Detection member for object to be detected and method for detecting object to be detected using same

Abstract

Provided is a detection member for an object to be detected, the detection member comprising a flocculable carrier on which a DNA aptamer that specifically binds to the object to be detected is supported, and an antibody that binds to the object to be detected, the antibody having a labeling group that emits light of a specific wavelength when irradiated with light of a prescribed wavelength. Also provided is a method for detecting an object to be detected using the detection member. The object to be detected may be a virus, a microbe, or a bacterium. The virus may be a coronavirus, an influenza virus, a norovirus, or an avian influenza virus.

Description

Detecting member for object to be detected and method for detecting object to be detected using the same

[0001] The present invention relates to a detection member for detecting an object to be detected and a method for detecting an object to be detected using the same.

[0002] Pathogenic viruses such as influenza and norovirus are so small that they cannot be seen in normal living environments. This invisibility of viruses increases anxiety and fear in society and spreads disease.

[0003] The fear of being "invisible to the naked eye" is particularly difficult to overcome when it comes to airborne viruses. If it were possible to visualize and detect pathogenic viruses present in the air in living spaces, offices, vehicles, etc., or attached to clothing or masks, easily and with high sensitivity without requiring specialized knowledge or operational skills in biochemical analysis, it would be possible to suppress and prevent viral infection and disease transmission.

[0004] In recent years, a relatively simple influenza virus testing technique using immunochromatography, as disclosed in Patent Document 1, has been used to detect such viruses.

[0005] JP 2008-275511 A

[0006] The method for detecting influenza viruses using immunochromatography is simple, but requires 10 3 ~10 4 Therefore, the detection sensitivity is not necessarily high when the amount of virus present is small.

[0007] In order to prevent infection with pathogens and the spread of disease, there is a strong demand for a method to quickly and sensitively visualize substances harmful to the human body, such as viruses present in the air in living spaces, offices, and vehicles, or attached to clothing and masks.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a detection element for an object to be detected that can visualize the presence of the object to be detected in a short time using a simple method without using complicated detection methods or devices, and a detection method for the object to be detected using the same.

[0009] In order to solve the above problems, the detection member for an object to be detected and the method for detecting an object to be detected using the same of the present invention employ the following means.

[0010] A first embodiment of the present invention provides a detection element for an analyte, comprising an agglutinable carrier carrying a DNA aptamer that specifically binds to the analyte, and an antibody that binds to the analyte and has a labeling group that emits light of a specific wavelength under predetermined conditions.

[0011] In the first embodiment, the substance to be detected may be a virus, a bacterium, or a bacteria. The virus may be a new coronavirus, an influenza virus, a norovirus, or an avian influenza virus.

[0012] In the first embodiment, the agglutinable carrier may be a magnetic bead or a gold nanoparticle.

[0013] In the first embodiment, the antibody having a labeling group that emits light of a specific wavelength under predetermined conditions may be an antibody modified with a fluorescent functional group.

[0014] In the first embodiment, the detection member may be an inner sheet of a mask provided with a detection patch containing a carrier and an antibody.

[0015] In the first embodiment, the wiper may contain a detection reagent containing a carrier and an antibody, and may be used to wipe a surface that has come into contact with a detection target that may contain or have the target substance attached thereto.

[0016] In the first embodiment, the detection member may be a detection reagent that is sprayed onto the wiper after wiping the surface that comes into contact with the object to be detected, which may contain or have the object to be detected attached thereto.

[0017] In the first embodiment, the inner sheet of the mask or the wiper may contain moisture at a predetermined rate.

[0018] In the first embodiment, the detection member may be a saliva test stick having a detection patch containing the carrier and the antibody.

[0019] In the first embodiment, the detection member may be a detection and display member attached to the air conditioner.

[0020] In the first embodiment, the detection member may be a detection and display member attached to the desk-mounted humidifier.

[0021] A second embodiment of the present invention provides a method for detecting an object to be detected, using the detection member according to the first embodiment.

