence image obtained by surface plasmon excitation of a quantum crystal are binarized, and the fluorescence points or particles that exceed a predetermined threshold are counted. meansThe fluorescence counter system is characterized by consisting of the following. In the present invention, in addition to the sandwich method, which utilizes the so-called antigen-antibody reaction in which the antigen is sandwiched between the antibody and the labeled antibody, the following methods are used: 1) the direct method, in which the virus and antigen are immobilized together with the antibody (usually diluted with buffer, the same applies hereinafter) (a state in which the antibody is aggregated on a metal substrate by the electrode potential difference with the metal substrate using the quantum crystal aggregation method), and then the virus antigen (usually inactivated with ethanol, etc., and may also be diluted with buffer, the same applies hereinafter) and the labeled antibody (labeled with a phosphor, usually diluted with buffer, the same applies hereinafter) are mixed and dropped onto the immobilized substrate, in which the antigen is sandwiched between the antibody and the labeled antibody. [Effects of the Invention]

[0009] Firstly, this invention efficiently enhances fluorescence by simultaneously immobilizing a virus or antibody-associated labeled fluorescent molecule captured by an antigen-antibody reaction using a plasmon metal complex, thereby enabling the quantitative determination of viruses and other samples as fluorescence points. Unlike conventional surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) immunoassay (Non-Patent Literature 1), which detects the fluorescence signal excited by localized field light induced on the surface of a gold film electrode, this method allows for the accurate quantification of viruses and antibodies by binarizing the fluorescence signal as points or granules in the fluorescence image observed with a fluorescence microscope and counting (counting) the fluorescence points or granules above a certain threshold, which correlates with the number of viruses and antibodies. This means that accurate quantification is possible, unlike conventional methods that measure fluorescence intensity. Furthermore, unlike qualitative PCR methods that only determine positive or negative results, this is a valuable quantitative test that can provide information on the onset, progression, and recovery status of a disease. In addition, unlike immunochromatography, which tests for immune antibodies, infection can be determined quickly and accurately by quantifying the amount of virus using fluorescence counting. Furthermore, compared to solid-state substrates excited by localized field light induced on the gold film electrode surface, this method eliminates the need for microchannels and also eliminates the false nature of fluorescence signals due to nonspecific reactions.

[0010] The difference between conventional fluorescence spectroscopy (SPFS) and the fluorescence counting method of the present invention lies in the fact that in the former, the sample, such as a virus, is immobilized on a gold thin film using organic molecules, while in the latter, it is formed and integrated by the aggregation of plasmon metal complex quantum crystals. Sample immobilization techniques are usually complex, and SPFS measurement, which is a substrate-type reaction field, is disadvantageous in terms of reaction efficiency. Microchannels are applied as a highly efficient reaction acceleration technique. However, the use of these microchannels makes surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) measurement complex and difficult. The present invention enables simple and rapid immobilization of viruses necessary for measurement by the aggregation of plasmon metal complex quantum crystals. That is, it provides a novel method that facilitates the immobilization of antibodies or antigens in the reaction field and enables highly reproducible surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) without the use of microchannels (Patent Document 1).

[0011] In other words, the inventors have found that this fluorescence counting method is excellent in quantitative detection of viruses. For example, when performing fluorescence spectroscopy on influenza virus using an antigen-antibody reaction (sandwich method), unlike conventional solid-phase deposition, the measured image, as shown in Figure 6, shows that when a virus is present, it is sandwiched between solid-phase antibody and labeled antibody on the quantum crystal, emitting numerous granular fluorescence. This granular fluorescence is the fluorescence of the labeled antibody that has sandwiched the virus, and counting the number of fluorescent particles above a certain threshold correlates with the number of viruses (Figure 6(a)). On the other hand, the inventors have newly discovered that when there is no virus, the numerous granular fluorescence do not appear (Figure 6(b)). Therefore, in further examination of the present invention, the following features were found. A plasmon metal complex in solution aggregates as a quantum crystal of the metal complex on a metal substrate having an electrode potential near the reduction potential, depending on the selection of the electrodeposition substrate potential (hereinafter referred to as the quantum crystal aggregation method). At that time, if an antigen or antibody is present in the solution, the antigen or antibody aggregates on the substrate or particles together with the metal complex, forming a solid-phase plasmon reaction field. Therefore, unlike conventional plasmon metal thin films, metal complex crystals of approximately 100 nm are arranged regularly, and antigens or antibodies are physically or chemically immobilized between these quantum crystals at regular intervals. This creates a structure similar to that of a microchannel, enabling surface plasmon excitation enhancement. As a result, in this surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) method, counting the number of granular fluorescence particles observed with a fluorescence microscope is effective for analyzing diseases by quantifying the number of viruses based on the presence or absence of viruses and the count.

[0012] The present invention provides a novel sample-immobilized fluorescence counting method for surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS), which excels in image retrieval observed with a fluorescence microscope and allows for the analysis of diseases by counting the number of granular fluorescence particles in the fluorescence image, determining the presence or absence of viruses and the number of counts.

[0013] In this invention, a plasmon metal complex is immobilized together with an antibody using a quantum crystal aggregation method to provide a surface plasmon excitation effect. Upon irradiation with excitation light, the fluorescence of the labeled complex, which is surface plasmon excited, can be observed as granular fluorescence in the fluorescence image, and the number of granular fluorescence particles can be detected as the amount of virus. Here, the quantum crystal aggregation method is a method in which a plasmon metal complex in solution aggregates as a quantum crystal of the metal complex on a metal substrate having an electrode potential near the reduction potential, depending on the selection of the electrodeposition substrate potential (hereinafter referred to as the quantum crystal aggregation method). At that time, if an antigen or antibody is present in the solution, the antigen or antibody aggregates on the substrate or particles together with the metal complex, forming a solid-phase plasmon reaction field. This aggregation method involves regularly arranged metal complex crystals of about 100 nm in size, with the antigen or antibody physically or chemically immobilized between these quantum crystals at regular intervals (see Japanese Patent Application Publication No. 2016-197114). In particular, this invention uses not only an Ag reagent (an aqueous solution of silver complex containing an aqueous solution of silver thiosulfate: typically a 1000-5000 ppm aqueous solution of silver thiosulfate with a pH of approximately 5), but also a buffer solution containing inactivated virus or antibody (typically a phosphate buffer solution with a pH of 7 or higher) mixed with this reagent. The resulting mixture typically shifts from neutral to weakly alkaline, and the buffering action causes the aggregation of quantum crystals and the sample on the metal substrate to tend to disperse. Furthermore, the pharyngeal swab samples used for PCR testing are collected using commercially available kits (swab swab and container with culture medium). Copan UTM is one example. The liquid culture medium of Copan UTM contains HEPES buffer, as well as sucrose and gelatin, making it suitable for the collection, storage, and transport of viruses, Chlamydia, Mycoplasma, and Ureaplasma. It also functions as a cryoprotective agent for clinical viruses, including cytomegalovirus and varicella-zoster virus. In this testing method, measurements are performed after inactivating the sample collected from a human. In this process, ethanol is usually used as the inactivation solution, but it is preferable to mix a buffer with the ethanol to inactivate the virus in a more stable manner. Specifically, a buffer (phosphate buffer pH 7.4) is mixed with 70% ethanol to create a 50% ethanol solution (buffer dilution), into which a human sample is immersed to inactivate the virus, and then used in this test method for detection.This allows the pH of inactivated samples collected from humans to be kept constant through the action of the buffer solution.

[0014] Furthermore, in another method of the present invention, the antigen can be immobilized using a quantum crystal aggregation method of plasmon metal complexes, the antigen and antibody can be reacted to form an antibody-immobilized substrate having gaps or microchannels between the quantum crystals, and a labeled secondary antibody labeled with a fluorescent substance can be further bound to it.

[0015] Furthermore, in this invention, metal powder may be used instead of a metal substrate. In this case, after washing, the remaining complex or labeled secondary antibody is irradiated with excitation light to surface plasmon-excite the quantum crystals, thereby enhancing the fluorescence of the complex or secondary labeled antibody. The fluorescence image is then observed, and the number of granular fluorescence particles in the image is counted for detection.

[0016] In the present invention, the antigen is usually sandwiched between fluorescently labeled antibodies (primary antibodies) during production. However, by capturing the antigen with a fluorescently labeled primary antibody and a fluorescently labeled secondary antibody, and then imaging and analyzing the fluorescence, it is possible to obtain fluorescence images more appropriately and accurately.

[0017] According to the present invention, the number of fluorescent particles of a viral antigen can be measured as the viral concentration, rather than the fluorescence intensity of the viral antigen. Moreover, since the quantum crystal forming the antibody or antigen-immobilized substrate has nm-sized gaps or microchannels between the quantum crystals, an interaction occurs between the photons incident by the excitation light and the free electrons of the plasmon metal particles forming the quantum crystal, causing surface plasmon excitation and enhancing the fluorescence of each complex or secondary labeled antibody. Therefore, instead of the overall fluorescence intensity, the granular fluorescence can be counted and detected with good reproducibility. Consequently, surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) can be used to perform rapid testing in a short time of 2 to 5 minutes, providing a highly accurate diagnostic result that replaces PCR testing, which has complicated pretreatment, low sensitivity due to primers, many protocols, and a long time to perform the test. Furthermore, it is groundbreaking because it can not only determine the presence or absence of disease, but also determine the severity of the disease, as the count corresponds to the number of viruses.

