Electrochemical detection of aerosolized pathogens using immuno-based biosensors

Abstract

The present disclosure is directed to a biosensor, method, and airborne detection device for analyzing aerosolized samples and detecting aerosolized pathogens in exhaled breath or environmental air samples. The biosensor includes an activated electrode containing nanobodies attached thereto. The airborne detection device further includes a sample collection device.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 486,282, filed February 22, 2023, which is incorporated herein by reference in its entirety.

[0002] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with government support under Grant No. U01AA029331 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The field of the disclosure relates generally to biosensors, methods, and devices for analyzing aerosolized samples and detecting aerosolized pathogens in exhaled breath or environmental air samples. More specifically, the biosensor includes an activated electrode containing nanobodies attached thereto. The airborne detection device further includes a sample collection device. [Background technology]

[0004] Inhalation of virus-containing aerosols exhaled by infected individuals is considered the primary route of transmission for respiratory viruses (e.g., SARS-CoV-2, influenza virus, rhinovirus, and respiratory syncytial virus (RSV)). Respiratory emissions during infection contain viral RNA of various aerosol sizes, with higher viral loads detected in aerosols smaller than 1 μm compared to larger aerosols. Submicron-sized viral aerosols are primarily generated via breathing. The generation of these aerosols involves the rupture of liquid membranes in the respiratory bronchioles of the lower airways of the human lung. Despite the demonstrated significance of aerosol-mediated disease transmission, direct and real-time detection of respiratory viral aerosols remains elusive.

[0005] The submicron size range of aerosols exhaled by infected individuals has been reported to contain the highest amount of SARS-CoV-2 viral genomes. Noninvasive screening of SARS-CoV-2 viral RNA in exhaled aerosols remains a technical challenge. Current testing requires exhaled breath condensate (EBC) collection followed by reverse transcription-polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 prevalence in various communities. This method limits its applicability for mass testing due to its long turnaround time (ranging from hours to days) and requires sophisticated equipment and skilled personnel. Indirect detection methods using electrochemical sensors also incorporate the quantification of volatile organic compounds (VOCs) in exhaled breath associated with COVID-19. These techniques identify distinct patterns or signatures of VOC emissions corresponding to specific variants. Therefore, the emergence of novel SARS-CoV-2 variants may hinder their implementation. This situation highlights the need for rapid testing methods capable of detecting variants in light of the need for early intervention and prevention in communities where the disease is endemic.

[0006] Thus, there is a need for straightforward, rapid, single-use, disposable, low-cost detection technologies and tests that are suitable for mass production and use in hospitals, airports, schools, and anywhere large numbers of people are expected to congregate. In particular, there is a need for electrochemical methods that offer improved limits of detection and greater accuracy than currently available "rapid" antigen tests. The embodiments described herein address at least these known deficiencies. Summary of the Invention

[0007] In certain aspects, the present disclosure is directed to an airborne detection device for analyzing an aerosolized sample in an exhaled breath or environmental air sample to detect an aerosolized pathogen, the device including an analysis vial and a biosensor electrode.

[0008] In another aspect, the present disclosure is directed to a method for detecting aerosolized pathogens, the method comprising: transporting a liquid sample from an external sampling device to an analysis vial of an aerosol collection device; adding a working fluid to the analysis vial of the aerosol collection device; detecting at least one pathogen; filling the external sampling device with the sample fluid; and transferring the analyzed sample from the analysis vial to a waste container of the aerosol collection device.

[0009] In yet another aspect, the present disclosure is directed to a system for detecting airborne pathogens, the system including an airborne detection device and an external sampling device.

[0010] In some embodiments, multiple pathogens can be detected simultaneously in a single test, and the multiple pathogens are selected from a combination of viruses, bacteria, parasites, fungi, and molds, or multiple viruses, multiple bacteria, or multiple species or strains of viruses, bacteria, parasites, fungi, or molds. In some embodiments, multiple variants of a pathogen can be detected in a single test, and the multiple variants include delta and omicron variants of SARS-CoV-2. In some embodiments, multiple pathogens are detected simultaneously in a multiplex test. [Brief explanation of the drawings]

[0011] The embodiments described herein can be better understood by reference to the following description in conjunction with the accompanying drawings.