[0022] The second embodiment includes the steps of contacting the detection member according to the first embodiment with a detection target that may contain or have attached thereto an analyte; agglutinating and recovering carriers that are contained in the contacted detection member and that carry DNA aptamers that specifically bind to the analyte; contacting the recovered carriers to which the analyte is bound with an antibody that binds to the analyte and that is labeled with a labeling group that emits light of a specific wavelength when irradiated with light of a specific wavelength, thereby forming an analyte-labeled antibody complex; and detecting the analyte by irradiating the formed analyte-labeled antibody complex with light of a specific wavelength and detecting the emitted light.

[0023] In the second embodiment, a tool having a magnet may be used in the step of recovering the agglomerated carriers.

[0024] In the second embodiment, in the step of detecting the substance to be detected, a detection patch containing a carrier and an antibody, which is provided on the inner sheet of the mask, may be irradiated with light of a predetermined wavelength.

[0025] In the second embodiment described above, the wiper contains an antibody that binds to the analyte and has a labeling group that emits light of a specific wavelength when irradiated with light of a predetermined wavelength, and after wiping a surface that has come into contact with the object to be detected, which may contain or have the analyte attached, a detection reagent containing a carrier carrying a DNA aptamer that specifically binds to the analyte may be sprayed onto the wiper to form an analyte-antibody complex.

[0026] By using the detection element for an analyte of the present invention, the presence of very small amounts of virus (subnanomolar) can be easily visualized without using complicated detection methods or large-scale equipment that is inconvenient to carry around, which makes it easy to detect the location of the virus and measure the amount of virus present even in environments such as living spaces and offices.

[0027] The design of the analyte detection element of the present invention allows the detection of multiple viruses using a single detection element, making it applicable as a first screening method for patients suspected of infection with a virus whose type cannot be identified based on symptoms alone.

[0028] 1 is a conceptual diagram of the mechanism for detecting an analyte using the detection element of the present invention. 2 is a diagram showing the predicted secondary structure of an example of a DNA aptamer carried on a carrier of the detection element of the present invention. 3 is a photograph of a pen-shaped device with a magnetic tip, which is an example of a device used to collect magnetic beads. 4 is a diagram showing an image of the inner sheet of a mask to which detection patches containing a carrier and an antibody are attached, where the round patches represent patches for coronavirus detection and the square patches represent patches for influenza virus detection. 5 is a diagram showing a desk-mounted humidifier equipped with a detection patch-containing sheet containing a carrier of the detection element of the present invention and an antibody. 6 is a diagram showing an air conditioner equipped with a detection patch-containing sheet containing a carrier of the detection element of the present invention and an antibody. 7 is a diagram showing an example of a detection patch-containing sheet containing a carrier of the detection element of the present invention and an antibody, and in this example, an image of a sheet containing patches for coronavirus detection and patches for influenza virus detection. These are fluorescence observation images (20x magnification) obtained by contacting a detection element of the present invention with a coronavirus and detecting the resulting coronavirus-fluorescently modified antibody complex. (a) is a fluorescence observation image when a spike-binding fluorescently modified antibody is used as the antibody, and (b) is a fluorescence observation image when a spike-free fluorescently modified antibody is used as the antibody. These images show magnetic beads that can be used as a carrier for the detection element of the present invention, recovered using a magnet attached to a slide glass. These are fluorescence observation images (10x magnification) obtained by contacting a detection element of the present invention with a coronavirus and detecting the resulting coronavirus-fluorescently modified antibody complex. (a) is a blank image, (b) is a 40-fold diluted solution of the spike protein stock solution, (c) is the spike protein stock solution, and (d) is a fluorescence observation image after coronavirus binding. This figure illustrates an example of using the detection element of the present invention as a wiper.

[0029] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of a detection member for an object to be detected and a detection method for an object to be detected using the same according to the present invention will be described with reference to the drawings.

[0030] In this embodiment, an aptamer sensor is provided that can detect pathogens such as viruses, germs, and bacteria with high sensitivity in a short time, such as a few seconds to a few tens of seconds, using simple tools such as the human eye or a smartphone camera, without requiring PCR probes or large-scale, expensive equipment or operations.

[0031] [First embodiment] A first embodiment of the present invention will be described below with reference to Fig. 1. The analyte detection member according to this embodiment includes an agglutinable carrier carrying a DNA aptamer that specifically binds to the analyte, and an antibody that binds to the analyte and has a labeling group that emits light of a specific wavelength under predetermined conditions.