[0018] According to the present invention, it is possible to provide an antibody test method that captures antibodies produced in the body that are effective against a specific virus. In this method, an antigen is captured by a primary antibody and a fluorescently labeled secondary antibody. When the primary antibody or the labeled secondary antibody is bound to the primary antibody or the labeled primary antibody, the fluorescence becomes stronger. Therefore, by adding the labeled secondary antibody, it becomes possible to detect fluorescence with higher sensitivity.

[0019] (Embodiment 1) A method for quantifying the target of measurement, namely, 1) inactivated viruses and antibodies The process consists of: 1) creating a solid-phase substrate by immobilizing Rus or its antibody; 2) a labeling step in which the immobilized virus or antibody is fluorescently labeled by an antigen-antibody reaction; 3) a fluorescence excitation step in which the fluorescently labeled virus or antibody is irradiated with excitation light to obtain a point-like fluorescence image of the fluorescently labeled virus or antibody by surface plasmon excitation; and 4) a fluorescence counting step in which the fluorescent points or particles in at least one field of view of the fluorescence image are binarized, and fluorescent points or particles above a predetermined threshold are selected and counted quantitatively. The number of fluorescent points correlates with the virus in the sample (Figures 7 and 8). (Embodiment 2) In the solid-phase formation step, inactivated virus or its antibody is collected in a buffer solution, mixed with an aqueous solution of a plasmon metal complex at 1000 ppm to 5000 ppm, preferably 1000 to 3000 ppm, to make it neutral, and then dropped onto a metal substrate. The virus in the sample is uniformly dispersed and solid-phase formed in the fluorescence image, and accurate measurement can be performed by measuring one field of view without even calculating the average value of two or more fields of view (Fig. 16). (Embodiment 3) The solid-phase formation target in the sample can be an inactivated virus that produces an antibody or its antibody, and the concentration in the sample can be 10 μg / ml or more. The sensitivity of the solid-phase formation substrate is improved by an increase in the antibody concentration to be solid-phase formed (Fig. 7). (Embodiment 4) In the fluorescence labeling in the present invention, the sandwich method is generally used. However, the virus antigen is solid-phase formed and then labeled with a labeled antibody, or the antibody is solid-phase formed and then labeled with a labeled antigen (labeled with a phosphor and including those that label a part of the antigen. The same applies hereinafter). 2) The direct method, or the virus antigen is solid-phase formed and then the antibody and the secondary antibody are sequentially bound for labeling, or the antibody is solid-phase formed and then the virus antigen is bound, and finally the antibody and the secondary antibody are sequentially bound for labeling. 3) The indirect method can also be used. The virus antigen is usually inactivated with ethanol or the like and preferably diluted with a buffer solution. The labeled antibody is labeled with a phosphor and is preferably diluted with a buffer solution usually. (Embodiment 5) When an excitation light is irradiated onto a plasmon metal complex quantum crystal aggregate (Fig. 9-1) of about 100 nm that has aggregated with an antigen or an antibody on a metal substrate, a surface plasmon excitation phenomenon occurs due to the quantum crystal, and the fluorescence labeling of the virus or its antibody solid-phase formed together with the quantum crystal is excited. As a result, measurement with less non-specific reaction is achieved, and the number of dot-like fluorescences having a luminance value equal to or higher than a predetermined threshold value is accurately obtained by surface plasmon excitation, which has a correlation with the virus or antibody concentration, enabling quantitative measurement (Fig. 11(b)). (Embodiment 6) In the fluorescence counting step according to the present invention, the quantification in one-field measurement can obtain a result equivalent to the average value of two or more field measurements. As a result, rapid quantitative measurement becomes possible (Figure 16). (Embodiment 7) In the solid-phase immobilization step, an antibody that binds to two or more different viruses through an antigen-antibody reaction is immobilized, and in the labeling step, it is labeled with a labeled antibody having a different fluorescence wavelength, so that quantification of two or more viruses in a specimen can be performed in one measurement (Figures 19A and B). (Embodiment 8) When Embodiment 7 is applied to influenza and the Covid-19 virus, the viruses can be detected separately in one measurement (Figures 19A and B).

Brief Description of the Drawings

[0020] [Figure 1] It is a schematic diagram consisting of steps (1) to (4) of the first method (sandwich method) of the present invention. [Figure 2A] In the first method of the present invention, it is a schematic diagram consisting of steps (1) to (3) when using metal powder instead of a metal substrate. [Figure 2B] In the first method of the present invention, it is a schematic diagram consisting of steps (4) to (5) when using metal powder instead of a metal substrate. [Figure 3] It is a schematic diagram consisting of steps (1) to (4) of the second method (indirect method) of the present invention. [Figure 4] It is a process diagram showing a method for manufacturing a quantum crystal substrate of the present invention. [Figure 5] It is a schematic diagram showing a virus detection method using a primary labeled antibody and a secondary labeled antibody that can bind to each other as labeled antibodies in the first and second methods of the present invention. [Figure 6] It is a fluorescence image (a) when there is an influenza virus and a fluorescence image (b) when there is no influenza virus. [Figure 7] It is a graph when quantifying the influenza virus using the fluorescence count number of the present invention. [Figure 8] It is a process explanatory diagram showing an analysis method of the fluorescence image of the present invention. [Figure 9-1] The images show 25,000x magnification SEM images of quantum crystal solid-phase substrates: (a) shows the solid-phase substrate when using 2000 ppm Ag reagent (aqueous solution of silver thiosulfate) and phosphate buffer; (b) shows the solid-phase substrate when using 2000 ppm Ag reagent and phosphate buffer containing influenza antibody (50 μg / ml); and (c) shows the solid-phase substrate when using 2000 ppm Ag reagent and phosphate buffer containing influenza virus (50 μg / ml). [Figure 9-2] The graphs show the quantum crystal state and component analysis results for each solid-phase substrate, including SEM images and component analysis graphs of the solid-phase substrates when using Ag reagent (silver thiosulfate aqueous solution) at 4000 ppm and phosphate buffer. [Figure 9-3] These are SEM images and component analysis graphs of a solid-phase substrate when using 4000 ppm Ag reagent and phosphate buffer containing influenza antibody (50 μg / ml). [Figure 9-4] These are SEM images and component analysis graphs of a solid-phase substrate when using Ag reagent at 4000 ppm and phosphate buffer containing influenza virus (50 μg / ml). [Figure 10] This image shows a diagram (a) illustrating the method for preparing an influenza antibody-immobilized substrate, and a clear view image (b) of the immobilized substrate. [Figure 11A] This is a test process diagram for the sensitivity of a solid-phase substrate. [Figure 11B] This graph shows the correlation between the concentrations of FITC influenza antibodies and the number of fluorescence spots in the fluorescence image. [Figure 12] This is an illustrative diagram showing the relationship between the immobilization of influenza antibodies and the quantum crystal concentration. [Figure 13] This is a process diagram for a method of fluorescence counting using the present invention method (direct method) after immobilizing inactivated influenza virus antigen onto a solid phase. [Figure 14] This is a process diagram showing a method for fluorescence counting of inactivated influenza virus antigen using the present invention method (sandwich method). [Figure 15] This is a process diagram showing a method for fluorescence counting of inactivated Covid-19 virus using patient samples with the method of the present invention (sandwich method). [Figure 16] This is a schematic diagram illustrating the analytical method of the quantum crystal solid-phase fluorescence counting method of the present invention. [Figure 17A] This is a schematic diagram of steps (1) to (4) of the third method (direct method) using the inactivated specimen of the present invention. [Figure 17B] This is a schematic diagram showing steps (5) to (7) of the third method of the present invention. [Figure 18A] This is a schematic diagram showing the collection process (1) to (3) using the sample inactive collection kit of the present invention. [Figure 18B] This is a schematic diagram showing the collection process (4) to (6) using the sample inactive collection kit of the present invention. [Figure 19A] This is a schematic diagram showing the (1) immobilization step and (2) the first half of the fluorescent labeling step when detecting two types of viruses using the sandwich method of the present invention. [Figure 19B] This is a schematic diagram of the sandwich method of the present invention for detecting two types of viruses, consisting of (3) the latter half of the fluorescent labeling step and (4) the surface plasmon excitation step. [Modes for carrying out the invention]

[0021] In this invention, a quantum crystal aggregation method is used to form the following multiple materials on a predetermined metal substrate and metal powder. Using such materials, a solid-state substrate can be easily created.