[0012] [Figure 1]1 is an exemplary embodiment of an exhaled aerosol analyzer system according to the present disclosure. The depiction includes sampling of exhaled aerosol produced from the lower respiratory tract during normal breathing, a diagram of the exhaled aerosol analyzer system (including an aerosol collector, an MIE biosensor, and a potentiostat module), a description of the mechanism of virus detection using the MIE biosensor, and an image of a 3D-printed exhaled aerosol collector and lid with a dosing straw. [Figure 2A] FIG. 2A is an exemplary embodiment of the specificity of an MIE biosensor for examining spike proteins of SARS-CoV-1 and SARS-CoV-2 according to the present disclosure. [Figure 2B] FIG. 2B is an exemplary embodiment of the sensitivity (or LoD) of a biosensor according to the present disclosure, assessed by serial dilutions of various SARS-CoV-2 mutants. [Figure 2C] Figure 2C shows an exemplary embodiment of the present disclosure (left) showing normalized oxidation current (Iox) measured by an MIE biosensor in a laboratory experiment. The dashed horizontal line indicates the limit of detection (LoD) of the system. (right) showing an exemplary embodiment of viral RNA copy numbers / mL determined using RT-qPCR for various aerosolized SARS-CoV-2 mutants. The difference in resulting viral RNA copy numbers between the four SARS-CoV-2 mutants was not statistically significant (t-test, p=0.17), indicating that the viral strain does not affect the viral collection efficiency of the exhaled aerosol collection device. The whiskers represent the range of the data, and the boxes indicate the interquartile range. [Figure 3A] FIG. 3A illustrates an exemplary embodiment of estimating the minimum breath rate for detection by an MIE biosensor according to the present disclosure, which shows predicted breath rates by estimating the number of viral copies per breath based on the expected range of viral loads in COVID-19 patients. [Figure 3B]FIG. 3B shows an exemplary embodiment of an estimation of the minimum number of exhalations required for detection by the MIE biosensor, showing the results of a clinical study demonstrating that SARS-CoV-2 viral particles can be detected after as few as two patient exhalations. [Figure 4] Figure 4 shows an exemplary embodiment of an experimental setup for aerosolization of SARS-CoV-2 mutants according to the present disclosure. Compressed air at 20 psi from the CHεST® and inert SARS-CoV-2 virus solution are delivered to the BLAM atomizer. The atomizer produces a stream of aerosolized particles, which are compressed using a conical connection to enhance impaction against the cooled breathalyzer surface. The virus-containing aerosol condenses and flows down, and an EBC sample is collected from the bottom of the exhaled breath aerosol collector. [Figure 5A] 5A is an exemplary embodiment of a probability density function (PDF) of a viral aerosol according to the present disclosure. Using various settings of the CHεST® device, virus-containing aerosols of various sizes in the submicron range (less than 1 μm) were generated to mimic a "deep" human exhale. [Figure 5B] FIG. 5B is an exemplary embodiment of modeling the collection efficiency of an EBC breathalyzer at various impingement surface temperatures according to the present disclosure. The collection efficiency of EBCs by a breathalyzer is highly dependent on the temperature difference between the warm breath and the temperature of the surface onto which it is blown. Breath collection was modeled with a hydrophobic impingement surface at various temperatures. The aerosol particle recovery by the EBC is approximately 18.6% at 4°C, compared to 43.6% at -20°C. [Figure 6] Figure 6 shows an exemplary embodiment of viral aerosol recovery (%) for various variants after aerosolization and condensation using a collection device according to the present disclosure. The range of viral aerosol recovery (%) values ​​for various variants depends on factors such as the volume of the EBC sample collected and viral RNA degradation due to the delay between collection and sample processing. Similar data distributions are observed for test fluid recovery using other commercially available EBC collection devices. [Figure 7]7 is an exemplary embodiment of normalized Iox values ​​for clinical data of eight patients according to the present disclosure. Columns with bold borders indicate false negative results. [Figure 8] Figure 8 shows an exemplary embodiment of a SARS-CoV-2 biosensor design according to the present disclosure. The CoV-2 biosensor uses square wave voltammetry to measure the oxidation of tyrosine amino acids in viral particles. Oxidation releases electrons that the sensor detects as a current. The biosensor uses nanobodies attached to the surface to provide specificity and to attract viral particles to the electrode for measurement. The electrode is blocked with albumin to limit nonspecific signal. [Figure 9] 9 is an exemplary embodiment of an AβMIE biosensor used in live mouse brain according to the present disclosure. ISFAβ40 concentrations over 180 minutes in APP / PS1 (n=6) and WT (n=3) mice. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure describes biosensors placed in a breathalyzer or environmental sensor device that detect exhaled breath or aerosolized viruses in a given space (e.g., any indoor or enclosed space, including open spaces or other spaces with potentially shared air environments) to determine whether viral particles (e.g., aerosolized CoV-2) are present. Also described are electrochemical, antibody-based biosensors that detect inactive viral particles of at least one respiratory virus (or several respiratory viruses) instead of, or in addition to, SARS-CoV-2 viral particles. In some embodiments, the biosensor detects at least one bacterial genus or species. In other embodiments, the device detects at least one parasite, fungus, or mold genus or species.