[0032] A DNA aptamer is a ligand molecule that specifically and strongly binds to a target substance due to the formation of a secondary or tertiary structure by complementary sequences within a DNA molecule. DNA aptamers have attracted attention in recent years as molecules that can inhibit, suppress, or enhance the activity of target molecules, or selectively detect or extract target molecules, by binding a DNA aptamer with a specific sequence.

[0033] Although DNA aptamers have a smaller molecular weight than antibodies, approximately 1 / 10 or less, they have high affinity and target selectivity comparable to antibodies. The present inventors have utilized this high target selectivity to discover a new approach to detecting analytes based on changes in fluorescence intensity or fluorescence polarization depending on the presence or absence of the analyte.

[0034] DNA aptamers are artificial DNA molecules with relatively short chain lengths (usually 100 bases or less) that can be obtained by chemical synthesis. Because DNA aptamers can be chemically synthesized, they have the advantages of low risk of biological contamination, generally low antigenicity, and, because they are DNA molecules, they are sufficiently stable at room temperature under near-neutral conditions in the absence of nucleic acid degrading enzymes (nucleases).

[0035] DNA aptamers can be chemically modified at any position in the sequence with a fluorescent functional group or a fluorescent quencher group to create a labeled probe, for example, a fluorescent probe. This high degree of freedom in molecular design is an advantage of using DNA aptamers, which is not available with antibodies.

[0036] Fluorescence polarization is a method for analyzing interactions between fluorescent molecules (including "molecules bound to fluorescent molecules") and target substances by measuring the degree of fluorescence polarization using a fluorescence polarimeter. Fluorescence polarization measurement does not require separation of bound and unbound fluorescent probes, and turbid solutions can be used as measurement samples, which has the advantage of shortening the preparation time for the measurement sample.

[0037] Fluorescence polarization refers to the degree to which a fluorescent molecule rotates between excitation and emission of fluorescence. When a three-dimensional structure is formed, the degree of freedom of rotation of the fluorescent molecule decreases, depending on the three-dimensional structure, and the rotation of the fluorescent molecule slows down. At this time, the fluorescence polarization increases. In this way, the fluorescent molecule's movement becomes limited due to the influence of the surrounding three-dimensional structure, or it becomes immobilized by adhering to a large amount of target substance. As a result, the rotation of the fluorescent molecule is strictly restricted, and the fluorescence polarization increases. Measuring fluorescence polarization allows for rapid analysis of the interaction between a fluorescent molecule and a target substance, even with a small sample volume.

[0038] As used herein, the term "analyte" refers to a substance that can be a specific binding target for a DNA aptamer. Examples of analytes that can be detected using the detection member of this embodiment include viruses, germs, and bacteria. Examples of viruses include, but are not limited to, the novel coronavirus (SARS-CoV-2), influenza virus, norovirus, and avian influenza virus.

[0039] A method for detecting an analyte will be described using the example shown in Figure 1. Aptamer beads 1, which have a plurality of DNA aptamers 3 that specifically bind to the analyte bound to them on an agglutinable carrier 2, are brought into contact with a detection target that may contain the analyte 4. Examples of detection targets include, but are not limited to, gases such as air, liquids that may contain the analyte, and solid substances such as furniture, equipment, and medical instruments.

[0040] When the substance to be detected contains an analyte 4, the analyte 4 binds to a DNA aptamer 3 that specifically binds to the analyte bound to the carrier 2, forming an aptamer-analyte complex 5. Figure 2 shows the predicted secondary structure of a 57-mer DNA aptamer, an example of a DNA aptamer according to this embodiment. The numbers in Figure 2 indicate the number of bases from the 5' end of the DNA aptamer.

[0041] The resulting aptamer-analyte complex 5 is contacted with a labeling antibody 6, which is an antibody 8 that binds to the analyte and is labeled with a labeling group 7 that emits light of a specific wavelength under predetermined conditions. In this embodiment, a fluorescent functional group or a luminescent functional group can be used as the labeling group that emits light of a specific wavelength under predetermined conditions.

