Method for detecting target substance and device for detecting target substance

Abstract

To provide a method for highly sensitive detection of an antigen such as a specific protein of a biological substance including a virus or a cell.SOLUTION: According to one embodiment of the present disclosure, a method for detecting a target substance is provided. The method includes: providing a metal oxide nanowire with a surface modified with gold; modifying the gold with a first antibody of the target substance; capturing the target substance with the first antibody; capturing the target substance with a second antibody of the target substance with an optical label; and detecting the optical label.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to techniques for detecting a substance of interest. [Background technology]

[0002] There is a demand for early detection of various diseases. For example, early detection of viral infections is highly desirable because they spread and spread rapidly.

[0003] As an example, dengue fever, a viral infection transmitted by the Aedes aegypti mosquito, has become a global public health concern due to its increasing prevalence in tropical and subtropical regions (Non-Patent Documents 1 and 2). However, there are currently no effective antiviral treatments for dengue fever (Non-Patent Document 3). Therefore, early diagnosis of dengue fever is essential for its effective management, especially in remote areas with limited access to diagnostic facilities.

[0004] Dengue nonstructural 1 protein (hereinafter referred to as "dengue NS1 protein") has emerged as a promising biomarker for the early detection of dengue virus infection. It has also been shown to play an important role in causing vascular leakage symptoms in severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Non-Patent Documents 7 and 8). Dengue NS1 protein exists in both membrane-bound and soluble secreted forms. Highly stable dimers and lipid-associated hexamers are soluble and present in infected body fluids (Non-Patent Documents 4 to 6). Therefore, detecting dengue NS1 protein at an early stage can improve early diagnosis and patient management at the point of care.

[0005] Non-invasive tests, such as urine tests, are generally safer than invasive tests that require inserting a needle or probe into the body. They can be easily collected and transported to a laboratory for analysis, reducing the discomfort experienced by patients. In the case of dengue fever, non-invasive diagnosis is particularly important because it is a viral infection that can affect large numbers of people, especially in resource-limited settings.

[0006] It has been shown that dengue NS1 can be detected in urine samples even when it is undetectable at serum levels (Non-Patent Document 9), and further studies have provided evidence that dengue NS1 protein in urine correlates with disease severity, which is the development of DHF in patients due to plasma leakage (Non-Patent Documents 10 and 11). However, both of these studies had to be designed and calibrated for serum or blood. Furthermore, application of enzyme-linked immunosorbent assay (ELISA) to urine samples, although showing relatively high specificity, currently has low sensitivity (Non-Patent Documents 9 to 12). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Bhatt, S. et al. The global distribution and burden of dengue. Nature 496, 504-507 (2013). [Non-patent document 3] World Health Organization, WPR Update on the Dengue situation in the Western Pacific Region. (World Health Organization, 2023). [Non-patent document 4] Paz-Bailey, G. et al. Dengue Vaccine: Recommendations of the Advisory Committee on Immunization Practices, United States, 2021. MMWR Recomm Rep 70, 1-16 (2021). [Non-patent document 4] Flamand, M. et al. Dengue Virus Type 1 Nonstructural Glycoprotein NS1 Is Secreted from Mammalian Cells as a Soluble Hexamer in a Glycosylation-Dependent Fashion. Journal of Virology 73, 6104-6110 (1999). [Non-Patent Document 5] Gutsche, I. et al. Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proceedings of the National Academy of Sciences 108, 8003-8008 (2011). [Non-Patent Document 6] Shu, B. et al. CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever. Nature Communications 13, 6756 (2022). [Non-Patent Document 7] Avirutnan, P. et al. Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement. The Journal of Infectious Diseases 193, 1078-1088 (2006). [Non-Patent Document 8] Beatty, P. R. et al. Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination. Sci Transl Med 7, 304ra141 (2015). [Non-Patent Document 9] Saito, Y. et al. Detecting Dengue Virus Nonstructural Protein 1 (NS1) in Urine Samples Using ELISA for the Diagnosis of Dengue Virus Infection. Japanese Journal of Infectious Diseases 68, 455-460 (2015). [Non-Patent Document 10] Chuansumrit, A. et al. Dengue nonstructural protein 1 antigen in the urine as a rapid and convenient diagnostic test during the febrile stage in patients with dengue infection. Diagnostic Microbiology and Infectious Disease 71, 467-469 (2011). [Non-Patent Document 11] Andries, A.-C. et al. Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens. PLOS Neglected Tropical Diseases 9, e0004100 (2015). [Non-Patent Document 12] Korhonen, EM, Huhtamo, E., Virtala, A.-MK, Kantele, A. & Vapalahti, O. Approach to non-invasive sampling in dengue diagnostics: Exploring virus and NS1 antigen detection in saliva and urine of travelers with dengue. Journal of Clinical Virology 61, 353-358 (2014). Summary of the Invention

[0008] The detection of dengue fever virus is just one example, and similar challenges are recognized for other infectious diseases and even non-infectious diseases such as cancer. Thus, there is a need for a highly sensitive method for detecting antigens such as specific proteins in biological materials such as viruses and cells.

[0009] In some embodiments of the present disclosure, a method for detecting a substance of interest is provided. The method includes providing a metal oxide nanowire having a gold-modified surface. The method includes modifying the substance of interest with a first antibody, allowing the first antibody to capture the substance of interest; and allowing a labeled second antibody to capture the substance of interest. In some embodiments, the method further includes detecting the label. [Effects of the Invention]

[0010] This makes it possible to detect target substances with high sensitivity. For example, gold (Au) can easily be bonded to biomolecules such as antibodies via thiol bonds, improving the detectability and sensitivity of target substances.