[0022] (Quantum crystal aggregation reaction) When agglomerating silver complex quantum crystals as a solid-state substrate, it is preferable to use copper and copper alloy substrates, particularly phosphor bronze substrates, as the aggregation substrate. The substrate having a plasmon metal quantum crystal region used in the method of the present invention is called a biochip. Its manufacturing method is as follows. 1) A metal complex aqueous solution is chemically reduced by electrode potential difference on a metal substrate with an electrode potential lower than the metal forming the complex (higher ionization tendency) to aggregate quantum crystals (nano-sized metal complex crystals). In the case of silver complexes, quantum crystals of the silver complex are formed by electrodeposition electrolysis by agglomerating a silver thiosulfate aqueous solution on copper or a copper alloy with an electrode potential lower than silver (higher ionization tendency). Specifically, the concentration of the metal complex in the aqueous solution should be determined mainly considering the size of the quantum crystals to be formed, and when using a dispersant, its concentration should also be considered. Usually, it can be used in the range of 100 ppm to 1000 ppm, but depending on the virus containing the antigen used in the antigen-antibody reaction or the antibody produced by the virus in the immune reaction, it is preferable to use a quantum crystal aqueous solution of 500 to 1000 ppm, preferably 1000 to 5000 ppm, preferably 1000 to 300 ppm, to prepare nano-sized nanoclusters of 50 to 150 nm. Furthermore, in the present invention, the antigen and antibody to be immobilized are mixed with an inactivation solution and a buffer solution and aggregated together with the quantum crystal aqueous solution. Therefore, unlike aggregation from the quantum crystal aqueous solution alone, the quantum crystals tend to disperse on the immobilized substrate (see Figures 9-1(a), (b) and (c)). 2) The metal complex that forms the quantum crystal is selected to have a complex stability constant (logβ) greater than or equal to that shown by equation (I), which correlates with the electrode potential E of the supported metal. Equation (I): E° = (RT / |Z|F)ln(βi) (Here, E° represents the standard electrode potential, R represents the gas constant, T represents the absolute temperature, Z represents the ionic valence, and F represents the Faraday constant.) Here, if the metal complex is a plasmon metal complex selected from Au, Ag, Pt, or Pd, it has a localized surface plasmon resonance enhancement effect with respect to excitation light. In particular, when the metal complex is a silver complex, it is preferably formed by the reaction of a silver complexing agent with a stability constant (formation constant) (logβi) of 8 or higher with a silver halide. Silver chloride is preferred as the silver halide, and one of the complexing agents selected from thiosulfate, thiocyanate, sulfite, thiourea, potassium iodide, thiosalicylate, and thiocyanurate is preferred. The silver complex has quantum dots consisting of nanoclusters with an average diameter of 5 to 20 nm, and the size of the quantum crystal is 50 to 150 nm. (Investigation of solid-phase concentration, part 1)

[0023] In solid-phase deposition technology using quantum crystals, the concentration of the quantum crystal reagent (Ag reagent) is extremely important. Therefore, we solidified Biotin by varying the concentration of the quantum crystal reagent and detected the FITC-labeled Avidin using an Avidin-Biotin bond with a fluorescence microscope. FITC-Avidin VEC “FLUORESCEIN AVIDIN D” CatNo.A-2001 Biotin, Wakosha, "(+)-Biotin", CatNo. 023-08711 Solid-state substrates were prepared by solidifying Biotin (5 μg / ml) at quantum crystal concentrations of 1000, 2000, 3000, 4000, and 5000 ppm (solidification time: 1 minute). Next, FITC-Avidin (5 μg / ml) was dropped onto the Biotin-solid-state substrate, and the Avidin labeled with FITC using Avidin-Biotin bonding was measured using a Keyence fluorescence microscope "BZ-X710," and the average brightness value of the obtained fluorescence image was calculated (reaction time: 1 minute). As a result, we can conclude that at 1000 ppm (average image brightness value 54), 2000 ppm (69), 3000 ppm (62), 4000 ppm (59), and 5000 ppm (59), a large amount of Biotin solidified, and that the most FITC-Avidin bound occurred at 2000 ppm (the concentration after adding an equal amount of Biotin), which had the highest average brightness. This is likely because a smaller amount of quantum crystals results in a smaller amount of solidified Biotin. It is thought that when there is a large amount of quantum crystals, the solid-phase biotin becomes buried, resulting in reduced detection of FITC-Avidin. The instruments used to measure each quantum crystal are as follows: Equipment used Equipment: Keyence BZ-X710 fluorescence microscope Light source: 80W metal halide lamp Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer (Investigation of solid-phase concentration, part 2)

[0024] Next, we investigated the optimal concentration of quantum crystal reagent for detecting influenza virus using antigen-antibody reactions. We immobilized influenza antibodies using different concentrations of quantum crystals, and measured the influenza virus and FITC-labeled influenza antibodies using a fluorescence microscope with an antigen-antibody reaction. We then counted the fluorescence spots from the resulting fluorescence images (antigen-antibody reaction-sandwich method). Influenza antibody: Hytest "Monoclonal Mouse anti-influenza A haemogglutinin H1" Cat No. 3AH1 Influenza virus: HyTest Corporation "Influenza A(H1N1) virus" CatNo.IN73-3 FITC influenza antibody (ARP Corporation "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3 Equal amounts of influenza antibody (100 μg / ml) at quantum crystal concentrations of 2000, 4000, and 6000 ppm were mixed with buffer and dropped onto a metal substrate to create a solid-phase substrate (solid-phase deposition time: 1 minute). Next, a complex formed by mixing inactivated influenza virus (10 μg / ml) and FITC-labeled influenza antibody (25 μg / ml) was dropped onto the solid-phase substrate (reaction time: 1 minute). Unbound complexes and FITC antibodies were washed away with water or buffer. This chip was measured using a Keyence fluorescence microscope "BZ-X710," and the fluorescence spots above a predetermined threshold in the obtained fluorescence image were counted. As a result, similar to the Avidin-Biotin binding case, the highest number of complexes containing influenza virus could be detected when influenza was solidified with 2000 ppm quantum crystal (1000 ppm overall). Figure 12 shows images of the results when influenza antibodies are immobilized at quantum crystal concentrations at concentrations of 2000, 4000, and 6000 ppm. Quantum crystal concentration (ppm) and count 2000 (228 counts), 4000 (159 counts), 6000 (47 counts) Measurement conditions: Threshold 62, no blur filter, single field measurement with a 10x lens (where, as shown in Figure 16, single field measurement refers to a method of acquiring only a portion of the chip, unlike in the case of Japanese Patent Application No. 2019-234330, which leads to a reduction in measurement time). Equipment used Equipment: Keyence BZ-X710 fluorescence microscope Light source: 80W metal halide lamp Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer (Preparation of solid-phase substrate)

[0025] The present invention's solid-phase substrate differs in that, in addition to an aqueous solution of a quantum crystalline metal complex, it is mixed with an inactivation solution and / or buffer containing a virus and antibody containing the antigen to be solidified, and solidifies into a quantum crystal. However, it is basically possible to solidify antibodies or antigens using a quantum crystal agglutination method (Japanese Patent Publication No. 2016-197114) for producing quantum crystals of plasmon metal complexes. Therefore, the method described in Japanese Patent Publication No. 2016-197114 is cited and referenced in this specification. However, the virus and the antibodies it produces are in the inactivation solution or buffer. The substance is added, mixed with a plasmon metal complex reagent (e.g., an aqueous solution of silver thiosulfate) for solidification, and then added to a solidification substrate where it aggregates. Unlike the case of quantum crystals alone, the solidification of antigens and antibodies using quantum crystals is thought to be affected by interactions with buffers and changes in pH. Figure 9 shows SEM images (Figure 9-1(a)) of a control (Figure 9-1(a)) prepared by mixing an equal volume of phosphate buffer with 2000 ppm silver thiosulfate aqueous solution (Ag reagent). The phosphate buffer was then mixed with 50 μg / ml each of antibody (influenza antibody: ARP Corporation "Anti-Influenza A virus (H1N1) FITC") CatNo. 12-6250-3) and antigen (inactivated influenza antigen: influenza virus: HyTest Corporation "Influenza A (H1N1) virus", CatNo. IN73-3), and then mixed with an equal volume of 2000 ppm silver thiosulfate aqueous solution (Ag reagent). Figures 9-1(a) and 9-1(b) show these results. It can be seen that when buffer is added, the quantum crystals diffuse throughout the solution, and the antibody and antigen bind to these quantum crystals, becoming a solid phase. It is believed that the dispersion and aggregation of such quantum crystals creates a situation in which fluorescence spot counting is possible in fluorescence imaging. As a result, when a substrate on which antigens and antibodies are immobilized is labeled, irradiation with excitation light causes surface plasmon excitation, and the fluorescence of the complex can be observed as granular fluorescence in its fluorescence image. The number of granular fluorescence particles can then be detected as the amount of virus. This is because, as shown in Figures 9-2, 9-3, and 9-4, in addition to the Cu and Sn components of the phosphor bronze substrate, the Ag component was detected in each quantum crystal, confirming that solidification was carried out by the quantum crystalline silver complex. Note that antibodies and antigens were not detected. (Preparation of influenza antibody-immobilized substrates and sensitivity)