[0014] The present disclosure represents a significant advance in the development of a device for real-time detection of aerosolized pathogens (including SARS-CoV-2) using an immune-based biosensor. The device is primarily intended for rapid diagnostic testing of infected individuals (both symptomatic and asymptomatic), with results expected to be obtained within 60 seconds of breathing into the device, eliminating the need for follow-up testing. A single-use, disposable exhaled breath condensate (EBC) collection device is used to capture exhaled aerosols. The patient breathes into the collection device, which has a chilled (approximately 10°C) hydrophobic film inside. The temperature difference between the exhaled breath and the cold surface triggers condensation growth and collection of aerosol particles. A test buffer is added, and the exhaled breath condensate is collected and applied to a biosensor for detection. In some embodiments, the exhaled breath condensate is applied to the biosensor and then analyzed within seconds or minutes of adding the test buffer. In other embodiments, the breath sample may be collected (e.g., condensed and washed with a testing buffer) and appropriately stored for a period of time (e.g., several hours, days, weeks, etc.) before contacting the biosensor for pathogen detection. For example, if an EBC collection device is provided for remote or home testing, the collected sample may require storage and / or transport before contacting the biosensor for analysis. Depending on the embodiment, breathalyzers may be deployed in hospitals, schools, airports, and military installations / vessels where rapid testing of large numbers of individuals is required. These devices may provide rapid results and serve as a platform for detecting other respiratory viruses and newly emerging pathogens.