[0042] The antibody 8 in the labeling antibody 6 that binds to the analyte binds specifically to the analyte 4 that is bound to the aptamer-analyte complex 5. This forms an aptamer-analyte-labeling antibody complex 9 in which the analyte 4 is bound to the agglutinable carrier 2 and the labeling antibody 6.

[0043] When the formed aptamer-analyte-labeling antibody complex 9 is exposed to predetermined conditions, it emits light of a specific wavelength. The presence of the analyte is detected by detecting this light of a specific wavelength. As an example, a case will be described in which a fluorescent functional group, fluorescein isothiocyanate (FITC), is used as a labeling group that emits light of a specific wavelength under predetermined conditions. In this case, 480 nm light is irradiated onto the aptamer-analyte-labeling antibody complex 9, and an amount of fluorescence proportional to the amount of the analyte present is detected at 520 nm. By measuring the amount of this fluorescence, it is possible to determine whether the analyte is present or not, and, if present, the amount of the analyte.

[0044] Applicable fluorescent functional groups include commonly used fluorescent molecules such as fluorescein, fluorescein isothiocyanate (FITC), Alexa Fluor (registered trademark) 488, Alexa Fluor (registered trademark) 514, Texas Red (trademark), Cy3 group, 5-carboxytetramethylrhodamine (TAMRA) group, Cy5 group, Cy5.5 group, ROX group, Alexa Fluor (registered trademark) 555, and Alexa Fluor (registered trademark) 647.

[0045] In the first step of the scheme shown in Figure 1, aptamer beads 1, which have multiple DNA aptamers 3 bound to an agglutinable carrier 2, are brought into contact with a target substance to be detected, which may contain an analyte 4, and once a complex between the aptamer beads 1 and the analyte 4 is formed, the complex may be recovered by utilizing the agglutination properties of the carrier. Magnetic beads or gold nanoparticles can be used as the carrier. When magnetic beads are used, the complex can be easily recovered using a device equipped with a magnet.

[0046] Commercially available magnetic beads that can be used for separating biological materials can be used. The particle size of the magnetic beads is preferably 50 nm to 5 μm, more preferably 0.5 μm to 2 μm. Activated functional groups on the magnetic beads are used to bind binding molecules such as streptavidin.

[0047] A simpler detection method involves recovering magnetic beads using a pen-shaped device 11 with a magnetic pen tip 12, as shown in Figure 3. After recovering the magnetic beads using the pen-shaped device 11, if the pen tip 12 glows under predetermined conditions, it is possible to confirm the presence or absence of corona in the treated detection object.

[0048] On the other hand, a DNA aptamer designed to specifically bind to an analyte is modified with a molecule capable of binding to the binding molecule on the magnetic beads. For example, if streptavidin is bound to the magnetic beads, biotin is bound to the DNA aptamer. This causes the streptavidin on the magnetic bead carrier to bind to the biotin on the DNA aptamer, resulting in aptamer beads 1.

[0049] When a binding molecule is bound to a DNA aptamer, it is bound to a portion that does not interfere with the aptamer's binding to the analyte, and preferably, the binding molecule is bound to the 3'- or 5'-end of the DNA aptamer.

[0050] As an example of the detection member of this embodiment, a fluorescent aptamer-based sensor for specifically detecting surface proteins of the novel coronavirus and influenza virus is provided. The aptamer is a protein that is specifically expressed by SELEX. １ Aptamers can be designed for the specific detection of a wide range of target molecules using techniques such as these. Therefore, the technology according to this embodiment can be extended to pathogens other than the novel coronavirus and influenza virus. Aptamers are antibodies. ２ The utility of this technology is advantageous compared to using antibodies because of their lower cost, ease of synthesis, and greater stability.

[0051] In Example 1 described below, the detection element according to this embodiment was used to detect the novel coronavirus, and as a result, the novel coronavirus was actually successfully detected with high sensitivity.

[0052] Once specifically bound to their target, DNA aptamers fold to form stable three-dimensional structures, and can also distinguish subtle molecular differences, such as those caused by protein mutations.３ This high discrimination ability allows the detection of an analyte with high sensitivity even when the analyte is in a low concentration.

[0053] Monoclonal antibodies (mAbs) are widely used in biotechnology and diagnostics, such as COVID-19 antigen self-test kits. However, mAbs have significant limitations, including poor stability, high manufacturing costs, and performance issues such as cross-reactivity and contamination in mass spectrometry-based proteomics analyses. The detection element according to the present embodiment, which uses DNA aptamers, can avoid these problems.