[0026] The above silver thiosulfate aqueous solution (Ag reagent) 2000 ppm (pH 5.2) and influenza antibody (50 μg / ml) 0.1 When an equal volume of mol / L phosphate buffer (pH 7.4) and the antibody (pH 7.2) was added to a phosphor bronze plate, it solidified in about 1 minute. The remaining liquid on the metal substrate was then blown off with air to obtain the antibody-solidified substrate (Figure 10(a)). Figure 10(b) is a clear image of the solidified substrate. Using the quantum crystal aggregation method described above, FITC-labeled influenza antibodies of varying concentrations were immobilized to create antibody-immobilized substrates, and the fluorescence points in the resulting fluorescence images were counted. FITC influenza antibody (ARP Corporation "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3) was used. Equal volumes of the above antibodies (250, 125, 62.5, 31.25 μg / ml) were mixed with a 2000 ppm silver thiosulfate aqueous solution (Ag reagent) and dropped onto a substrate to create various solid-phase substrates (Figure 11(a)). The required time was approximately 1 minute. The fluorescence sensitivity of these solid-phase substrates was measured using a fluorescence microscope (Keyence BZ-X710) as shown in Figure 11(b), by counting the fluorescence spots above a predetermined threshold. As a result, it was found that the count of the solid-phase substrates of the present invention increased in a concentration-dependent manner. That is, the antibodies are quantitatively immobilized on the solid-phase substrate according to the present invention and can be quantified as shown in Figure 12. Therefore, it can be seen that antigens and antibodies can be quantitatively immobilized according to the present invention. (Virus immobilization, part 1)

[0027] Figure 13 shows the direct method testing process for influenza virus (immobilization of inactivated influenza virus). In this method, an inactivated influenza virus is immobilized using quantum crystal agglutination, and an influenza antibody labeled with FITC is dropped onto the substrate. The fluorescence image obtained is then analyzed by counting the fluorescence spots (this is called the direct method using antigen-antibody reaction). Influenza virus: HyTest Corporation "Influenza A(H1N1) virus" CatNo.IN73-3 FITC influenza antibody (ARP Corporation "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3 An equal volume mixture (pH 7.4) of the above silver thiosulfate aqueous solution (Ag reagent) at 2000 ppm and early influenza virus (50 μg / ml) in phosphate buffer was added to a phosphor bronze plate. A Russ-based solid-phase substrate is prepared (solidification takes approximately 1 minute). For comparison, a virus-free buffer is mixed with a quantum crystal Ag reagent to form a solid-phase substrate. Next, FITC-labeled influenza antibody (25 μg / ml) is dropped onto the two solid-phase substrates (reaction time is only 1 minute). Unbound complexes and FITC antibodies are washed away with water or buffer. The remaining liquid on the metal substrate is blown off with air to obtain an antibody-based solid-phase substrate (Figure 10(a)). Figure 10(b) is a fluorescence image of the solid-phase substrate. (Immunization of antibodies, part 1)

[0028] Figure 14 shows the sandwich method testing process for influenza viruses (immobilization of influenza antibodies). Influenza antibodies are immobilized on a substrate using quantum crystal agglutination. FITC-labeled influenza antibodies and inactivated influenza viruses are then combined onto this substrate, and the fluorescence points obtained from the antigen-antibody reaction are counted. Influenza antibody: Hytest "Monoclonal Mouse anti-influenza A haemogglutinin H1" Cat No. 3AH1 Influenza virus: HyTest Corporation "Influenza A(H1N1) virus" CatNo.IN73-3 FITC influenza antibody (ARP Corporation "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3 An equal-volume mixture (pH 7.2) of 2000 ppm of the above-mentioned silver thiosulfate aqueous solution (Ag reagent) and influenza antibody (50 μg / ml) in phosphate buffer is added to a phosphor bronze plate to prepare an antibody-immobilized substrate (immobilization takes approximately 1 minute). Next, FITC-labeled influenza antibody (25 μg / ml) and influenza virus are mixed to form a complex, which is then dropped onto the immobilized substrate (reaction time is only 1 minute). Unbound complexes and FITC antibodies are washed away with water or buffer. This measurement chip is measured using a Keyence fluorescence microscope "BZ-X710," and the fluorescence points above a predetermined threshold in the obtained fluorescence image are counted. The measurement conditions and equipment used are the same as those for virus immobilization. (Immunization of antibodies, part 2)

[0029] Figure 15 shows the testing process. A complex of an inactivated COVID-19 patient sample and a FITC-labeled COVID-19 antibody is dropped onto a substrate on which a COVID-19 antibody has been immobilized using a quantum crystal, and the fluorescence points are counted from the resulting fluorescence image. COVID-19 antibody: GeneTex “SARS-COV-2 antibody spike” CatNo.GTX135356 FITC-labeled COVID-19 antibody: GeneTex's "SARS-CoV-2 spike antibody" CatNo. GTX135356 with FITC labeling (labeling index 8.64) An antibody-immobilized substrate is prepared by mixing an equal volume of COVID-19 antibody (50 μg / ml) with 2000 ppm quantum crystal reagent (silver thiosulfate) and dropping it onto a phosphor bronze substrate. Next, a pharyngeal swab obtained from a COVID-19 patient is inactivated with 70% ethanol, and a complex formed by mixing it with FITC-labeled COVID-19 antibody (34.5 μg / ml) is dropped onto the immobilized substrate. Unbound complexes and FITC antibodies are washed away with water or buffer solution. This measurement chip is measured using a Keyence BZ-X710 fluorescence microscope, and the fluorescence spots in the resulting fluorescence image that exceed a predetermined threshold are counted. The measurement conditions and equipment used are the same as those for virus immobilization, part 1. As a result, the virus was detected in both samples taken from pharyngeal swabs of two COVID-19 patients. The count number reflected the patients' symptoms. A 70% ethanol solution was used for the blank sample. The results were: Blank: count 8 (relative value 0), Sample 1: count 16 (relative value 8), Sample 2: count 51 (relative value 43). The relative value is calculated with the blank count set to 0. Even when the sample is saliva, the count is slightly lower, but the results are similar.

[0030] (Manufacturing of quantum crystals) When a 2000 or 4000 ppm aqueous solution of silver thiosulfate was prepared, one drop was placed on a phosphor bronze plate, left for about 1 minute, and then the solution was blown off, quantum crystals were observed to have formed in the SEM image. The photographs showing various SEM images of the nanoparticle aggregates (quantum crystals) produced in Example 1 show thin hexagonal prismatic crystals of about 100 nm, with surface irregularities on the order of several nanometers. Facets characteristic of metallic nanocrystals could not be confirmed. The correlation between the standing time after dropping onto the phosphor bronze plate and the shape of the quantum crystals is shown. First, hexagonal quantum crystals were formed and were observed to grow while maintaining their shape. In the graph showing the results of the EDS spectrum (elemental analysis) of the quantum crystals, silver and elements derived from complex ligands were detected in the crystals formed on the phosphor bronze plate. However, when a 1000 ppm aqueous solution of silver thiosulfate was prepared on a copper plate, one drop was placed on it, left for about 3 minutes, and then the solution was blown off, only silver was detected.

[0031] (Quantum crystal agglomeration theory) When a 2000 or 4000 ppm aqueous solution of silver thiosulfate complex is dropped onto a phosphor bronze plate and left for 1 minute, quantum crystals form in the shape of hexagonal prisms of approximately 100 nm. SEM images confirmed that each hexagonal prism quantum crystal has irregularities on the order of several nanometers. However, no facets characteristic of metal nanocrystals were observed, and EDS elemental analysis detected elements derived from silver and complex ligands. Therefore, it is presumed that the whole is a nanocrystal of the silver complex, and the irregularities appearing on its surface are caused by silver in the complex forming quantum dots as clusters and spreading out. Observing the phenomenon where the silver complex quantum crystals of the present invention are formed on a phosphor bronze plate, while only silver nanoparticles are deposited on a copper substrate, it is thought that only silver (0.80) is deposited on the copper substrate because the equilibrium potential of the silver thiosulfate complex is 0.33, which is equivalent to the electrode potential of copper (0.34). In the case of phosphor bronze, the electrode potential is slightly lower at 0.22, so it is thought that silver complex crystals are deposited. Therefore, in order to create quantum crystals, it is important that 1) the complex aqueous solution is in the dilute range of 500 to 2000 ppm, 2) the electrode potential of the supported metal is slightly negative relative to the equilibrium potential of the metal complex aqueous solution, and 3) the metal complex aggregates due to the electrode potential difference. However, it was found that for the immobilization of antigens and antibodies, it is preferable to use a thiosulfate Ag quantum crystal reagent with a higher concentration of 2000 ppm or more. The substrate can be polished with sandpaper to physically remove the surface oxide film, and then a silver thiosulfate solution can be dropped onto it to form a solid-state substrate through the aggregation of quantum crystals. The physical state of the substrate surface can affect the formation state of quantum crystals, which in turn can affect the measured values. Therefore, in order to keep the quantum crystal formation region constant, as shown in Figure 4, 1) a circular groove (etching) is made in the substrate liquid dropping region of the phosphor bronze plate, 2) the region is used as is or polished with sandpaper, electrolytically, or chemically, 3) an Ag reagent solution (2000-4000 ppm silver thiosulfate solution) is dropped onto it and the liquid accumulates in the circular groove due to surface tension, and 4) it is then removed to ensure the aggregation state of the quantum crystals. By observing the aggregation state and examining the variability of the measurement results, it was found that there was variability in the measurement results when using the substrate as is, electrolytically polished, chemically polished, or sandpaper polished.