[0015] Viral particles detectable by the biosensors of the present disclosure include, but are not limited to, chikungunya fever, cholera, Crimean-Congo hemorrhagic fever, Ebola virus disease, Hendra virus infection, influenza (pandemic, seasonal, zoonotic), Lassa fever, Marburg virus disease, meningitis, MERS-CoV, monkeypox, Nipah virus infection, novel coronavirus (2019-nCoV), plague, Rift Valley fever, SARS, smallpox, tularemia, yellow fever, Zika virus disease, viruses related to Ebola, and Marburg virus (family Filoviridae); Ross River virus, chikungunya virus, Sindbis virus, Eastern equine encephalitis virus (family Togaviridae, genus Alphavirus), vesicular stomatitis virus (family Rhabdoviridae, genus Vesiculovirus), Amapari virus, and the like.virus), Pichinde virus, Tacaribe virus, Junin virus, Machupo virus (family Arenaviridae, genus Mamuarenavirus), West Nile virus, dengue virus, yellow fever virus (family Flaviviridae, genus Flavivirus); human immunodeficiency virus type 1 (family Retroviridae, genus Lentivirus); Moloney murine leukemia virus (family Retroviridae, genus Gammaretrovirus); influenza A virus (family Orthomyxoviridae); respiratory syncytial virus (family Paramyxoviridae, family Pneumoviridae, genus Pneumovirus); vaccinia virus (family Poxviridae, subfamily Chordopoxvirinae, genus Orthopoxvirus); herpes simplex virus type 1, herpes simplex virus Human cytomegalovirus (Herpesviridae, Betaherpesvirinae, Cytomegalovirus genus); Autographa californica nuclear polyhedrosis virus (Baculoviridae, Alphabaculoviridae) (insect virus); Ebola and Marburg viruses (Filoviridae); Semliki Forest virus, Ross River virus, Chikungunya virus, O'nyong-nyong virus, Sindbis virus, Eastern / Western / Venezuelan equine encephalitis virus (Togaviridae, Alphavirus genus); Rubella (German measles) virus (Togaviridae, Rubivirus genus); Rabies virus, Lagos bat virusvirus), Mokola virus (family Rhabdoviridae, genus Lyssavirus); Amapari virus, Pichinde virus, Tacaribe virus, Junin virus, Machupo virus, Guanarito virus, Sabia virus, Lassa virus (family Arenaviridae, genus Mamuarenavirus); West Nile virus, Dengue virus, Yellow fever virus, Zika virus, Japanese encephalitis virus, Saint Louis encephalitis virus, Tick-borne encephalitis virus, Omsk hemorrhagic fever virus, Kyasanur Forest disease virus (family Flaviviridae, genus Flavivirus); Human hepatitis C viruses (family Flaviviridae, genus Hepacivirus); human immunodeficiency virus type 1 (family Retroviridae, genus Lentivirus); influenza A / B viruses (family Orthomyxoviridae, common "influenza" virus genus); respiratory syncytial virus (family Paramyxoviridae, family Pneumoviridae, genus Pneumovirus); Hendra virus, Nipah virus (family Paramyxoviridae, subfamily Paramyxovirinae, genus Henipavirus); measles virus (family Paramyxoviridae, subfamily Paramyxovirinae, genus Morbillivirus); smallpox (family Variola Variola major (smallpox) virus (family Poxviridae, subfamily Chordopoxvirinae, genus Orthopoxvirus); human hepatitis B virus (family Hepadnaviridae, genus Orthohepadnavirus); hepatitis delta virus (hepatitis D virus) (family Unclassified, genus Deltavirus); herpes simplex virus type 1, herpes simplex virus type 2 (family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplexvirus); human cytomegalovirus (family Herpesviridae, subfamily Betaherpesvirinae, genus Cytomegalovirus), adeno-associated virus, genus Dependovirus, family Parvoviridae, Aichi virus, genus Kobuvirus, family Picornaviridae, Australian bat lyssavirus, family Rhabdoviridae, BK polyomavirus (BKpolyomavirus, family Polyomaviridae, Banna virus, genus Seadornavirus, family Reoviridae, Barmah forest virus, genus Alphavirus, family Togaviridae, Bunyamwera virus, genus Orthobunyavirus, family Bunyaviridae, La Crosse bunyavirus, genus Orthobunyavirus, family Bunyaviridae, snowshoe hare bunyavirus, genus Orthobunyavirus, family Cercopithecine herpesvirus, genus Lymphocryptovirus, family Herpesviridae, Chandipura virus, genus Vesiculovirus, family Rhabdoviridae, Chikungunya virus Alphavirus, Togaviridae, Cosavirus A, Cosavirus, Picornaviridae, Cowpox virus, Orthopoxvirus, Poxviridae, Coxsackievirus, Enterovirus, Picornaviridae, Crimean-Congo hemorrhagic fever virus, Nairovirus, Bunyaviridae, Dengue virus, Flavivirus, Flaviviridae, Dhori virus, Thogotovirus, Orthomyxoviridae, Dugbe virus, Nairovirus, Bunyaviridae, Duvenhage virus, Lyssavirus, Rhabdoviridae, Eastern equine encephalitis virusAlphavirus, Togaviridae, Ebolavirus, Filoviridae, Echovirus, Enterovirus, Picornaviridae, Encephalomyocarditis virus, Cardiovirus, Picornaviridae, Epstein-Barr virus, Lymphocryptovirus, Herpesviridae, European bat lyssavirus, Rhabdovirus, GB virus C / Hepatitis G virus, Pegivirus, Flaviviridae, Hantaan virus, Hantavirus, Bunyaviridae, Hendra virus, Henipavirus, Paramyxoviridae, Hepatitis A virus, Hepatovirus, Picornaviridae, Hepatitis B virus Orthohepadnavirus (Hepadnaviridae), Hepatitis C virus (Hepacivirus), Flaviviridae, Hepatitis E virus (Hepacivirus), Unclassified, Hepatitis Delta virus (Deltavirus), Unclassified, Horsepox virus (Orthopoxvirus), Poxviridae, Human adenovirus (Mastadenovirus), Adenoviridae, Human astrovirus (Mamastrovirus), Astroviridae, Human coronavirus (Alphacoronavirus), Coronaviridae, Human cytomegalovirus (Herpesviridae), Human enterovirus 68, 70 70) Enterovirus, Picornaviridae, Human herpesvirus type 11) Simplexvirus (family Herpesviridae, human herpesvirus type 2, family Simplexvirus, family Herpesviridae, human herpesvirus type 6, family Roseolovirus, family Herpesviridae, human herpesvirus type 7, family Roseolovirus, family Herpesviridae, human herpesvirus type 8, family Rhadinovirus, family Herpesviridae, human immunodeficiency virus (family Lentivirus, family Retroviridae), family Human papillomavirus type 1, family Mupapillomavirus, family Papillomaviridae, family Human papillomavirus type 2, family Alphapapillomavirus, family Papillomaviridae, family Human papillomavirus types 16 and 18, family Alphapapillomavirus, family Papillomaviridae, family Human parainfluenza virus (family Human papillomavirus) Parainfluenza (Respirovirus, Paramyxoviridae), Human parvovirus B19 (Erythrovirus, Parvoviridae), Human respiratory syncytial virus (Orthopneumovirus, Pneumoviridae), Human rhinovirus (Enterovirus, Picornaviridae), Human SARS coronavirus (Betacoronavirus, Coronaviridae), Human spumaretrovirus (Spumavirus, Retroviridae), Human T-lymphotropic virus (Deltaretrovirus, Retroviridae), Human torovirus (Coronaviridae), Influenza A virus (Influenzavirus, Influenzavirus)A) Orthomyxoviridae, Influenza B virus (Influenza virus B genus, Orthomyxoviridae), Influenza C virus (Influenza virus C genus, Orthomyxoviridae), Isfahan virus (Vesiculovirus genus, Rhabdoviridae), JC polyomavirus (Polyomaviridae), Japanese encephalitis virus (Flavivirus genus, Flaviviridae), Junin arenavirus (Arenaviridae), KI polyomavirus (Kunjin virus genus, Flaviviridae), Lagos bat virus (Lyssavirus genus, Rhabdoviridae), Lake Victoria marburgvirus (Marburgvirus genus, Filoviridae), Langat virus (Flavivirus genus, Flaviviridae), Lassa virus (Arenavirus genus, Arenaviridae), Lordsdale virus Norovirus (Norovirus) Caliciviridae, Looping ill virus (Flavivirus) Flaviviridae, Lymphocytic choriomeningitis (Arenaviridae), Machupo virus (Arenaviridae), Mayaro virus (Alphavirus) Togaviridae, MERS coronavirus (Betacoronavirus), Measles virus (Morbillivirus) Paramyxoviridae, Mengo encephalomyocarditis virus (Cardiovirus) Picornaviridae, Merkel cell polyomavirus (Polyomaviridae), Mokola virus (Lyssavirus) Rhabdoviridae, Molluscum contagiosumMolluscipoxvirus (genus Molluscipoxvirus), family Poxviridae, Monkeypox virus (genus Orthopoxvirus), family Poxvirus Family: Mumps virus, Rubulavirus, Paramyxoviridae, Murray Valley encephalitis virus, Flavivirus, Flaviviridae, New York virus, Hantavirus, Bunyaviridae, Nipah virus, Henipavirus, Paramyxoviridae, Norwalk virus, Norovirus, Caliciviridae, O'nyongnyong virus, Alphavirus, Togaviridae, Orf virus, Parapoxvirus, Poxviridae, Oropouche virus, Orthobunyaviridae, Bunyaviridae, Pichinde virus, Arenavirus, Arenaviridae, Poliovirus, Enterovirus, Picornaviridae, Punta Toro phlebovirus, Bunyaviridae, Puumala virus Hantavirus (Bunyaviridae), Rabies virus (Lyssavirus), Rhabdoviridae, Rift Valley fever virus (Phlebovirus), Rosavirus A (Picornaviridae), Ross River virus (Alphavirus), Togaviridae, Rotavirus A (Reoviridae), Rotavirus B (Reoviridae), Rotavirus C (Reoviridae), Rubella virus (Rubella), Sagiyama virus (Alphavirus), Togaviridae, Salivirus A (Salivirus), Picornaviridae, Sicilian sandfly fever virus (Phlebovirus), Sapporo virus Sapovirus, Caliciviridae, Semliki Forest virusAlphavirus, Togaviridae, Seoul virus, Hantavirus, Bunyaviridae, Simian foamy virus, Spumavirus, Retroviridae, Simian virus 5, Rubulavirus, Paramyxoviridae, Sindbis virus, Alphavirus, Togaviridae, Southampton virus, Norovirus, Caliciviridae, St. Louis encephalitis virus, Flavivirus, Tick-borne powassan virus, Flaviviridae, Torque teno virus, Alphatorquevirus, Anelloviridae, Toscana virus, Phlebovirus, Bunyaviridae, Uukuniemi virus, Phlebovirus, Bunyaviridae, Vaccinia virus Orthopoxvirus (Poxviridae), Varicella-zoster virus, Varicellovirus (Varicellovirus), Herpesviridae, Variola virus, Orthopoxvirus (Poxviridae), Venezuelan equine encephalitis virus, Alphavirus (Togaviridae), Vesiculovirus (Rhabdoviridae), Western equine encephalitis virus, Alphavirus (Togaviridae), WU polyomavirus (Polyomaviridae), West Nile virus, Flavivirus (Flaviviridae), Yaba monkey tumor virus, Orthopoxvirus (Poxviridae), Yaba-like disease virus, Orthopoxvirus (Poxviridae), Yellow fever virusThese include the Flavivirus genus Flaviviridae family and the Zika virus Flavivirus genus Flaviviridae family.