[0011] The above effects are not necessarily limiting, and any of the effects described in this specification, or other effects that can be understood from this specification, may be achieved in addition to or instead of the above effects. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows a schematic diagram illustrating a process of a method for detecting a target substance according to an embodiment. [Figure 2] 1 shows a nanowire surface before gold modification (a), a STEM image of the nanowire surface after gold modification (b), and the size distribution of gold nanoparticles (c), according to one example. [Figure 3] 1 shows an optical photograph of a well plate for a plate reader with nanowire structures placed thereon, according to one embodiment. [Figure 4] 1 shows calibration curves for an untreated (bare) quartz substrate ("bare substrate") (a), an Au-unmodified ZnO nanowire substrate (ZnO) (b), and an Au-modified ZnO nanowire substrate (ZnO / Au) (c), according to one example. [Figure 5] 1 shows a calibration curve for a comparative example. [Figure 6] Fluorescence intensities are shown for one example of a bare quartz substrate (no Dengue NS1 sample) "Bare No target", gold-unmodified ZnO nanowires (with Dengue NS1 sample) "ZnO w / target", gold-modified ZnO nanowires (without Dengue NS1 sample) "ZnO / Au No target", and gold-modified ZnO nanowires (with Dengue NS1 sample) "ZnO / Au w / target". [Figure 7] 1 shows the optical density of a urine sample using an ELISA according to a comparative example (a) and the fluorescence intensity of a urine sample according to an example (b). [Figure 8] 1A-1C show perspective views of a substrate to schematically illustrate a process for growing nanowires on a substrate, including patterning, according to one embodiment. [Figure 9] 1 shows SEM images of random, non-epitaxial branched nanowires according to one embodiment and epitaxial branched nanowires according to one embodiment. [Figure 10] 1 shows an SEM image of Au nanoparticles formed on the surface of ZnO nanowires according to one example. [Figure 11]1A shows a schematic diagram and (B) an optical photograph of a fluidic device according to one embodiment. [Figure 12] 1 shows a bright-field image (a), a fluorescent image (b), a composite image (c), and a three-dimensional image (d) of Dengue NS1 protein captured by a nanowire, according to one embodiment. [Figure 13] Fluorescence intensities are shown for Au-unmodified random branched nanowires (Rnd-BZnO), Au-unmodified epitaxial branched nanowires (Epi-BZnO), Au-modified random branched nanowires (Rnd-BZnO / Au), and Au-modified epitaxial branched nanowires (Epi-BZnO / Au). [Figure 14] 1 shows the fluorescence intensity for each configuration, illustrating the effect of various factors on the fluorescence intensity, according to one example. [Figure 15] 1 shows the concentration dependence of fluorescence intensity for epitaxial branched nanowires, according to one example. [Figure 16] 1 shows the fluorescence intensity of each urine sample according to one example. [Figure 17] 1 shows visible light absorption spectra of ZnO nanowires not modified with gold, ZnO nanowires modified with gold, and ZnO nanowires modified with a first antibody and modified with gold, according to one example. DETAILED DESCRIPTION OF THE INVENTION

[0013] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced with one or more of these specific details. In other instances, features and procedures well known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0015] <Target substances> As used herein, the term "substance of interest" refers to an antigen that can be bound by an antibody that can be used in the technology of the present disclosure, or a biological substance that has an antigen. Examples of antigens include, but are not limited to, molecules or molecular structures (parts of molecules) such as proteins, peptides, polysaccharides, lipids, and nucleic acids.

[0016] As used herein, the term "biological material" refers collectively to high molecular weight organic compounds contained in living organisms or artificially synthesized that function in biological phenomena. Examples of biological materials include, but are not limited to, peptides, proteins, lipids, nucleic acids, hormones, sugars, amino acids, extracellular organelles, vesicles, cells, bacteria, fungi, viruses, and pathogens. Biological materials may be complexes of biomolecules, such as protein complexes or multiprotein complexes.

[0017] Examples of extracellular organelles include, but are not limited to, mitochondria. Examples of vesicles include, but are not limited to, vacuoles, lysosomes, transport vesicles, secretory vesicles, gas vesicles, extracellular matrix vesicles, extracellular vesicles, etc. Examples of extracellular vesicles include, but are not limited to, exosomes, exotomes, shedding microvesicles, microvesicles, membrane particles, plasma membranes, apoptotic blebs, etc.

[0018] Examples of cells include, but are not limited to, cells having a cell membrane structure, specifically blood cells such as granulocytes, lymphocytes, reticulocytes, red blood cells, white blood cells, and platelets.

[0019] Examples of bacteria include, but are not limited to, bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Shigella, Bacillus anthracis, Mycobacterium tuberculosis, Clostridium botulinum, Clostridium tetani, Streptococcus, and the like, melioidosis, and the like.

[0020] Examples of fungi include, but are not limited to, mushrooms, molds, yeasts, and the like, and specifically include Trichophyton, Candida, Aspergillus, Saccharomyces cerevisiae, and the like.

[0021] Examples of viruses include, but are not limited to, dengue virus, norovirus, rotavirus, influenza virus, adenovirus, coronavirus, measles virus, rubella virus, hepatitis virus, herpes virus, human immunodeficiency virus (HIV), Zika virus, and the like.

[0022] Examples of pathogens include those endemic to Southeast Asia and other regions.

[0023] In this specification, the target substance to be captured and detected by the nanowire is described as a "biological substance," but it is not limited to this as long as it can be captured by an antibody. The target substance may also be an artificial substance, a non-biological substance, etc.

[0024] As used herein, biological material may be in a liquid, gas, vapor, aerosol, or in the breath of a subject. The subject material may be from an animal, including a human, mouse, rat, rabbit, cat, dog, cow, horse, pig, monkey, etc.

[0025] In some embodiments, the target substance may be provided in a solution. A solution containing the target substance may be introduced into the nanowire. The solution may be a body fluid or a body-derived liquid (e.g., a dilution solution or a treatment solution). The solution may be a non-body fluid (non-body fluid-derived) solution, an artificially prepared liquid, or a mixture of a body fluid or a body-derived solution and a non-body fluid-derived solution. The solution may be a solution used for sample measurement or a solution used for calibration measurement. The solution may be used as is, or may be a diluted or concentrated version of the original solution. The solution may be a standard solution or a calibration solution. The sample to be measured may be a specimen. The solution may contain a physiological buffer solution, such as phosphate-buffered saline (PBS) or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES), containing the substance to be recovered. The body fluid may contain additives, such as stabilizers or pH adjusters.