[0054] As a modification of the detection member or detection reagent applied to the first embodiment, an aptamer sensor that utilizes fluorescence resonance energy transfer (FRET) between two fluorescent molecules can also be used. FRET occurs, and energy is transferred to the acceptor fluorescent molecule by exciting the donor fluorescent molecule. As a result, the acceptor fluorescent molecule exhibits fluorescence. Therefore, the binding of the analyte to the fluorescently labeled DNA aptamer can be detected as a change in the degree of fluorescence polarization at the detection wavelength of the acceptor fluorescent molecule, or an increase or decrease in fluorescence intensity.

[0055] Second Embodiment A second embodiment of the present invention will now be described.

[0056] As described above, the detection member described in the first embodiment brings the aptamer beads 1 into contact with a detection target that may contain the analyte 4. The detection target may be a gas such as air, a liquid that may contain the analyte, or a solid substance such as furniture, equipment, or medical instruments. Therefore, the detection member may take various forms.

[0057] Specific embodiments of the detecting member according to this embodiment will be described below.

[0058] (A) Application to the inner sheet of a mask The novel coronavirus and influenza virus are detected inside a face mask via an inner sheet containing a fluorescent aptamer sensor.

[0059] One example of a fluorescent aptamer for detecting COVID-19 and influenza is a DNA aptamer that binds to the receptor-binding domain of the spike protein of the novel coronavirus with high affinity, comparable to the affinity of commercially available antibodies that bind to the spike protein.

[0060] Mutations in the spike protein of the novel coronavirus, especially in the receptor-binding domain of the spike protein, affect the rate at which the virus invades host cells, generating variants of concern (VoC). Aptamers need to be designed to recognize the latest coronaviruses currently circulating.

[0061] An inner sheet containing multiple detection reagents is inserted into the face mask to simultaneously detect multiple viruses, such as the novel coronavirus and influenza virus. Viruses detected on the inner sheet are detected either through the wearer's breath or exposure to viruses in the external air. In either case, fluorescence observed on the inner sheet indicates the need for further diagnostic testing. By using the detection member of this embodiment as the inner sheet, the face mask becomes a real-time virus detector.

[0062] A virus sensor patch, specifically a cellulose filter containing an aptamer sensor bound to a fluorescent molecule specific to each virus, is printed on a cellulose sheet that serves as the base material for the inner sheet 21. By binding different fluorescent molecules, sensors for different viruses, such as the novel coronavirus and influenza virus, can be distinguished by different colors, as shown by 21-1 and 21-2 in Figure 4.

[0063] The example in Figure 4 shows an image of inner sheet 21-1 that detects the presence of virus A, and inner sheet 21-2 that detects the presence of virus B. If both virus A and virus B are present in exhaled or inhaled breath, they can be detected by color changes in multiple types of virus sensor patches. It is also possible to change the shape of the sensor patch for each virus to be detected, as shown by the reference numerals 25 and 26 on the inner sheet 21 in Figure 4. The inner sheet 21 is inserted into the mask 22 as shown in Figure 4.

[0064] (B) Detection indicator (indicator) for attachment to air conditioners, humidifiers, and other common indoor surfaces The virus sensor patch described in (A) can be attached to the surface of an air conditioner or humidifier via adhesive. By using this sensor patch as an indicator, it is possible to monitor whether indoor air is contaminated with airborne viruses. The cellulose sheet containing the patch can be attached to any surface of the device via a device such as double-sided tape, as shown in Figures 5 and 6.

[0065] 5 shows a desk-mounted humidifier 31 with detection patch-containing sheets 32 and 33 containing the detection member of this embodiment attached to its surface. By simultaneously attaching detection patches targeting different viruses, it is possible to detect the presence or absence of one virus or both. The presence or absence of a virus can be easily detected by irradiating the detection patch with light of a wavelength at which the labeling group contained in the patch fluoresces.