[0016] Bacterial genera and species that can be detected with the biosensors of the present disclosure include, but are not limited to, Xanthomonas, Pseudomonas, Salmonella, Shigella, Chlamydia, Helicobacter, Yersinia, Bordetella, Pseudomonas, Neisseria, Vibrio, Haemophilus, and the like. lus, Mycoplasma, Streptomyces, Treponema, Coxiella, Ehrlichia, Brucella, Streptobacillus, Fusospirocheta, Spirillum, Ureaplasma, Spirochaeta, Mycoplasma, Actino Actinomycetes, Borrelia, Bacteroides, Trichomonas, Branhamella, Pasteurella, Clostridium, Corynebacterium, Listeria, Bacillus, Erysipelothrix, Rhodococcus ccus), Escherichia, Klebsiella, Pseudomonas, Enterobacter, Serratia, Staphylococcus, Streptococcus, Legionella, Mycobacterium, Proteus, Campylobacter, Enterococcus,Bacteria related to the genera Acinetobacter, Morganella, Moraxella, Citrobacter, Rickettsia, and Rochlimeae, as well as bacterial species such as: Pseudomonas aeruginosa; E. coli, P. cepacia, Staphylococcus epidermis, E. faecalis, Streptococcus pneumoniae, Staphylococcus aureus, N. meningitidis, S. pyogenes, Pasteurella multocida, and Treponema pallidum. Genera and species of Gram-negative bacteria that can be detected with the biosensors of the present disclosure include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp., and Vibrio spp. Gram-negative bacterial genera and species detectable with the biosensors of the present disclosure include, but are not limited to, Salmonella, Escherichia coli, Yersinia pestis, Klebsiella, and Shigella, Proteus, Enterobacter, Serratia, and Citrobacter.