[0026] The "body fluid" may be a solution. The body fluid may be in a liquid state or in a solid state, such as a frozen state. The solution may contain a target substance to be recovered, such as a biomolecule, or may not contain the target substance, and may contain a substance for measuring the target substance to be recovered.

[0027] The body fluid may be that of an animal. The animal may be a reptile, a mammal, or an amphibian. The mammal may be a dog, a cat, a cow, a horse, a sheep, a pig, a hamster, a rat, a squirrel, or a primate, such as a monkey, a gorilla, a chimpanzee, a bonobo, or a human.

[0028] Body fluids may be intracellular fluids or extracellular fluids such as blood, lymph, tissue fluids (interstitial fluid, intercellular fluid, interstitial fluid), and body cavity fluids (serous cavity fluid, pleural fluid, peritoneal fluid, pericardial fluid, cerebrospinal fluid, synovial fluid, aqueous humor, etc.). Body fluids may be digestive fluids such as saliva, gastric juice, bile, pancreatic juice, and intestinal juice, as well as sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, and milk.

[0029] "Urine" refers to the liquid waste produced by the kidneys. Urine may be a liquid or substance excreted externally through the urethra or accumulated in the bladder. "Saliva" refers to a secretion secreted into the oral cavity by the salivary glands.

[0030] Body fluids may be collected, extracted, collected, etc. invasively (hereinafter sometimes simply referred to as collection) or non-invasively. They may be extracted, collected, or harvested from the body using an extractor such as a syringe. The solution may be the body fluid of a healthy subject, the body fluid of a subject with a specific disease, the body fluid of a subject suspected of having a specific disease, or the body fluid of a subject being tested for disease.

[0031] For example, although it is said that no virus is present in a urine sample, proteins such as NS1 are present. Also, although viruses may be present in blood, proteins such as NS1 are also present. Therefore, target substances (e.g., proteins) contained in or floating in blood or urine may be captured with nanowires.

[0032] The raw solution containing the target substance may be processed after collection. For example, cells, viruses, bacteria, etc. may be lysed using a cell lysis solution (such as a lysis buffer). The target substance released from the interior or surface of the cells, etc., can be captured and detected by the nanowire.

[0033] In some embodiments, the disease may be an infectious disease. Examples of infectious diseases and their pathogens include, but are not limited to: Bacterial infections: Streptococcus (group A beta-hemolytic streptococcus, pneumococcus, etc.), Staphylococcus aureus (methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA)), Staphylococcus epidermidis, Enterococcus faecalis, Listeria, Neisseria meningitidis, Neisseria gonorrhoeae, pathogenic Escherichia coli (O157:H7, etc.), Klebsiella pneumoniae, Proteus, Bordetella pertussis, Pseudomonas aeruginosa , Serratia marcescens, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, tuberculosis and non-tuberculous mycobacteria, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, syphilis, tetanus, leprosy, Legionnaires' pneumonia (legionnaires' disease), leptospirosis, salmonella, typhoid, paratyphoid, Lyme disease, tularemia, Q fever, etc. Rickettsia infections: typhus, scrub typhus, Japanese spotted fever, etc. Chlamydia infections: Chlamydia pneumonia, trachoma, genital chlamydia infection, psittacosis, etc. Fungal infections: aspergillosis, candidiasis, cryptococcosis, tinea mycosis, histoplasmosis, Pneumocystis pneumonia (formerly known as Pneumocystis carinii pneumonia), etc. Parasitic protozoan infections: amoebic dysentery, malaria, toxoplasmosis, leishmaniasis, cryptosporidium, etc. Parasitic helminth infections: echinococcosis, schistosomiasis japonicum, filariasis, ascariasis, diphyllobothriasis, etc. Viral infections: influenza, viral pneumonia, viral hepatitis, viral meningitis, viral gastroenteritis, viral conjunctivitis, acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, yellow fever, common cold syndrome, rabies, cytomegalovirus infection, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), coronavirus disease (COVID-19), novel bunyaviridae virus, severe fever with thrombocytopenia syndrome, progressive multifocal leukoencephalopathy, chickenpox, shingles, herpes simplex, hand, foot and mouth disease, dengue fever, Zika fever, Japanese encephalitis, erythema infectiosum, infectious mononucleosis, smallpox, rubella, acute poliomyelitis (polio), measles, pharyngoconjunctival fever (swimming pool fever), Marburg hemorrhagic fever, hemorrhagic fever with renal syndrome, Lassa fever, mumps, West Nile fever, herpangina, chikungunya fever, etc. Prion diseases / transmissible spongiform encephalopathies: Bovine spongiform encephalopathy (BSE), kuru, Creutzfeldt-Jakob disease, fatal familial insomnia (FFI), Gerstmann-Straussler-Scheinker syndrome (GSS), etc.

[0034] In some embodiments, the disease may be cancer. The cancer may be a solid cancer or a blood cancer. The solid cancer may be an epithelial cancer or a non-epithelial cancer. The cancer may be, for example, but not limited to, hematopoietic cell malignancies, leukemia, lymphoma, multiple myeloma, brain cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck cancer (such as laryngeal cancer, oral cancer, salivary gland cancer, and paranasal sinus cancer), thyroid cancer, kidney cancer, lung cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, prostate cancer, testicular tumor, renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer, anal cancer, and the like, or may be selected from the group consisting of any two or more thereof.

[0035] <Nanowire> As used herein, a "nanowire" generally refers to a linear member having an elongated direction, the size of which is on the nanometer level in the direction perpendicular to the elongated direction. A "nanowire" may also be a "whisker."

[0036] The nanowire may have branches or branches. The nanowire may have a trunk nanowire and branch nanowires extending from the trunk wire. The nanowire may have a multi-branched structure. The nanowire may have a single, unbranched structure (only a trunk nanowire) with no branched chains. The multi-nanofibers may include branched nanowires and unbranched nanowires.

[0037] The length of the nanowires can be greater than or equal to values such as, but not limited to, 500 nm, 1 μm, 1.5 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 17 μm, 20 μm, etc. The length of the nanowires can be less than or equal to values such as, but not limited to, 1 μm, 1.5 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 17 μm, 20 μm, 50 μm, 100 μm, 200 μm, etc.