[0066] FIG. 6 shows an indoor air conditioner 41 on whose surface a base 42 is mounted a detection patch-containing sheet 43, 44 containing the detection member of this embodiment. By simultaneously mounting detection patches 43, 44 targeting different viruses, it is possible to detect the presence or absence of one virus or both. Detection of the presence or absence of a virus can be easily observed by irradiating the patch with light of a wavelength at which the labeling group contained in the detection patch fluoresces. The example in FIG. 7 shows the state in which the color of the detection patch 44 changes to 44' upon the presence of the target substance (virus) 45 targeted by the detection patch 44. This allows for easy detection of whether a virus is floating in the room.

[0067] (C) Saliva Testing Device The fluorescent aptamer sensor for the novel coronavirus and influenza virus is used in a saliva testing device for detecting COVID-19 and influenza. This allows for a much faster response time than commonly used antigen testing kits for COVID-19, enabling detection in seconds rather than minutes.

[0068] The DNA aptamers used in this device configuration bind to viral surface proteins in the presence of moisture, which is found in respiratory droplets when detecting airborne viruses.

[0069] (D) Application to Wipes For detecting viruses on surfaces, a wipe that enables wiping, sterilization, and detection in one step is used as the detection element. Surfaces that come into contact with the target object include, but are not limited to, desks, vehicle seats, and clothing surfaces. For surface detection using a wipe, a wipe containing an aptamer detection patch is pre-moistened with a reagent solution, or the wipe is sprayed with the reagent solution.

[0070] By using it as a wipe, it is possible to easily detect whether or not the target virus is present in living spaces, offices, vehicle seats, and on the surface of clothing.By using it in the form of a wet wipe, it can be carried around.

[0071] By using the detection member according to this embodiment, it is possible to detect viruses present on the surfaces of furniture in living spaces, offices, and vehicles, as well as viruses absorbed into filters inserted into face masks, thereby reducing social anxiety and fear due to the invisibility of viruses and suppressing the spread of disease.

[0072] Detecting SARS-CoV-2 and influenza using a face mask is more efficient and cost-effective than undergoing antigen testing. Applying the detection element of this embodiment to detect airborne viruses in offices, residential spaces, and cars can prevent infection in these spaces and, if a virus is detected, can suppress its spread by quickly decontaminating the area.

[0073] Example 1: Method for detecting coronavirus using magnetic beads Magnetic beads bound to DNA aptamers were prepared, and the magnetic beads were brought into contact with coronavirus to allow the coronavirus to bind to the aptamer on the beads, and the beads were then collected. A spike-binding fluorescent antibody was bound to the coronavirus bound to the collected beads to form a complex, and its fluorescence was measured.

[0074] The sequences of the DNA aptamers used in this example are shown below. An aptamer (57 mer) having biotin at the 5' end and an aptamer (54 mer) having biotin at the 3' end were prepared and used independently in the experiment.

[0075] Test Method 1. Magnetic Beads The following magnetic beads were used in this example. Magnetic Beads MyOne C1: Streptavidin-bound superparamagnetic polymer beads with a particle size of 1.0 μm and a hydrophilic surface, based on carboxyl-activated beads. Bead particle size: 1.0 μm, concentration: 10 mg / mL (7-12 × 10 ９ beads / mL).

[0076] 2. Method for preparing aptamer-bound magnetic beads 1) Magnetic bead (MB) solution (20 μL) was washed and dispersed in 200 μL of 2x BW buffer. (BW: 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) 2) 4 μL of aptamer (100 μM) was diluted with 100 μL of pure water. 3) The MB solution prepared in 1) and the aptamer solution prepared in 2) were mixed and left at room temperature for 15 minutes. The MB was then recovered and redispersed in 200 μL of HEPES (20 mM) (concentration: 200 μg / 200 μL).

[0077] 3. Aptamer-bound magnetic beads Aptamer binding: It was confirmed that 8 pmol of aptamer was present in 20 μL of magnetic beads.

[0078] Binding of coronavirus to magnetic beads followed by binding with fluorescent antibodies (1) 5 μL of heat-inactivated coronavirus dispersion (SARS related Coronavirus 2 culture fluid, Zeptmetrix, concentration unknown) or Spike solution (SARS-CoV-2 (2019-nCoV) Spike S1 (D614G)-His Recombinant Protein, HPLC-verified, Shino Biological, 0.25 mg / mL) was added to 20 μL of aptamer-bound magnetic beads, and the mixture was shaken at room temperature for 20 minutes to allow binding. The magnetic beads were collected using a magnet and washed twice with 100 μL of HEPES (20 mM).