[0032] (Target of solidification) The targets of immobilization include not only viruses, bacteria, and fungi that induce antibody production through the immune system of humans and animals, but also heavy metals and proteins. Furthermore, it includes antibodies produced by such viruses. Antibodies include monoclonal antibodies of five classes (IgA, IgD, IgE, IgG, IgM) from animal species such as rats, mice, chickens, rabbits, and humans; polyclonal antibodies containing the five classes (IgA, IgD, IgE, IgG, IgM) from animal species such as rabbits, guinea pigs, goats, sheep, rats, mice, and chickens; antibodies such as immunoglobulins; fragmented antibody parts such as Fc region, Fab region, heavy chain, light chain, antigen-binding site, and hinge region; recombinant antibodies and fragmented parts; and human antibodies obtained by infecting human B lymphocytes with a virus (such as EBV), proliferating them, and cloning the antibody gene.

[0033] Viruses include animal viruses that infect animals, such as coronaviruses, influenza viruses, avian influenza viruses, and adenoviruses; plant viruses that infect plants, such as tobacco mosaic virus; and bacterial viruses that infect bacteria, such as bacteriophages. It also includes fragmented parts of viruses, such as spikes on the surface of viruses and nucleocapsids, as well as recombinant viruses and fragmented parts.

[0034] In this invention, specimens collected from humans or animals, and specimens containing inactivated viruses or fragmented parts are included as objects to be immobilized, and bodily fluids collected from humans or animals, such as pharyngeal swabs, saliva, blood, and urine, are included as specimens.

[0035] (Example 1 of solid-state substrate) Examples of solidifying a quantum crystal with biotin, which has the ability to bind to avidin, are given. A solution is prepared by mixing an Ag reagent (1000 ppm, 20 μl) for creating quantum crystals with biotin (5 μg / ml, 20 μl), and this solution is dropped onto a phosphor bronze plate, allowing the biotin to solidify onto the purified quantum crystals on the substrate. Next, avidin labeled with FITC, which has the ability to bind to biotin, is dropped onto a biotin-solid-state substrate. The biotin and the FITC-labeled avidin then bind, and fluorescence microscopy reveals the fluorescence of granular FITC. This demonstrates that by mixing quantum crystals and biotin and dropping them, a solid-state substrate with biotin solidified on the quantum crystals can be formed. From this, it was found that molecular compounds and other materials can be solidified on a quantum crystal substrate. (The Ag reagent ranges from 500 ppm to 10000 ppm, and the biotin that is immobilized ranges from 1 pg / ml to 1 g / ml.)

[0036] (Example 2 of solid-state substrate) A solution was prepared by mixing equal volumes of Ag reagent (2000 ppm, 12.5 μl) for creating quantum crystals and hemagglutinin H1 influenza A antibody (25 μg / ml, 12.5 μl). This solution was dropped onto a phosphor bronze plate, and the hemagglutinin H1 influenza A antibody was immobilized onto the purified quantum crystals on the substrate. Next, a complex of H1N1 influenza virus, which binds to the hemagglutinin H1 influenza A antibody in an antigen-antibody reaction, and H1N1 influenza virus antibody labeled with FITC was dropped onto the hemagglutinin H1 influenza A antibody-immobilized substrate. The complex of influenza virus and FITC-labeled antibody then bound to the immobilized antibody on the substrate, and fluorescence microscopy revealed granular fluorescence from FITC. As a result, it was found that by mixing quantum crystals and hemagglutinin H1 influenza A antibody and dropping them, an immobilized substrate was created in which the hemagglutinin H1 influenza A antibody was immobilized onto the quantum crystals. This indicates that antibodies and other substances can be immobilized on quantum crystal substrates. (The Ag reagent ranges from 500 ppm to 10000 ppm, and the immobilized hemagglutinin H1 influenza A antibody ranges from 1 pg / ml to 1 g / ml.)

[0037] (Example 3 of solid-state substrate) Next, a solution is prepared by mixing equal volumes of Ag reagent (4000 ppm, 12.5 μl) for creating quantum crystals and hemagglutinin H1 influenza A antibody (100 μg / ml, 12.5 μl). This solution is then dropped onto a phosphor bronze plate, and the hemagglutinin H1 influenza A antibody is immobilized onto the purified quantum crystals on the substrate. Next, a complex of H1N1 influenza virus (100 μg / ml, 5 μl) that binds to hemagglutinin H1 influenza A antibody via an antigen-antibody reaction and FITC-labeled H1N1 influenza virus antibody (50 μg / ml, 5 μl) was dropped onto a hemagglutinin H1 influenza A antibody-immobilized substrate. The complex of influenza virus and FITC-labeled antibody bound to the immobilized antibody on the substrate, and when observed with a fluorescence microscope, granular fluorescence of FITC was observed as shown in the image below. The equipment used is as follows: Equipment: Keyence BZ-X710 fluorescence microscope Light source: 80W metal halide lamp Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer

[0038] (Method for analyzing fluorescence images) The fluorescence images were analyzed as shown in Figures 8(1) to (3). Step (1) is the image acquisition step, in which the fluorescence image obtained by measurement is imported into the image analysis software "BZ-X Analyzer" and analyzed (fluorescence is observed as round particles). Here, as shown in Figure 16, if only one 10x objective lens is photographed (1 field measurement), and then binarized and counted, the shooting time is reduced to 3.5 seconds, but multiple images may also be taken. Step (2) is a binarization step in which all fluorescence particles with a brightness value greater than or equal to the set value are extracted from the entire range of the fluorescence image and binarized (red particles are the extracted fluorescence particles with a brightness value greater than or equal to the set value). Step (3) is a counting step in which the number of fluorescent particles that are above the set brightness value extracted is counted (calculating the number of fluorescent particles that are red (dashed lines)).

[0039] "Fluorescence Image Acquisition Process" Here, we can refer to a technique in which fragmented DNA trapped on the plasmon metal nanocrystal substrate is irradiated with excitation light to enhance the autofluorescence of the trapped fragmented DNA by the surface plasmon enhancement effect, and the fluorescent colonies are obtained as a fluorescence image (see fluorescence measurement method in Japanese Patent Application No. 2019-234330).

[0040] (Example 4) Similarly, a solution was prepared by mixing an Ag reagent (2000 ppm, 5 μl) used to create quantum crystals with FITC-labeled H1N1 influenza virus antibody (250 μg / ml, 5 μl). This solution was then dropped onto a phosphor bronze plate, and the purified quantum crystals on the substrate were immobilized with the FITC-labeled H1N1 influenza virus antibody. When this immobilized substrate was observed with a fluorescence microscope, fluorescence from granular FITC was observed. This indicates that fluorescent labels such as FITC can be immobilized onto quantum crystal substrates. (The Ag reagent ranges from 500 ppm to 10000 ppm, and the FITC-labeled H1N1 influenza virus antibody ranges from 1 pg / ml to 1 g / ml.)