[0038] The diameter (or size in the direction of thickness) of the nanowires can be greater than or equal to values such as, but not limited to, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, etc. The diameter (or size in the direction of thickness) of the nanowires can be less than or equal to values such as, but not limited to, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, 1 μm, etc.

[0039] As used herein, a "nanowire" is composed at least in part or entirely of an oxide of a metal or semiconductor (collectively referred to as a "metal oxide"). Examples of nanowire materials include, but are not limited to, main group metals (alkali metals: Li, Na, K, Rb, Cs; alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg, aluminum group elements: Al, Ga, In, rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu, tin group elements: Ti, Zr, Sn, Hf, Pb, Th, iron group elements: Fe, Co, Ni, earth elements: V, Nb, Ta, chromium group elements: Cr, Mo, W, U, manganese group elements: Mn, Re, precious metals (copper group, coinage metals): Cu, Ag, Au, platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, naturally occurring radioactive elements: radioactive decay products of U and Th: U, Th, Ra, Rn, actinides, transuranium elements: Np, Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No, etc., elements after uranium, or alloys thereof, etc. The nanowires may be or contain an oxide of any one of the above metals or alloys, or an alloy or mixture thereof. The material of the nanowires or at least the surface (e.g., coating) of the nanowires may be, but is not limited to, ZnO, SiO2, Li2O, MgO, Al2O3, CaO, TiO2, Mn2O3, Fe2O3, CoO, NiO, CuO, Ga2O3, SrO, In2O3, SnO2, Sm2O3, and EuO.

[0040] Examples of methods for growing nanowires or coating surfaces include, but are not limited to, pulsed laser deposition, physical vapor deposition such as VLS (Vapor-Liquid-Solid), ALD (Atomic Layer Deposition), CVD (Chemical-Vapor-Deposition), arc discharge, laser evaporation, metal organic vapor phase selective deposition, hydrothermal synthesis, reactive ion etching, baking, melting, sputtering, and the like.

[0041] Nanowires may be grown, for example, by hydrothermal synthesis. For example, when ZnO fine particles are used, they may be grown by hydrothermal synthesis. Specific examples include, but are not limited to, zinc nitrate hexahydrate (Zn(NO3)2·6HO), hexamethylenetetramine (CH6H6), and the like. 12 N4) is immersed in a precursor solution dissolved in deionized water, and the solution is heated and maintained at a constant temperature. This allows ZnO nanowires to grow from the growth origin (catalytic member or surface structure). The precursor solution may contain ammonia.

[0042] As used herein, the terms "gold modification" or "Au modification" refer to at least partially covering the surface of a metal oxide nanowire with gold, or to forming gold nanostructures (also referred to as "nanoparticles") on the surface of the metal oxide nanowire, or to a gold layer or gold nanoparticles formed on the nanowire surface. In the present disclosure, gold is primarily used as the metal for modifying the surface of the metal oxide nanowire, but this is not a limitation. Metals other than gold, such as, but not limited to, other noble metals (platinum, palladium, etc.), may also be used as long as they have a similar effect.

[0043] In some embodiments, metal nanoparticles may be disposed on the surface of the metal oxide nanowires. As used herein, the term "nanoparticle" generally refers to a structure having a size on the nanometer level. "Nanoparticles" generally have a diameter and thickness of a few nanometers, tens of nanometers, or hundreds of nanometers. In some embodiments, a thin metal film having a thickness on the nanometer level may be disposed on the surface of the metal oxide nanowires.

[0044] <Sandwich method> In this disclosure, the so-called sandwich method is applied. The detection method for a target substance in this disclosure will be described with reference to the schematic diagram in Figure 1. A first antibody (also called a capture antibody) 11 for the target substance is immobilized on a gold-modified nanowire surface 10 (Figure 1(a)). Next, the target substance 12 is introduced into the nanowire system and captured by the first antibody 11 on the nanowire surface 10 (Figure 1(b)). A second antibody 13 for the target substance, which is a labeled antibody (labeled antibody), is introduced into the nanowire system and binds to the target substance 12 captured by the first antibody 11 on the nanowire surface 10 (Figure 1(c)). This forms an antigen-antibody complex on the nanowire surface. In this state, the label 13 of the second antibody is detected using a predetermined detection device 15 (Figure 1(d)).

[0045] The order of the steps is not limited to the above. Other orders are also possible as long as an antigen-antibody complex is ultimately formed and the label can be detected. The formed antigen-antibody complex may be retained on the nanowire surface during detection, or may be removed from the nanowire surface and detected. Furthermore, other steps, such as washing, may be performed between each step. For example, washing can remove impurities or unwanted substances, reducing adverse effects on subsequent steps or increasing the sensitivity of final detection.

[0046] The first antibody and the second antibody must recognize different sites on the target substance. In some embodiments, the first antibody and the second antibody may recognize different types of antigens expressed on the target substance. In some embodiments, the first antibody and the second antibody may recognize the same type of antigen expressed on the target substance. For example, both the first antibody and the second antibody may comprise an anti-NS1 antibody and recognize the NS1 protein of dengue virus.

[0047] In some embodiments, the "label" of the labeled antibody may be an optical label. In some embodiments, the label may be a fluorescent label. For example, immunofluorescent staining, flow cytometry, and the like may be applied. Examples of fluorescent labels include, but are not limited to, fluorescent dyes such as fluorescein isothiocyanate (FITC), Cy dye, and AlexaFluor® dye, and fluorescent proteins such as phycoerythrin (PE) and allophycocyanin (APC). In some embodiments, the label may be an enzyme label. For example, Western blotting, immunostaining, ELISA, and the like may be applied. Examples of enzyme labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), and the like. Other methods such as biotin and immunoprecipitation may also be used.

[0048] Example 1: Unbranched ZnO nanowires decorated with Au nanoparticles In this example, we used unbranched stem nanowires with Au nanoparticles on their surfaces grown on a quartz substrate. This nanowire substrate was placed in a well for a plate reader. Dengue virus nonstructural protein 1 (hereinafter referred to as "dengue NS1 protein") attached to the ZnO / Au nanowires in the well was measured by sandwich ELISA. The details are described below.