[0079] (2) The obtained aptamer-bound magnetic bead coronavirus was dispersed in 100 μL of HEPES, and 5 μL of AntiSpike Antibody (SARS-CoV-2 Spike Protein (RBD) Recombinant Human Monoclonal Antibody (P05DHu), Alexa Fluor® 488, eBioscience 0.5 mg / mL) was added, followed by shaking at room temperature for 30 minutes.

[0080] (3) The magnetic beads were collected, washed with HEPES, dispersed in 200 μL of HEPES, and the fluorescence was measured.

[0081] The measurement results of fluorescence detection using the obtained aptamer-captured spike-antibody complex are shown in Table 3. "Blank" in Table 3 indicates the value for a sample that does not contain spike protein or coronavirus.

[0082] The fluorescence observation image (×20) obtained by dispersing the sample on black filter paper is shown in FIG.

[0083] Improving sensitivity by collecting with a magnet Magnetic beads were collected using a 3 mm diameter neodymium magnet to concentrate the magnetic beads (concentrate coronaviruses). By agglomerating the magnetic beads with a magnet, it was confirmed that detection at locally high concentrations was possible, as shown in Figure 9.

[0084] The obtained fluorescence observation images (10x magnification) are shown in Figure 10. The four images in Figure 10 are fluorescence observation images (10x magnification): (a) is a blank, (b) is a 40-fold diluted solution of the spike protein stock solution, (c) is the spike protein stock solution, and (d) is a fluorescence observation image after coronavirus binding, in which the resulting coronavirus-fluorescently modified antibody complex was detected after contact with the coronavirus. This example confirmed that viruses can be detected using a desk-sized measuring device, without the need for large, expensive measuring equipment.

[0085] Example 2: Surface detection using a wiper A wiper that enables wiping, disinfection, and detection in one step is created. A cellulose sheet containing a virus detection patch is used as the wiper. The wiper can take the following two forms: (A) and (B).

[0086] (A) An example of a cellulose sheet containing a virus detection patch is shown in FIG. 11A. As shown in (a), a cellulose sheet containing a virus detection patch is used to wipe the surface of an object on which a virus may be present. Next, as shown in (b), a solution containing a detection reagent is sprayed onto the wiped sheet. Finally, (c) the sheet sprayed with the detection reagent is irradiated with light of a predetermined wavelength, and the presence or absence of a virus is detected based on the presence or absence of fluorescence or color development on the sheet.

[0087] (B) Figure 11B shows an example of a form in which a cellulose sheet containing a virus detection patch is pre-moistened with a solution containing a detection reagent. As shown in (a), a cellulose sheet containing a virus detection patch is pre-moistened with a solution containing a detection reagent and stored as a wet sheet. Next, as shown in (b), a sheet impregnated with the detection reagent is used to wipe the surface of an object where the virus may be present. Finally, (c) the sheet sprayed with the detection reagent is irradiated with light of a predetermined wavelength, and the presence or absence of the virus is detected based on the presence or absence of fluorescence or color on the sheet.

[0088] The detection member or detection reagent used in Example 2 can also be an aptamer sensor that utilizes fluorescence resonance energy transfer (FRET) between two fluorescent molecules. FRET occurs, and energy is transferred to the acceptor fluorescent molecule by exciting the donor fluorescent molecule. As a result, the acceptor fluorescent molecule exhibits fluorescence. Therefore, the binding of the analyte to the fluorescently labeled DNA aptamer can be detected as a change in the degree of fluorescence polarization or an increase in fluorescence intensity at the detection wavelength of the acceptor fluorescent molecule.

[0089] (References) 1. Sefah, K. , Shangguan, D. , Xiong, X. et al. Development of DNA aptamers using Cell-SELEX. Nat Protoc 5:1169-1185 (2010). 2. Thiviyanathan V, Gorenstein DG. Aptamers and the next generation of diagnostic reagents. Proteomics Clin Appl 6:563-573 (2012). 3. Conrad R, Ellington AD. Detecting immobilized protein kinase C isozymes with RNA aptamers. Anal Biochem. 242:261-265 (1996).