[0017] Fungi that can be detected with the biosensors of the present disclosure include, but are not limited to, Cryptococcus neoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis; Histoplasma capsulatum; fungi related to Coccidioides immitis; Candida species (including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, and C. krusei), Aspergillus species (including A. fumigatus), and the like. fumigatus, A. flavus, and A. niger), Rhizopus species; Rhizomucor species; Cunninghamella species; Apophysomyces species (including A. saksenaea, A. mucor, and A. absidia); Sporothrix schenckii, Paracoccidioides brasiliensis; Pseudallescheria boydii, Torulopsis glabrata glabrata); Trichophyton species, Microsporum species, and Dermatophyres species, as well as any other yeast or fungus now known or later identified as pathogenic.

[0018] Parasites that can be detected with the biosensor of the present disclosure include, but are not limited to, the genera Anaplocephala, Ancylostoma, Necator, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Cyathostomum, Cylicocyclus, and Cyli. codontophorus, Cylicostephanus, Craterostomum, Dictyocaulus, Dipetalonema, Dipylidium, Dracunculus, Echinococcus, Enterobius, Fasciola, Filaroides, Habronema onema, Haemonchus, Metastrongylus, Moniezia, Nematodirus, Nippostrongylus, Oesophagostomum, Onchocerca, Ostertagia, Oxyuris, Parascaris, Schistosoma, Str Parasites associated with the genera Toxocara, Taenia, Toxocara, Strongyloides, Toxascaris, Trichinella, Trichuris, Trichostrongylus, Triodontophorus, Uncinaria, and Wuchereria, leishmaniasis, human African trypanosomiasis, and Chagas disease.Parasites belonging to the phylum Apicomplexa, such as antigens from species of the genera Babesia, Toxoplasma, Plasmodium, Eimeria, Isospora, Atoxoplasma, Cystoisospora, Hammondia, Besniotia, Sarcocystis, Frenkelia, Haemoproteus, Leucocytozoon, Theileria, Perkinsus, and Gregarina; carinii; parasites belonging to the phylum Microspora, such as the genera Nosema, Enterocytozoon, Encephalitozoon, Septata, Mrazekia, Amblyospora, Ameson, Glugea, Pleistophora, and Microsporidium species; and parasites belonging to the phylum Ascetospora, such as Haplosporidium species, as well as Plasmodium falciparum, P. vivax, P. ovale, and the like. ovale), Plasmodium malariae (P. malariae), Toxoplasma gondii, Leishmania mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania donovani, Trypanosoma cruzi,Trypanosoma brucei (T. brucei), Schistosoma mansoni (S. mansoni), Schistosoma haematobium (S. haematobium), Schistosoma japonium (S. japonium); Trichinella spiralis (Trichinella spiralis); Wuchereria bancrofti (Wuchereria bancrofti); Brugia malayi (Brugia malayi); Entamoeba histolytica (Entamoeba histolytica); Enterobius vermicularis (Enterobius vermicularis); Taenia solium (Taenia solium), Taenia saginata (Taenia saginata), Trichomonas vaginalis (T. vaginalis), Trichomonas hominis (T. hominis), Trichomonas tenax (T. tenax); Giardia lamblia (Giardia lamblia); Cryptosporidium parvum (Cryptosporidium parvum; Pneumocystis carinii, Babesia bovis, B. divergens, B. microti, Isospora belli, L. hominis; Dientamoeba fragilis; Onchocerca volvulus; Ascaris lumbricoides; Necator americanus; Ancylostoma duodenale; Strongyloides stercoralis; Capillaria philippinensis; Angiostrongylus cantonensis; Hymenolepis nana; Diphyllobothria tapeworm latum); Echinococcus granulosus, Echinococcus multilocularis; Paragonimus westermani,Species including Paragonimus caliensis (P. caliensis); Clonorchis sinensis (Clonorchis sinensis); Opisthorchis felineus (G. viverini), Fasciola hepatica (Fasciola hepatica), Sarcoptes scabiei (Sarcoptes scabiei), Pediculus humanus (Pediculus humanus), Phthirus pubis (Philus pubis), and Dermatobia hominis (Dermatobia hominis), as well as any other parasites now known or later identified as pathogenic.

[0019] Additional pathogens that can be detected with the biosensors of the present disclosure include, but are not limited to, Coronaviridae (e.g., MERS, SARS-CoV-2), Bunyavirales (e.g., Lassa virus, Junin virus, Rift Valley fever virus, Andes virus, Sin Nombre virus, LaCrosse virus, California Encephalitis virus, Crimean Congo Hemorrhagic Fever virus), Filoviridae (e.g., Ebola virus, Marburg virus), Flaviviruses (e.g., Dengue virus, Zika virus, West Nile virus), and the like. Nile), Paramyxoviridae (e.g., Nipah virus, Hendra virus), Picornaviridae (e.g., EV-D68, EV-A71), Togaviridae (e.g., Chikungunya, EEE, VEE, WEE), Bacillus anthracis (including genotypic resistance markers), Yersinia pestis (including genotypic resistance markers), Francisella tularensis (including genotypic resistance markers), Burkholderia spp. (including genotypic resistance markers), Botulinum toxin (including identification and classification of related serotypes), ESKAPE pathogens with genotypic resistance markers (e.g., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, baumannii), Pseudomonas aeruginosa, Enterobacter spp., Lassa virus, Nipah virus, Rift Valley fever virus, Enterovirus D68 virus, Candida auris, Coccidioides spp., and novel coronaviruses.