[0049] The limit of detection (LOD) was calculated using the following formula:

number

[0050] The fluorescence intensity F as a function of Dengue NS1 protein concentration follows the formula:

number

[0051] <Preparation of nanowire device> On a quartz glass slide (Crystal Base, 15.4 mm diameter, 0.13 mm thickness), a ZnO seed layer was applied onto the substrate using radio frequency sputtering (Sanyu Electron).

[0052] ZnO nanowires were then grown in a hydrothermal growth solution at 95 °C for 3 h. The hydrothermal synthesis solution contained 30 mM hexamethylenetetramine (HMTA) (Wako Pure Chemical Industries, Ltd.) and zinc nitrate hexahydrate (Zn(NO3)2·6H2O) (Thermo Fisher Scientific Inc.).

[0053] After drying, Au nanoparticles were deposited on the ZnO nanowire surface for 5 minutes using a direct current (DC) sputter (SVC-700TMSG, Sanyu Electronics Co., Ltd.).

[0054] Figure 2 shows STEM images of Au-unmodified ZnO nanowires (Fig. 2(a)) and Au-modified ZnO (referred to as "ZnO / Au") nanowires (Fig. 2(b)). Au nanoparticles dispersed on the surface of the ZnO / Au nanowires were observed. STEM (scanning transmission electron microscope) / EDS (energy dispersive X-ray spectroscopy) mapping also confirmed that these were Au nanoparticles (not shown). The diameters of the Au nanoparticles ranged from 1 to 13 nm, with an average of 4 nm (Fig. 2(c)).

[0055] The slide glass with the ZnO / Au nanowires was placed in a 24-well plate. Figure 3 shows an optical photograph of the well plate with the nanowire substrate placed on it.

[0056] <Example 1-1: Measurement of artificially prepared dengue NS1 protein solution> <Sandwich method> A filtered PBS solution (pH 7.2) containing 100 μg / mL of anti-NS1 antibody was prepared. 100 μL of this solution (i.e., 10 μg of anti-NS1 antibody) was added to each well and incubated at room temperature in the dark for 3 hours. This allowed the antibody to modify the surface of the Au nanoparticles. The solution was then drained, and the wells were washed with filtered PBS. The modification of the gold nanoparticles by the antibody was confirmed by visible light spectroscopy.

[0057] Next, 100 μL of Dengue NS1 protein solution was introduced into the wells and allowed to incubate at room temperature in the dark for 1 hour, after which the solution was drained and the wells were washed with filtered PBS.

[0058] Next, an aqueous solution containing 10 μg of fluorophore-conjugated anti-NS1 antibody was added to the wells and incubated for 1 hour at room temperature in the dark. Finally, the wells were washed with filtered PBS. After that, filtered PBS was added to the wells and left for 10 minutes before detecting the fluorescent signal.

[0059] <Fluorescence intensity measurement> Fluorescence intensity was measured using a well plate reader (Tecan Trading AG). The fluorescence measurement area of the well was divided into 12 sections, and one section was selected and used to detect the fluorescence intensity of all samples.

[0060] Figure 4 shows the calibration curves for (a) bare quartz substrate ("bare substrate"), (b) unmodified ZnO nanowire substrate (ZnO), and (c) modified ZnO nanowire substrate (ZnO / Au). The vertical axis represents the fluorescence intensity (arbitrary units), and the horizontal axis represents the concentration of dengue NS1 protein.

[0061] The concentration of dengue NS1 protein in serum is said to vary from 600 ng / mL to 15 μg / mL. Furthermore, the amount of dengue NS1 protein in urine is 1 / 10 of that in serum. 5 From 1 / 10 3 Therefore, the concentration of the dengue NS1 samples in this experiment was set to 0.65 pg / mL to 10 ng / mL.

[0062] From these graphs, the LOD was 7.32 ng / mL for the "bare quartz substrate," 6.89 pg / mL for the "ZnO," and 1.35 pg / mL for the "ZnO / Au" substrate, using the average values and the above formula 1. Thus, the "ZnO / Au" substrate showed the lowest LOD.

[0063] <Comparative Example: Measurement of Dengue Virus NS1 Using a Commercially Available ELISA Kit> The dengue virus stock solution (aqueous solution of virus) was confirmed using a commercially available dengue virus NS1 ELISA kit (Arigo Laboratories). This method is a sandwich ELISA, measuring the color change caused by an enzymatic reaction. The prepared samples were stored at -80°C. Adding 50 μL of quenching solution before measurement changed the color of the horseradish peroxidase (HRP)-labeled antibody from blue to yellow. Optical density measurements were immediately performed at 450 nm using a plate reader.

[0064] The calibration curve is shown in Figure 5. The vertical axis represents optical density (OD), and the horizontal axis represents the concentration of the dengue virus NS1 sample. In this experiment, concentrations ranging from approximately 1 ng / mL to approximately 10 ng / mL were used. The LOD was 6.19 ng / mL.

[0065] Thus, it was revealed that "ZnO / Au" exhibited an extremely low LOD.

[0066] Figure 6 shows the fluorescence intensity for a bare quartz substrate (no Dengue NS1 sample) ("Bare No target"), uncoated ZnO nanowires (with Dengue NS1 sample) ("ZnO w / target"), gold-coated ZnO nanowires (without Dengue NS1 sample) ("ZnO / Au No target"), and gold-coated ZnO nanowires (with Dengue NS1 sample) ("ZnO / Au w / target"). The sample concentration was 650 pg / mL for the "with Dengue NS1 sample" and "w / target" cases. In the figure, "ns" indicates p-value > 0.05, meaning no significance, "***" indicates p-value < 0.001, and "****" indicates p-value < 0.0001.