[0090] REFERENCE SIGNS LIST 1 Aptamer beads 2 Carrier 3 DNA aptamer 4 Analyte 5 Aptamer-analyte complex 6 Labeling antibody 7 Labeling group 8 Antibody that binds to the analyte 9 Aptamer-analyte-labeling antibody complex 11 Pen-shaped device 12 Pen tip 21 Inner sheet 21-1 Inner sheet when virus A is present 21-2 Inner sheet when virus B is present 22 Mask 25 Sensor patch 26 Sensor patch 31 Humidifier 32 Sheet containing detection patch 33 Sheet containing detection patch 41 Air conditioner 42 Base 42' Base after use 43 Sheet containing detection patch 44 Sheet containing detection patch 44' Sheet on which the analyte has been detected 45 Analyte

Claims

1. A detection element for an analyte, comprising: an agglutinable carrier carrying a DNA aptamer that specifically binds to the analyte; and an antibody that binds to the analyte and has a labeling group that emits light of a specific wavelength under specified conditions.

2. The detection member for an analyte according to claim 1, wherein the analyte is a virus, germ or bacteria.

3. The detection member for an object to be detected according to claim 2, wherein the virus is a coronavirus, an influenza virus, a norovirus, or an avian influenza virus.

4. The detection member for an analyte according to claim 1, wherein the agglutinable carrier is a magnetic bead or a gold nanoparticle.

5. A member for detecting an analyte according to claim 1, wherein the antibody having a labeling group that emits light of a specific wavelength when irradiated with light of a predetermined wavelength is an antibody modified with a fluorescent functional group.

6. The detection member for detecting an analyte according to claim 1, which is an inner sheet of a mask provided with a detection patch containing said carrier and said antibody.

7. A detection member for detecting an analyte as described in claim 1, which is a wiper that contains a detection reagent containing the carrier and the antibody and that wipes a surface that comes into contact with a detection target that may contain or have the analyte attached thereto.

8. A detection member for detecting an analyte as described in claim 1, which is a detection reagent containing the carrier and the antibody and which is sprayed onto a wiper after wiping a surface that has come into contact with a detection target that may contain or be attached to the analyte.

9. The detection member for detecting an analyte according to claim 1, which is a saliva test stick having a detection patch containing said carrier and said antibody.

10. The object detection device according to claim 1, which is a detection and display device to be attached to an air conditioner.

11. The object detection member according to claim 1, which is a detection and indicating member attached to a desk-mounted humidifier.

12. A method for detecting an analyte using a detection member for an analyte, the detection member comprising: an agglutinable carrier carrying a DNA aptamer that specifically binds to the analyte; and an antibody that binds to the analyte and has a labeling group that emits light of a specific wavelength under specific conditions, the method comprising the steps of: bringing the detection member into contact with an object to be detected that may contain or be attached to the analyte; agglutinating and recovering the carrier carrying the DNA aptamer that specifically binds to the analyte, the carrier being contained in the contacted detection member and that is contained in the contacted detection member; bringing the recovered carrier to which the analyte is bound into contact with an antibody that binds to the analyte and that is labeled with a labeling group that emits light of a specific wavelength when irradiated with light of a specific wavelength, to form an analyte-labeled antibody complex; and detecting the analyte by irradiating the formed analyte-labeled antibody complex with light of a specific wavelength and detecting the emitted light.

13. The method for detecting an analyte according to claim 12, wherein in the step of recovering the agglutinated carriers, the carriers are recovered using an instrument having a magnet.

14. A method for detecting an analyte as described in claim 12, wherein in the step of detecting the analyte, a detection patch containing the carrier and the antibody and provided on an inner sheet of a mask is irradiated with light of a predetermined wavelength.

15. A method for detecting an analyte as described in claim 12, in which, in the step of forming an analyte-antibody complex, the antibody is contained in a wiper, and an analyte-antibody complex is formed by spraying a detection reagent containing a carrier carrying a DNA aptamer that specifically binds to the analyte onto the wiper that has wiped a surface that has come into contact with the object to be detected, which surface may contain or have the analyte attached thereto.