[0020] Immuno-based biosensors and environmental detection of airborne pathogens

[0021] In an exemplary embodiment, the biosensor is mounted on an environmental detector for rapid (e.g., real-time or near-real-time) continuous measurement of sampled air. The disclosed environmental biosensor for detecting target organisms is surprisingly based on an ultrasensitive electrochemical technique used in vivo (e.g., brain, tissue, interstitial fluid, etc.) to detect target macromolecules in Alzheimer's disease research.

[0022] The first microimmunoelectrode (MIE) biosensor was developed to detect amyloid-β (Aβ) peptides in Alzheimer's disease. The electrochemical sensor uses voltammetry to measure the oxidation of tyrosine amino acids in proteins. Oxidation results in the release of electrons, which are detected as a change in current at a carbon fiber electrode. The amount of current is proportional to the amount of protein present. The biosensor uses antibodies covalently bound to the surface to provide specificity and to attract proteins to the electrode for measurement.

[0023] The disclosed biosensor is based on a similar design to the MIE. Some embodiments herein describe a CoV-2 nanobody (produced in llamas) with 5 nM affinity for the repeat binding domain (RBD) of the SARS-CoV-2 spike protein and very high selectivity for the CoV-1 spike protein. The present disclosure demonstrates an initial sensitivity of 2 fg / ml for the CoV-2 biosensor. In contrast, conventional CoV-2 antigen tests have a sensitivity in the low pg / mL range. Development of the environmental sensors, biosensors, and methods disclosed herein includes mimicking real-world environmental conditions, particularly with respect to atmospheric aerosols, which is essential for testing and optimizing biosensor performance for field deployment.

[0024] As disclosed herein, immuno-based electrochemical biosensors provide real-time and continuous measurement of CoV-2 aerosols for use in airborne environmental detection and diagnostics. In some embodiments, the sensors target CoV-2. In other embodiments, the designs and methods are adapted for multiple airborne or respiratory pathogens. The airborne detectors described herein monitor public gathering places for environmental risks and provide warnings for evacuation and / or increased disinfection.

[0025] In exemplary embodiments, the CoV-2 biosensor has moderate sensitivity to recombinant spike protein and is adaptable depending on the viral (pathogen) particle, antibody type, lifetime, concentration, and electrode surface orientation. In some embodiments, the biosensor is applicable to the detection of inactive CoV-2 viral particles. In these embodiments, specificity controls include other viral surface proteins and viral particles. Depending on the embodiment, the electrode design is optimized for the size and type of material that has the greatest impact on specificity and oxidation properties.

[0026] Airborne transmission of CoV-2 is driven by the spread of droplet nuclei (aerosols), which remain infectious even over long distances and for extended periods of time. Observational studies of CoV-2 and other infectious viruses have systematically evaluated the performance of the disclosed biosensor in terms of sensitivity, detection limit, and lifetime, demonstrating the sensitivity of aerosolized viral particles to relevant environmental parameters, including relative humidity, temperature, and atmospheric residence time. Biosensor performance is also tested under conditions mimicking actual indoor and urban air conditions, where aerosolized viral droplets mix with particulate pollutants (e.g., volatile organics, dust, soot). In some embodiments, an environmental sensor device sampling a given space detects CoV-2 in real time or near real time for at least about 12 to about 24 hours.

[0027] Diagnostic tests

[0028] As noted above, there is a need for enhanced and improved testing capabilities for pathogens, including CoV-2. While detection and diagnosis of both symptomatic and asymptomatic individuals are needed to isolate and / or quarantine individuals and reduce community transmission, an equally important, yet evolving, measure is real-time (or near-real-time) monitoring of airborne viruses in public gatherings, which may result in the closure of the space or extensive disinfection of the area. In some embodiments described herein, airborne pathogen sensors are used in conjunction with aerosol disinfectant sprayers as a rapid clean to limit the spread of detected viruses and / or pathogens.

[0029] Immunobiosensors for aerosolization and airborne detection

[0030] Disclosed herein is an ultrasensitive immuno-based electrochemical biosensor that detects pathogens (in an exemplary embodiment, the spike protein on the surface of CoV-2). The CoV-2 biosensor disclosed herein is applicable to detecting airborne viruses (and / or pathogens) using environmental sampling and detection systems that can be applied to large spaces (e.g., airports, hospitals, conference centers, or school environments). Depending on the embodiment, a biosensor specifically optimized for the deployment situation and the long-term nature of sampling and monitoring is implemented. Depending on the embodiment, the sampling platform may be modified to detect other pathogens and / or combinations of pathogens.