[0041] (Measurement Example 1) This invention applies surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) to detect viruses in patients, using pharyngeal swabs, saliva, urine, or feces containing viral antigens. Figure 1 shows the method consisting of steps (1) to (4). Step (1) involves preparing an antibody-immobilized substrate using quantum crystal aggregation. Specifically, a viral antibody is added to a buffer (phosphate buffer at pH 7.4) to prepare an immobilized antibody solution. An equal volume of 1000 to 10000 ppm, preferably 2000 to 4000 ppm, aqueous solution of a plasmon metal complex is added to this to prepare a complex aqueous solution of the plasmon metal complex and the viral antibody. The plasmon metal complex solution containing the viral antibody is then dropped onto a metal substrate having an electrode potential near the reduction potential of the plasmon metal complex to aggregate the plasmon metal complex quantum crystals to which the antibody is bound, thereby preparing a viral antibody-immobilized substrate. Here, one plasmon metal is selected from palladium, platinum, gold, silver, and copper, and a metal substrate having an electrode potential near the redox potential of the plasmon metal complex is selected. When using a quantum crystal of the silver thiosulfate complex, copper or a copper alloy, particularly phosphor bronze, is selected as the substrate. Here, as antibodies, influenza virus antibodies include monoclonal antibodies of five classes IgA, IgD, IgE, IgG, and IgM from animal species such as rats, mice, chickens, rabbits, and humans, produced from viral antigens and hybridomas, or polyclonal antibodies containing five classes IgA, IgD, IgE, IgG, and IgM from animal species such as rabbits, guinea pigs, goats, sheep, rats, mice, and chickens, as well as antibodies such as immunoglobulins, Fc region, Fab region, and heavy chain. This includes fragmented antibody parts such as light chains, antigen-binding sites, and hinge regions, recombinant antibodies or fragmented parts, and human antibodies obtained by infecting human B lymphocytes with a virus (such as EBV), allowing them to proliferate, and cloning the antibody gene. Examples of coronavirus antibodies include monoclonal antibodies of five classes (IgA, IgD, IgE, IgG, IgM) from animal species such as rats, mice, chickens, rabbits, and humans, created from viral antigens or hybridomas; polyclonal antibodies containing the five classes (IgA, IgD, IgE, IgG, IgM) from animal species such as rabbits, guinea pigs, goats, sheep, rats, mice, and chickens; antibodies such as immunoglobulins; fragmented antibody parts such as Fc regions, Fab regions, heavy chains, light chains, antigen-binding sites, and hinge regions; recombinant antibodies or fragmented parts; and human antibodies obtained by infecting human B lymphocytes with a virus (such as EBV), allowing them to proliferate, and cloning the antibody gene. In step (2), an antigen-antibody reaction is used to form a complex between a viral antibody labeled with a fluorescent substance and the viral antigen in the sample. The sample can be a pharyngeal swab, saliva, urine, or feces. Examples of fluorescent substances used to label the viral antibody include those with excitation light wavelengths of 400nm to 436nm, such as Pacific Blue, FITC, 453 to 505nm, TRITC, 485 to 566nm, APC, 488 to 706nm, and IRDye800, 732 to 784nm. In step (3), the antigen-antibody reaction is used to drop the above complex onto the antibody-immobilized substrate, allowing the complex to bind to the antibody on the substrate. Unbound complexes and antibodies are then washed with pure water or a buffer solution. Here, a neutral phosphate buffer was used as the buffer solution, but PBS, HEPES, TRIS, BIS-TRIS, CAPS, CAPSO, Glycylglycine, MES, MOPS, PIPES, etc., can also be used. In step (4), the labeled antibody-antigen complex remaining on the substrate is irradiated with excitation light, and the fluorescence image is observed with a fluorescence microscope or fluorescence reader by surface plasmon excitation. From any range of the obtained fluorescence image or the entire image, fluorescence particles with a brightness value of any value or higher are binarized, and the number of obtained particles is counted. To binarize and count fluorescence particles in the fluorescence image that are above a certain threshold, the fluorescence measurement method of Japanese Patent Application No. 2019-234330 of the same applicant can be used, and this fluorescence measurement method is hereby referenced. In this invention, the single-field measurement conditions shown in Figure 16 are: threshold 62, no blur filter, and single-field measurement with a 10x lens (wherein, as shown in Figure 16, single-field measurement refers to a method of acquiring only one part of the chip, unlike in the case of Japanese Patent Application No. 2019-234330. When using the solid-phase substrate according to the present invention, the average value of two or more single-field measurements is almost equivalent, so it has been found that the results of the single-field measurement can be used without adopting the average value. This indicates that the solid-phase formation of the antigen or antibody is almost uniform due to the quantum crystal).

[0042] In the above embodiment, a substrate with an antibody immobilized on it was used, but by using the same type of metal powder, viral antigens can be detected by utilizing the antigen-antibody reaction in liquid. Pharyngeal swabs, saliva, urine, and feces containing viral antigens are used, and the process consists of steps (1) to (5) as shown in Figure 2. Step (1) uses quantum crystal agglutination to create antibody-immobilized metal powder. Specifically, viral antibody is added to an aqueous solution of plasmon metal complex at a concentration of 500 to 10000 ppm, and a carrier metal powder is added and mixed thereto. The plasmon metal complex, along with the viral antibody, agglutinates with the metal powder having an electrode potential near the reduction potential of the plasmon metal complex, forming a viral antibody-immobilized metal powder in which the viral antibody, plasmon metal complex, and carrier metal powder are integrated. On the other hand, in step (2), a first antigen-antibody reaction is used to form a complex between a viral antibody labeled with a fluorescent substance and the viral antigen in the sample. Here, the sample and fluorescent substance are the same as in the first method. Next, in step (3), the complex is added to the antibody-immobilized powder solution, and the antibody-immobilized powder and the complex are bound together using the second antigen-antibody reaction. In step (4), the combined product of the antibody-immobilized powder and the complex is filtered, and the unbound complex and antibody are washed with pure water or buffer solution. Finally, in step (5), the labeled antibody-antigen complex remaining on the substrate is irradiated with excitation light, and its fluorescence image is observed with a fluorescence microscope or fluorescence reader by surface plasmon excitation. Then, from any range of the obtained fluorescence image or the entire image, the fluorescence particles with a brightness value greater than or equal to any value are binarized, and the number of obtained particles is counted.

[0043] The second method of the present invention involves immobilizing a viral antigen or a part thereof (a part of a non-infectious antigen, for example, a fragmented part of the virus such as spikes on the surface of the virus or the nucleocapsid) and capturing antibodies produced in the body. As shown in Figure 3, the antigen or a part thereof is immobilized using a quantum crystal aggregation method of plasmon metal complexes, and then the antigen or a part thereof is reacted with an antibody to form an antigen or a part thereof immobilized substrate with gaps or microchannels between the quantum crystals. At the same time, an antibody labeled with a fluorescent substance is dropped onto the antigen or a part thereof immobilized substrate to bind the two, the unbound labeled antibody is washed away, and the remaining labeled antibody on the substrate is irradiated with excitation light to surface plasmon-excite the quantum crystals, thereby enhancing the fluorescence of the labeled antibody, and the fluorescence is detected. This method involves using a quantum crystal agglutination method of plasmon metal complexes to immobilize an antigen or a part thereof, then reacting the antigen or a part thereof with an antibody to form an antigen or a part thereof immobilized substrate with gaps or microchannels between the quantum crystals. Simultaneously, labeled antibodies labeled with a fluorescent substance are dropped onto the antigen or a part thereof immobilized substrate to bind them together, and after washing away the unbound labeled antibodies, the remaining labeled antibodies on the substrate are irradiated with excitation light to surface plasmon-excite the quantum crystals. The resulting fluorescence image is observed with a fluorescence microscope or fluorescence reader, and fluorescence particles with a brightness value or higher than a desired value are binarized from an arbitrary range or the entire image of the obtained fluorescence image, and the number of obtained particles is counted for detection. Specifically, in step (1), a substrate immobilized with an antigen or a part thereof is prepared using the quantum crystal agglutination method. More specifically, a viral antigen or a part thereof is added to an aqueous solution of a plasmon metal complex at a concentration of 500 to 10000 ppm. Here, a pharyngeal swab, saliva, urine, feces, or a part of a non-infectious antigen is used as the viral antigen or a part thereof. For the treatment of viral antigens and some of them, methods such as autoclaving (high-pressure steam sterilization at 121°C for 15 minutes or more), immersion in 0.01% or higher sodium hypochlorite solution for 1 hour or more, immersion in 4% formaldehyde solution, or immersion in 70% ethanol can be used as reference. A complex aqueous solution of a plasmon metal complex and a viral antigen or a part thereof is prepared. The plasmon metal complex solution containing the viral antigen or a part thereof is dropped onto a metal substrate having an electrode potential near the reduction potential of the plasmon metal complex. This aggregates the plasmon metal complex quantum crystal to which the antigen or a part thereof is bound, thereby preparing a substrate on which the viral antigen or a part thereof is immobilized. Next, in step (2), the first antigen-antibody reaction is used to form a complex between the immobilized antigen or a portion thereof and viral antibodies in the blood. Here, blood, serum, or plasma can be used as the sample containing viral antibodies. In step (3), a labeled antibody is prepared and dropped onto an antigen-antibody immobilized substrate using the second antigen-antibody reaction. The complex is then bound to the antibody on the substrate, and the unbound labeled antibody is washed with pure water or buffer solution. However, the sample from step (2) and the labeled antibody from step (3) can also be mixed beforehand. In step (4), the labeled antibody-antigen complex remaining on the substrate is irradiated with excitation light, and the fluorescence image is observed with a fluorescence microscope or fluorescence reader by surface plasmon excitation. From any range of the obtained fluorescence image or the entire image, fluorescence particles with a brightness value of any value or higher are binarized, and the number of obtained particles is counted and detected.