[0067] The fluorescence intensity of "ZnO / Au w / target" was by far the highest among the other systems. The intensity of "ZnO / Au w / target" with sample was higher than that of "ZnO / Au no target" without sample. The fluorescence intensity of "ZnO / Au no target" without sample was higher than that of "bare no target." This is likely due to the presence of FITC-Anti-NS1 residues in the void spaces of the ZnO / Au nanowires. Nevertheless, the intensity of "ZnO / Au w / target" with sample was significantly higher than that of "ZnO / Au no target" without sample. These results confirmed that the ZnO nanowire system with Au nanoparticles efficiently captured dengue NS1 protein and enhanced its detection sensitivity.

[0068] <Example 1-2: Measurement of real urine samples> Blood and urine samples were obtained from patients with dengue fever symptoms (specifically, fever) at a hospital in Thailand. A commercially available lateral flow test kit was used to determine whether IgG, IgM, and NS1 in the blood samples were positive or negative. If at least one of IgM and NS1 was positive, the patient was considered dengue fever "positive." If neither IgM nor NS1 was positive, the patient was considered dengue fever "negative" (see Table 1). Additionally, three healthy individuals, i.e., individuals without at least dengue fever symptoms, were selected, and blood and urine samples were obtained from these three individuals.

[0069] The three "dengue positive" patients or samples from them are called P1, P2, and P3. The three "dengue negative" patients or samples from them are called N1, N2, and N3. The three healthy individuals or samples from them are called H1, H2, and H3.

[0070] First, as a comparative example, urine samples H1-H3, N1-N3, and P1-P3 were measured using a commercially available dengue virus NS1 ELISA kit. Figure 7(a) shows the optical density (OD) at 540 nm. The optical density was below the LOD for all samples, including the "positive" samples P1-P3.

[0071] On the other hand, the same urine sample was measured using the ZnO / Au nanowire-FLISA. First, the urine sample was diluted 100-fold with 1% Triton-X-100 in PBS, which disrupted the virus and isolated the NS1 protein.

[0072] Figure 7(b) shows the fluorescence intensities of H1 to H3, N1 to N3, and P1 to P3 measured using the ZnO / Au nanowire-FLISA of the present disclosure. First, the quantitative detection of urine using this method can be said to be in the range of 2 to 8 pg / mL (Table 1). [Table 1]

[0073] The intensities of the urine samples from healthy individuals (H1 to H3) were lower than the LOD of this measurement system and were judged to be "not detected."

[0074] The intensities of the "positive" urine samples (P1-P3) were higher than the LOD, confirming the detection of dengue NS1 protein. This was significantly different from the commercial kit. The concentrations were calculated as 5.37 pg / mL (P1), 7.67 pg / mL (P2), and 2.03 pg / mL (P3).

[0075] In the "negative" urine samples (N1-N3), the mean intensity of N1 was lower than the LOD, but the intensities of N2 and N3 were higher than the LOD. Based on these results, patients with N2 and N3 are judged to be positive. As a trend, N1-N3 can be said to have shown a possible positive result. Considering that the LOD of the ZnO / Au nanowire-FLISA measurement system is at the picogram level, N2 and N3 can be judged to be positive for dengue fever.

[0076] Patients N1 to N3 showed symptoms consistent with dengue fever at the hospital. Their blood samples tested negative, while their urine samples tended to be positive. There are two possible reasons for this. First, the commercially available lateral flow strip kit used for the blood test may have been non-quantitative and insufficiently sensitive. Second, the presence of dengue NS1 protein in urine does not necessarily correlate with its presence in blood. However, these considerations are the inventors' current speculations, and the results should not be interpreted as being limited to these. Other scientific or rational considerations may also be possible.

[0077] Example 2: Au-modified branched ZnO nanowires In this example, a branched trunk nanowire with Au nanoparticles on the surface grown on a patterned quartz substrate was used.

[0078] <Preparation of nanowire device> The substrate surface was patterned to define the areas for growing stem ZnO nanowires. A specific example is described with reference to Figure 8. A THMR ip3300 photoresist was applied to the surface of a quartz glass substrate (Figure 8(a)) to a thickness of 0.8 μm (Figure 8(b)). Photolithography was then performed using a photomask and i-line UV irradiation (Figure 8(c)). This resulted in the formation of multiple linear patterns with a width of 1 μm and spaced at intervals of 40 μm (Figure 8(d)). A ZnO seed layer was applied by RF sputtering (Figures 8(e) and 8(f)). The substrate was then immersed in acetone and ultrasonically removed the photoresist. As a result, multiple linear ZnO seed layer patterns with a width of 1 μm and spaced at intervals of 40 μm were formed on the surface of the quartz glass substrate (Figure 8(g)). Branched ZnO nanowires were then grown on the surface of the linear ZnO seed layer patterns (Figure 8(h)). The SEM image from the 50° direction is shown in Figure 8(h).

[0079] The branch nanowires were grown by two methods: first, random growth and second, epitaxial growth.

[0080] <Formation of randomly branched nanowires> 1) Growth of ZnO nanowires In this example, trunk ZnO nanowires were grown by hydrothermal synthesis. An aqueous solution containing 10 mM Zn precursor, 10 mM HMTA, and 600 mM ammonia was prepared. The substrate with the ZnO seed layer was immersed in the solution at 95°C for 6 hours. As a result, trunk ZnO nanowires with lengths of approximately 8.5 μm to 10 μm were formed. Figure 9(A) shows an SEM image of the side view.

[0081] 2) Formation of seed layer for branch nanowire growth A seed growth solution (a 30 mM NaOH ethanol solution and a 10 mM Zn precursor ethanol solution were mixed and heated at 60°C for 2 hours to prepare a seed solution. See the method described in Non-Patent Document 2) was prepared for the trunk nanowire. This seed solution was spin-coated onto the trunk nanowire substrate to form a seed layer on its surface.

[0082] 3) Growth of branch nanowires Aqueous solutions containing 40 mM Zn precursor, 40 mM HMTA, and 400 mM ammonia were prepared, and the substrate with the ZnO seed layer was immersed in the solutions at 95° C. for 6 hours.

[0083] <Formation of epitaxial branched nanowires> In the formation of the randomly branched nanowires, step 2) was performed immediately after step 1) without forming a seed layer. The branch nanowires grew epitaxially with respect to the crystals of the trunk nanowire.