[0044] In the sandwich method described above, a primary labeling antibody and a secondary labeling antibody that can bind to each other are used simultaneously as labeled antibodies to enhance fluorescence intensity. The method shown in Figure 4 uses a primary labeling antibody and a secondary labeling antibody that can bind to each other simultaneously in steps (1) to (4) of the first method of the present invention. In step (1), an antibody-solidified substrate is prepared using the quantum crystal aggregation method. This is the same as step (1) in Figure 17. In step (2), the antigen-antibody reaction is used to label the viral antibody with a fluorescent substance, which is then used as the first The first-order labeled antibody and the second-order labeled antibody are used simultaneously to form a complex with the viral antigen in the sample. Alternatively, the primary labeled antibody may be attached first, followed by the secondary labeled antibody. The samples used here include pharyngeal swabs, saliva, urine, and feces. Examples of fluorescent substances used to label viral antibodies include those with excitation wavelengths of 400nm-436nm, such as Pacific Blue, FITC, 453-505nm, TRITC, 485-566nm, APC, 488-706nm, and IRDye800, 732-784nm. As a combination of primary labeling antibody and secondary labeling antibody A secondary labeling antibody that recognizes the animal species from which the primary labeling antibody originates is combined. For example, when using a primary labeling antibody derived from a mouse, a secondary labeling antibody that recognizes the mouse antibody is used, and the same combination is applied to other animal species. In step (3), the antigen-antibody reaction is used to drop the above-mentioned complex onto the antibody-immobilized substrate, the complex is bound to the antibody on the substrate, and the unbound complex and antibody are washed with pure water or buffer. Here, PBS, HEPES, TRIS, BIS-TRIS, CAPS, CAPSO, Glycylglycine, MES, MOPS, PIPES, etc. can be used as buffer. In step (4), the labeling antibody-antigen complex remaining on the substrate is irradiated with excitation light, and the fluorescence image is observed with a fluorescence microscope or fluorescence reader by surface plasmon excitation. From any range of the obtained fluorescence image or the entire image, fluorescence particles with a brightness value of any value or higher are binarized, and the number of obtained particles is counted. (Measured by antigen-antibody reaction of influenza virus)

[0045] Equal volumes of Ag reagent (500-10000 ppm), which forms the basis of quantum crystals, and influenza antibody (5-1000 μg / ml) are mixed. The mixture is then dropped onto a phosphor bronze plate, and the quantum crystals and antibody are immobilized on the phosphor bronze plate. Next, equal amounts of influenza virus (5-1000 μg / ml) and FITC-labeled influenza antibody (5-1000 μg / ml) are mixed and dropped onto the quantum crystal substrate mentioned earlier. Examples of reagents • Influenza antibody: HyTest Monoclonal mouse anti-Influenza A haemagglutinin H1 • Influenza virus (HyTest Corporation) Influenza A (H1N1) Virus • The FITC influenza antibody, IBL's Anti-Influenza A Virus (H1N1) FITC, was used. Excess FITC-labeled influenza antibodies were washed with pure water, irradiated with light from a light source (metal halide lamp 80W), and then scanned using a fluorescence microscope (Keyence BZ-X710). Measurement is performed using [tool name]. Images were observed with a fluorescence microscope and analyzed with BZ-X Analyer. The results are shown in Figure 6(a). In contrast, the case without influenza antigen is shown in Figure 6(b). Surface plasmon excitation is performed and the resulting fluorescence image is observed with a fluorescence microscope or fluorescence reader. From any range of the obtained fluorescence image or the entire image, fluorescence particles with a brightness value of any value or higher are binarized, and the number of obtained particles is counted for detection. When a virus is present, it is sandwiched between immobilized antibodies on the quantum crystal and labeled antibodies, emitting numerous granular fluorescence. This granular fluorescence is the fluorescence of the labeled antibodies that have sandwiched the virus (Figure 6(a)), whereas when no virus is present, the numerous granular fluorescence do not appear (Figure 6(b)). Using influenza antibodies (25 μg / ml) and FITC influenza antibodies (25 μg / ml), the virus concentration and the brightness value of fluorescent particles above 57 in the fluorescence image were analyzed using Keyence BZ-X710 with a 10x objective lens. The results shown in Figure 7 were obtained by binarizing and counting these values. The graph in Figure 7 shows a linearized version of this result. The obtained image shows a relative relationship between the count and the virus concentration. (Testing of samples collected on-site)

[0046] The present invention is suitable for rapid on-site virus testing during immigration inspections and hospital diagnoses, and may include inactivating or weakening antigens in human specimens (pharyngeal swabs, saliva, sputum, nasopharyngeal fluid, urine, etc.). The inactivated antigens are then immobilized on a substrate using quantum crystal agglutination, and labeled antibodies are bound to the immobilized antigens by an antigen-antibody reaction. The unbound labeled antibodies are then washed with buffer or pure water, excitation light matching the antibody label (fluorescent substance) is irradiated from a light source, and the fluorescent particles on the substrate are counted using a fluorescence microscope. Here, quantum crystal agglutination refers to the agglutination method for producing quantum crystals of plasmon metal complexes as shown in Japanese Patent Application Publication No. 2016-197114, which refers to a method in which plasmon metal complexes in solution agglutinate as quantum crystals of metal complexes on a metal substrate having an electrode potential near the reduction potential, depending on the selection of the electrodeposition substrate potential. In this case, an antibody may be immobilized on a substrate using quantum crystal agglutination beforehand. The antigen in the collected sample is then inactivated and bound to the substrate by an antigen-antibody reaction. The label is then attached to the substrate using a labeled antibody, and the unbound labeled antibody is washed with buffer or pure water. The substrate is then irradiated with excitation light that matches the antibody label (fluorescent substance) from a light source, and the fluorescent particles on the substrate are counted using a fluorescence microscope.

[0047] In this invention, the viruses to be inactivated in a sample are basically composed of either nucleic acid DNA or RNA and a protective shell protein (capsid), and are classified into two types: those enclosed in a lipid-containing membrane called an envelope, and small spherical viruses that do not have an envelope. Therefore, the difference in whether or not a virus is susceptible to inactivation by a drug depends on whether or not it has an envelope, but generally, viruses with an envelope are susceptible to disinfectants, so the use of a drug is preferable. Other inactivation methods that are effective against most viruses include boiling (98°C or higher) for 15-20 minutes, 2 w / v% glutaraldehyde, 0.05-0.5 w / v% (500-5,000 ppm) sodium hypochlorite, 76.9-81.4 v / v% disinfectant ethanol, 70 v / v% isopropanol, 2.5 w / v% povidone-iodine, 55 w / v% phthalaldehyde, and 0.3 w / v% peracetic acid. Furthermore, many viruses are inactivated by the denaturation of their capsid proteins at 56°C for 30 minutes, and enveloped viruses are easily inactivated by lipid solvents such as ether, chloroform, and fluorocarbon. Additionally, by using antibodies that recognize nucleosides, nucleotides, and nucleocapsids present inside the virus, it is possible to detect inactivated viral antigens that have been broken down by destroying the membrane and shell. Therefore, from the viewpoint of minimizing or no impact on the antigen-antibody reaction, the inactivation method according to the present invention can utilize drug methods using ethanol, formalin, and AVL buffer, as well as inactivation methods such as heat treatment, SD treatment (chemical treatment), acid treatment, alkali treatment, and radiation treatment. In this invention, the product can also be manufactured using metal powder instead of a substrate. Furthermore, in the above method, by simultaneously using a fluorescently labeled primary antibody and a fluorescently labeled secondary antibody as labeled antibodies and performing imaging and analysis, fluorescence images can be obtained more appropriately and accurately.

[0048] The present invention is applicable to on-site collection and on-site testing, and it is preferable to use a sample inactivation collection kit, and it is preferable to inactivate the sample using a drug. As shown in Figure 18(1), a tube 10 containing a drug solution L such as ethanol and a rod-shaped sample collection part 20 are set together, and the sample collection part is made of an absorbent material such as nonwoven fabric or gauze. Next, (2) a sample is collected with the rod-shaped sample collection part. Saliva, sputum, pharyngeal swab, nasopharyngeal fluid, etc. are used as the sample S. (3) After collecting the sample S, the collection part 20 is placed inside the tube 10. (4) The back of the tube 10 is a narrow section, and when the sample collection part 20 is inserted, the sample collection part 20 is compressed by the narrow wall, and the sample S (saliva) is dispersed in the drug solution L such as ethanol. (5) When everything except the sample collection part 20 is removed, the sample collection part 20 remains inside the tube 10. (6) The sample S is inactivated by the drug L and remains inside the sample collection part 20.

[0049] According to the present invention, viral antigens are collected on-site, inactivated, immobilized on a substrate by quantum crystal agglutination, and labeled antibodies are bound to them, or the viral antigens inactivated by antigen-antibody reaction on a substrate on which antibodies have been immobilized in advance are bound to labeled antibodies, and the fluorescence intensity is measured by the virus The number of fluorescent particles of the antigen can be counted to measure the virus concentration. Moreover, the quantum crystal forming the antibody or antigen-immobilized substrate undergoes interaction between photons incident by excitation light and the free electrons of the plasmon metal particles forming the quantum crystal, causing surface plasmon excitation and enhancing the fluorescence of the labeled antibody. Therefore, instead of the overall fluorescence intensity, the granular fluorescence can be counted and detected with high reproducibility. Consequently, surface plasmon excitation-enhanced fluorescence spectroscopy (SPFS) can be used to perform rapid testing in a short time of 2 to 5 minutes, providing a highly accurate diagnostic result as an alternative to PCR testing, which has complicated pretreatment, low sensitivity due to primers, many protocols, and a long time to perform the test. Furthermore, it is groundbreaking because it can not only determine the presence or absence of disease, but also determine the severity of the disease, as the count corresponds to the number of viruses.

[0050] According to the sample inactivation collection kit of the present invention, the collected virus can be inactivated and secured in a tube, so it can be sent to the required testing site and retrieved for testing at any time. In the present invention, the inactivated antigen is directly immobilized using quantum crystal agglutination, but the antibody may be immobilized beforehand, and the inactivated antigen may be bound to the antibody labeled by an antigen-antibody reaction for detection. Direct, sandwich, and indirect methods of immunofluorescence can be employed. (Detection of viruses from inactive samples)

[0051] A solution was prepared by mixing equal volumes of Ag reagent (2000 ppm, 12.5 μl) for creating quantum crystals and hemagglutinin H1 influenza A antibody (25 μg / ml, 12.5 μl), and this solution was dropped onto a phosphor bronze plate, where it aggregated on the substrate. Hemagglutinin H1 influenza A antibody is immobilized together with quantum crystals. Next, inactivation A complex of an inactivated sample (25 μg / ml, 5 μl) and FITC-labeled H1N1 influenza virus antibody (25 μg / ml, 5 μl) was dropped onto a hemagglutinin H1 influenza A antibody-immobilized substrate. If H1N1 influenza virus was present in the inactivated sample, the complex of influenza virus and FITC-labeled antibody bound to the immobilized antibody on the substrate, and fluorescence microscopy revealed fluorescence of granular FITC. If influenza virus was not present in the sample, fluorescence of granular FITC was not observed. When the measurement images obtained by fluorescence microscopy were analyzed using "Keyence Analysis Software: BZ-X Analyzer" with a threshold of 57, a significant difference was found in the count value of granular FITC fluorescence. As a result, when counting the fluorescence particles from the fluorescence image, the count was 4 in the virus-free case, compared to 145 in the virus-present case. The equipment used is as follows: Equipment: Keyence BZ-Z710 fluorescence microscope Light source: 80W metal halide lamp Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer

[0052] (Example 1 of patient virus measurement) The fluorescence counting method of the present invention is applied to the detection of the virus in patients, and pharyngeal swabs, saliva, sputum, nasopharyngeal fluid, urine, and feces containing viral antigens are used. Figure 17 shows the method consisting of steps (1) to (7). In step (1), an inactivated sample is prepared using the sample inactivation collection kit shown in Figure 18. In step (2), the inactivated antigen in the sample is mixed with Ag reagent (silver thiosulfate complex solution). In step (3), a solid-phase substrate of the inactivated antigen is prepared using the quantum crystal agglutination method. Specifically, the inactivated antigen is added to an aqueous solution of a plasmon metal complex at a concentration of 2000 to 6000 ppm to prepare a complex aqueous solution of the plasmon metal complex and the inactivated antigen, and the plasmon metal complex solution containing the inactivated antigen is dropped onto a metal substrate having an electrode potential near the reduction potential of the plasmon metal complex to agglutinate the plasmon metal complex quantum crystals and prepare a solid-phase substrate on which the inactivated antigen is immobilized (step (4)). Here, as the plasmon metal A metal substrate is selected from palladium, platinum, gold, silver, and copper, having an electrode potential near the redox potential of the plasmon metal complex. When using a quantum crystal of a silver thiosulfate complex, copper or a copper alloy, particularly phosphor bronze, is selected as the substrate. The method described in Japanese Patent Publication No. 2016-197114 is cited and referenced as the method for preparing the quantum crystal. In step (5), the antigen-antibody reaction is used to label the viral antigen in the immobilized sample with a viral antibody labeled with a fluorescent substance. The sample can be a pharyngeal swab, saliva, sputum, nasopharyngeal fluid, urine, or feces. Examples of fluorescent substances used to label the viral antibody include those with excitation light wavelengths of 400nm to 436nm, such as Pacific Blue, FITC, 453 to 505nm, TRITC, 485 to 566nm, APC, 488 to 706nm, or IRDye800, 732 to 784nm. In step (6), the unbound complexes and antibodies are washed from the substrate with pure water or a buffer solution. In addition to neutral phosphate buffer, PBS, HEPES, TRIS, BIS-TRIS, CAPS, CAPSO, Glycylglycine, MES, MOPS, PIPES, etc. can be used as buffer solutions. In step (7), the labeled antibody-antigen complex remaining on the substrate is irradiated with excitation light, and the fluorescence image is observed with a fluorescence microscope or fluorescence reader by surface plasmon excitation. From any range of the obtained fluorescence image or the entire image, fluorescence particles with a brightness value of any value or higher are binarized, and the number of obtained particles is counted. Fluorescence particles with a threshold of a certain level in the fluorescence image are binarized and counted. According to the present invention, highly accurate detection of viruses comparable to the PCR method can be performed simply and quickly. Therefore, rapid on-site testing is possible in immigration inspections, hospitals, etc. (Detection of two types of viruses)

[0053] The following method describes a measurement technique that detects two types of viruses using a single measurement chip. 1) Mix equal volumes of the aforementioned Ag reagent (3000 ppm aqueous solution of silver thiosulfate), influenza antibody buffer (containing multiple antibodies as influenza antibodies), and coronavirus (Covid-19) antibody buffer to prepare a solid-phase sample containing 1000 ppm of Ag reagent. Alternatively, prepare the sample by mixing equal volumes of all three sequentially. Drop the mixture onto a metal substrate to create a solid-phase substrate (see Figure 19A(1)). 2) Next, a sample taken from a human (pharyngeal swab or saliva, etc.) that has been inactivated with ethanol, etc., is mixed with a mixture of green-labeled coronavirus antibody and red-labeled influenza antibody. If either virus is present in the sample, it will form a complex with the labeled antibody. Then, this complex is dropped onto a solid-phase substrate. 3) The complex binds to the antibody immobilized on the substrate via an antigen-antibody reaction (see Figure 19A(2)). Unbound complexes and labeled antibodies are washed away with water or buffer (see Figure 19B(3)). Here, coronavirus antibodies are labeled with labels in the green region such as FITC or Cy2, and influenza antibodies are labeled with labels in the red region such as Cy5 or APC, and the labels are selected so that their respective fluorescence regions do not overlap. The sample is irradiated with either green or red excitation light from a light source. If influenza virus is present in the sample, no fluorescence is observed with green excitation, but fluorescence is observed from red-labeled influenza antibodies with red excitation. On the other hand, if coronavirus is present in the sample, no fluorescence is observed with red excitation, but fluorescence is observed from green-labeled coronavirus antibodies with green excitation (see Figure 19B(4)). In this way, two fluorescence images obtained from the two excitation lights are acquired using a fluorescence microscope, and the fluorescence spots or particles on the images are counted and quantified. Note: The green region filter has an excitation wavelength of 470±20nm and an fluorescence wavelength of 525±25nm. The red region filter was set to an excitation wavelength of 620±20nm and an fluorescence wavelength of 700±37.5nm.

Claims

1. A measuring device that performs a method of quantitative analysis by fluorescently labeling an antigen or antibody to be measured in a sample, capturing it in an antigen-antibody reaction using a solid-phase substrate of antibody or antigen solidified with a plasmon metal complex quantum crystal, then enhancing the labeled fluorescence of the target to be measured formed by excitation light irradiation with the surface plasmon enhancement effect of the plasmon metal complex quantum crystal, and counting the fluorescence, comprising: 1) a solid-phase substrate for capturing the antigen or antibody to be measured in an antigen-antibody reaction; 2) means for fluorescently labeling the antigen or antibody to be measured in the sample; 3) means for dropping a sample containing the fluorescently labeled antigen or antibody onto the solid-phase substrate and capturing it; 4) a light source for irradiating the antigen or antibody to be measured captured on the solid-phase substrate with excitation light; 5) a fluorescence microscope or fluorescence reader for obtaining a point-like fluorescence image of the fluorescently labeled antigen or antibody to be measured; and 6) fluorescence counting analysis software for binarizing and counting the fluorescence points in the fluorescence image. A fluorescence counting and quantitative device utilizing an antigen-antibody reaction, characterized in that the solid-phase substrate enhances the labeling fluorescence of the antigen or antibody in the captured sample by the surface plasmon enhancement effect of the plasmon metal complex quantum crystal, allowing the acquisition of a point-like fluorescence image with a fluorescence microscope or fluorescence reader, and enabling quantitative analysis by binarizing the fluorescence points in the fluorescence image with fluorescence counting and analysis software and selecting fluorescence points above a predetermined threshold, thereby enabling quantification by counting those fluorescence points.

2. The fluorescence counting apparatus according to claim 1, wherein the solid-phase substrate is manufactured by collecting an antibody or antigen that captures the antigen or antibody to be measured in a sample by an antigen-antibody reaction in a buffer solution, then mixing it with a 1000 to 3000 ppm aqueous solution of a plasmon metal complex to neutralize it, and dropping this onto a metal substrate.

3. The fluorescence counting and quantitative apparatus according to claim 1 or 2, wherein the solid-phase substrate is a phosphor bronze plate, the plasmon metal complex quantum crystals aggregated on the phosphor bronze plate are in a region of silver thiosulfate quantum crystals ranging from 50 nm to 150 nm, and the fluorescence label of an antigen or antibody solidified together with the quantum crystals appears as a fluorescent spot in a fluorescence image and is counted.

Images