[0084] Figure 9 shows SEM images of randomly branched nanowires (Figure 9(A)) and epitaxially branched nanowires (Figure 9(B)).

[0085] Au nanoparticles were deposited on the ZnO nanowire surface for 5 minutes using a direct current (DC) sputter (SVC-700TMSG, Sanyu Electronics Co., Ltd.). Figure 10 shows a scanning electron microscope (SEM) image. It was confirmed that Au nanoparticles with an average size of 11 nm were formed (not shown).

[0086] PDMS was used to form the substrate 1101 with the flow channels to be bonded to the nanowire substrate. A 130-μm-deep cavity 112 with a herringbone structure was formed using stamp and stick bonding (Figure 11(A)). Next, the PDMS substrate 111, coated with a wet film, was bonded to the nanowire substrate 115, left at room temperature for 15 minutes, and then heated at 120°C for 10 minutes. This resulted in a tight bond between the two substrates. 0.5-m PEEK tubing (Japan Micro Chemical Technology Laboratory) was connected to the inlet 113 and outlet 114 at both ends of the flow channel in the PDMS substrate and sealed with prepolymer, forming the inlet and outlet for the fluid flow channel. Figure 11(B) shows an optical photograph of the nanowire fluidic device fabricated in this way, with four parallel flow channels.

[0087] <Example 2-1: Measurement of artificially prepared dengue NS1 protein solution> <Sandwich method> The fluorescent immunoassay was performed in three steps. First, a solution of antibody to mouse dengue NS1 protein was introduced into the flow channel, and Au nanoparticles were attached to the anti-NS1 antibody. Next, a solution of the target substance, dengue NS1 protein, was introduced into the flow channel, and the anti-NS1 antibody was allowed to capture the NS1 protein. Finally, a solution of FITC-anti-NS1 antibody (F Anti-NS1) was introduced, and the FITC-anti-NS1 antibody was allowed to recognize the NS1 protein.

[0088] <Fluorescence measurement> In this experiment, the PDMS substrate was removed from the nanowire substrate and observed under a fluorescence microscope. Figure 12 shows a bright-field image (a), a fluorescent image (b), a composite image (c), and a three-dimensional image (d). The presence of FITC-anti-NS1 antibody was confirmed in the linear region where the nanowires were arranged.

[0089] Figure 13 shows the fluorescence intensity for the Au-unmodified random branched nanowires (Rnd-BZnO), Au-unmodified epitaxial branched nanowires (Epi-BZnO), Au-modified random branched nanowires (Rnd-BZnO / Au), and Au-modified epitaxial branched nanowires (Epi-BZnO / Au). In the figure, "ns" means p-value > 0.05, not significant, and "****" means p-value < 0.0001.

[0090] Thus, the Au-modified branched nanowires ("Rnd-BZnO / Au" and "Epi-BZnO / Au") clearly showed higher sensitivity than the non-Au-modified branched nanowires ("Rnd-BZnO" and "Epi-BZnO"). This confirmed that the Au-modified branched nanowires have high sensitivity.

[0091] Next, among the Au-modified branched nanowires ("Rnd-BZnO / Au" and "Epi-BZnO / Au"), the fluorescence intensity of "Epi-BZnO / Au" was clearly higher than that of "Rnd-BZnO / Au".

[0092] As shown in Figure 14, it was confirmed that the Au modification (Figure 14(A)), the use of sandwich antibodies (Figure 14(B)), and the branched structure of the branched nanowires (Figure 14(C)) all play important roles in achieving high sensitivity. In the figure, "****" indicates a p-value of <0.0001.

[0093] Figure 15 shows the concentration dependence of fluorescence intensity for epitaxial branched nanowires. The vertical axis represents fluorescence intensity, and the horizontal axis represents the NS1 solution. The NS1 concentration was 0 to 0.7 ng / mL. Using formula (1), the LOD was 18.46 fg / mL. This demonstrates that NS1 can be detected with extremely high sensitivity by using the method and device of the present disclosure.

[0094] The LOD for each method is shown in Table 2. As is clear from this, the method and device of the present disclosure can be fully applied not only to invasive samples but also to non-invasive samples. [Table 2]

[0095] <Example 2-2: Measurement of real urine samples> Urine samples were collected in the same manner as in Examples 1-2, classified according to the blood samples, and diluted and adjusted.

[0096] Among the "positive" subjects P01 to P09, blood samples from P03, P04, and P07 to P09 did not show NS1 positivity. Furthermore, urine samples from P03 to P08 were either negative (P04, P06, and P07) or undetectable (P03, P05, and P08) using commercially available ELISA kits. In contrast, with this method, urine samples from healthy subjects H01 to H03 and negative subject N01 showed fluorescence intensities below the LOD, and urine samples from all positive subjects P01 to P09, as well as all urine samples from P01 to P09, showed fluorescence intensities above the LOD (Table 2, Figure 16).

[0097] A comparison with other methods is shown in Table 3. As is clear from this comparison, it was confirmed that the LOD of the method of the present disclosure is overwhelmingly low. [Table 3]

[0098] As in Example 1, this example clearly demonstrates that highly sensitive antigen detection is possible by combining Au-modified nanowires and branched nanowires with sandwich antibodies (ZnO / Au nanowire-FLISA).

[0099] <Plasmon resonance> There is no single mechanism that enables highly sensitive detection. However, one mechanism that the inventors hypothesize is plasmon resonance. Figure 17 shows the visible light absorption spectra of unmodified ZnO nanowires, gold-modified ZnO nanowires, and gold-modified ZnO nanowires modified with a primary antibody. While the unmodified ZnO nanowire showed no absorption band in the visible light range, the two gold-modified ZnO nanowires showed absorption bands in the 500-550 nm range. This may suggest that the gold nanoparticles absorb and scatter light due to collective oscillations of incident light with conduction electrons, resulting in surface plasmon resonance.

[0100] A distance of 10 nm or more between the gold nanoparticles and the fluorophore is necessary to prevent plasmonic quenching. Dengue NS1 protein exists in dimers, tetramers, hexamers, and other forms. These are approximately 5 nm to 10 nm in size. Therefore, in the above example, when the sizes of the first and second antibodies are added, the distance from the gold nanoparticles to the fluorophore is greater than the distance at which plasmonic quenching occurs. This may be why the fluorescent signal was enhanced.

[0101] In some embodiments, the detection method may involve plasmon resonance. Plasmon resonance may well increase signal strength and sensitivity. However, this disclosure itself should not be construed as being limited to one mechanism. The above is part of a scientific consideration. The distance between the gold nanoparticle and the fluorophore should also not be limited to 10 nm or the distances known in the scientific literature at the time of filing. If not limited to plasmon resonance, other mechanisms and distances are possible.

[0102] The present disclosure also includes the following embodiments: A001 1. A method for detecting a substance of interest, comprising: Providing metal oxide nanowires having a gold-modified surface; modifying the gold with a first antibody to the substance of interest; allowing the first antibody to capture the substance of interest; allowing a second antibody of the target substance having an optical label to capture the target substance; and detecting the optical label; A method for providing the above. A011 A001, or the method of any embodiment, comprising: The gold is gold nanoparticles. method. A021 A001, or the method of any embodiment, comprising: The metal oxide is ZnO. method. A031 A001, or the method of any embodiment, comprising: the optical label is a fluorescent label; method. A032 A031, or the method of any embodiment, comprising: the optical label comprises a fluorophore; method. A041 A001, or the method of any embodiment, comprising: The nanowire has a trunk nanowire and branch nanowires branching from the trunk nanowire. method. A042 A041, or the method of any embodiment, comprising: the branch nanowires are formed from the trunk nanowire by epitaxial growth; method. A043 A041, or the method of any embodiment, comprising: the branch nanowires are formed by growing in random directions from the trunk nanowire; method. A051 A001, or the method of any embodiment, comprising: The target substance is a biological substance. method. A052 A051, or the method of any embodiment, comprising: The target substance is a virus or a cell. method. A053 A052, or the method of any embodiment, comprising: the first antibody (capture antibody) is an antibody against a first protein expressed on the surface of the virus or the cell, the second antibody (labeled antibody) is an antibody against a second protein expressed on the surface of the virus or the cell; method. A054 A053, or the method of any embodiment, comprising: the second antibody is optically labeled; method. A055 A053, or the method of any embodiment, comprising: the substance of interest is a dengue virus or a dengue NS1 protein, the first antibody and the second antibody are anti-NS1 antibodies; method. B001 1. A device for detecting a substance of interest, comprising: a metal oxide nanowire having a gold-modified surface; a fluid reservoir that accommodates the metal oxide nanowires; Equipped with the gold-modified surface is modified with a first antibody to the substance of interest; allowing the first antibody to capture the substance of interest; allowing a second antibody of the target substance having an optical label to capture the target substance; Devices configured to: B011 B001, or the device of any embodiment, The device is a cartridge for a plate reader, the fluid reservoir is a well of the cartridge; device. B021 B001, or the device of any embodiment, the device has a flow path that includes the fluid storage portion at least in part thereof; The flow path includes an inlet for introducing a solution and an outlet for waste liquid. device.

[0103] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Any or any aspect of the embodiments disclosed herein may be independently, partially, or in whole combined in any manner with other embodiments and aspects described herein. For example, one, two, or three or more embodiments may be combined in whole or in part.

[0104] As used herein, the terms "comprises" and "comprising" are intended to mean that compositions and methods include the recited elements, but do not exclude others. Embodiments defined by each of these transition terms are within the scope of the present invention. Thus, methods, compositions, and compositions can "comprise" additional steps and components.

[0105] A group of items connected by the conjunction "and" should not be read as requiring all of those items to be present in the group, but should be read as "and / or" unless otherwise stated. Similarly, a group of items connected by the conjunction "or" should not be read as requiring mutual exclusivity between the items in the group, but should be read as "and / or" unless otherwise stated.

[0106] Although the present invention has been described with reference to the above specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. Accordingly, it is contemplated that the present invention encompasses all such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for detecting a substance of interest, comprising: providing metal oxide nanowires having a gold-modified surface; modifying the gold with a first antibody to the substance of interest; allowing the first antibody to capture the target substance; allowing a second antibody of the target substance having an optical label to capture the target substance; and detecting the optical label; A method for providing the above.

2. 10. The method of claim 1, The gold is gold nanoparticles. method.

3. 10. The method of claim 1, The metal oxide is ZnO. method.

4. 10. The method of claim 1, The optical label is a fluorescent label. method.

5. 5. The method of claim 4, the optical label comprises a fluorophore; method.

6. 10. The method of claim 1, The nanowire has a trunk nanowire and branch nanowires branching from the trunk nanowire. method.

7. 7. The method of claim 6, the branch nanowires are formed from the trunk nanowire by epitaxial growth; method.

8. 7. The method of claim 6, the branch nanowires are formed by growing in random directions from the trunk nanowire; method.

9. 10. The method of claim 1, The target substance is a biological substance. method.

10. 10. The method of claim 1, The target substance is a virus or a cell. method.

11. 11. The method of claim 10, the first antibody (capture antibody) is an antibody against a first protein expressed on the surface of the virus or the cell, the second antibody (labeled antibody) is an antibody against a second protein expressed on the surface of the virus or the cell, and is an optically labeled antibody; method.

12. 12. The method of claim 11, the substance of interest is a dengue virus or a dengue NS1 protein, the first antibody and the second antibody are anti-NS1 antibodies; method.

13. 1. A device for detecting a substance of interest, comprising: a metal oxide nanowire having a gold-modified surface; a fluid reservoir that accommodates the metal oxide nanowires; Equipped with the gold-modified surface is modified with a first antibody to the substance of interest; allowing the first antibody to capture the target substance; allowing a second antibody of the target substance having an optical label to capture the target substance; Devices configured to:

14. 14. The device of claim 13, The device is a cartridge for a plate reader, the fluid reservoir is a well of the cartridge; device.

15. 14. The device of claim 13, the device has a flow path that includes the fluid storage portion at least in part thereof; The flow path includes an inlet for introducing a solution and an outlet for waste liquid. device.

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