[0031] The innovations of the systems, methods, and devices disclosed herein are based, at least in part, on immune-based biosensors, specificity-conferring nanobodies, and / or sampling and processing of aerosolized pathogen particles (e.g., CoV-2 viral particles) to match real-world environmental conditions prior to biosensor testing.

[0032] CoV-2 Biosensor

[0033] The microimmunoelectrode (MIE) technology disclosed herein utilizes square-wave voltammetry to measure the oxidation of tyrosine amino acids in specific proteins. In CoV-2 biosensor embodiments, sensitivity was observed down to 2 fg / mL of CoV-2 spike RBD protein, in contrast to conventional CoV-2 immunoadsorption assays, which have biosensor sensitivity in the low pg / mL range. In some embodiments, the biosensor utilizes recombinant spike protein. In other embodiments, the biosensor utilizes CoV-2 virions.

[0034] As disclosed herein, target specificity is based on covalently binding antibodies to the electrode surface. Oxidation of the antibody-bound CoV-2 spike protein was measured as a direct indicator of the protein's presence. Importantly, tyrosine oxidation is irreversible, meaning that the protein bound to the antibody on the electrode surface is measured only once. This contrasts with many conventional electrochemical sensors, which measure the impedance of the electrode surface; essentially, they measure the binding event rather than the actual protein. Impedance measurements can suffer from specificity issues because nonspecific proteins or molecules can be present on the electrode surface and still emit a signal, a phenomenon often referred to as "fouling."

[0035] Anti-CoV-2 nanobodies

[0036] Anti-CoV-2 nanobodies were derived from camelids (including llamas). These animals produce a subclass of IgG with an unpaired heavy chain variable region (known as nanobodies). The nanobodies designed and described herein were sequenced to allow for rapid and inexpensive production in cells for large-scale production and for recombinant molecular biology modifications, if necessary, for example, to increase affinity or for placement on an electrode surface. Nanobodies are generally more durable than antibodies; they can withstand dehydration and a wide temperature range, although this may vary depending on the sampling environment of the airborne detector described herein in combination with a biosensor electrode. Airborne detection under realistic environmental conditions.

[0037] Aerosol transmission is an important route of CoV-2 transmission based on clinical observations in closed spaces. Currently, there are uncertainties regarding the aerodynamic properties and transmission routes of CoV-2 in aerosols due to challenges associated with sampling in real environments and quantifying variable particle sizes and concentrations. These real-world environmental factors directly impact the integrity and measurability of the virus. Biosensor characterization includes a wide range of viral aerosol sizes and concentrations, including, but not limited to, different particle size distributions corresponding to different routes of airborne release via speaking, coughing, and sneezing. Aerosol samples are injected into an environmental chamber and transported through a realistic, contaminated indoor environment. The biosensor development described herein encompasses a revolutionary understanding of how airborne CoV-2 viral particles change with environmental conditions.

[0038] CoV-2 detector

[0039] Immune-based CoV-2 biosensors offer ultra-high sensitivity for real-time pathogen detection. In some embodiments, the sensors detect CoV-2. Other embodiments include similar sensors developed using antibodies for other pathogens in multi-electrode arrays. Airborne detectors would enable continuous and immediate feedback on viral threats in the environment. They could subsequently alert spaces to evacuation or increased disinfection. They could also be coupled with disinfectant aerosol sprays for immediate response, keeping crowds safe in real time and minimizing disruption to ongoing activities. Importantly, airborne detectors could alert crowds to positive COVID-19 cases, prompting individual testing within the population and enabling identification and isolation on a much larger scale than currently possible.

[0040] Environmental detection of airborne CoV-2

[0041] SARS-CoV-2 spreads via several routes, including aerosols and droplets, which remain suspended in the air long enough to be inhaled. When aerosols or droplets containing respiratory fluids and microorganisms are released into unsaturated air, i.e., air with a relative humidity (RH) below 100%, they partially or completely evaporate and reach equilibrium with the surrounding environment. This process reduces particle size, thereby extending their airborne suspension time. Evaporation also increases the concentration of free H+ ions in the aerosol, thereby decreasing the pH. Meanwhile, solutes such as salts and proteins remain intact. Within the shrinking droplets, salt interactions, changes in pH, temperature, and RH interact dynamically. When distributed to aerosol surfaces, enveloped viruses such as SARS-CoV-2 can be damaged by increased surface tension, shear stress, and conformational changes induced by these dynamic interactions. Peptide unfolding and subsequent protein denaturation can occur at the air-liquid interface of the droplets. Therefore, proper consideration of environmental parameters such as RH and temperature is necessary in determining biosensor design, as they affect the transmission kinetics (e.g., particle size, residence time, and travel distance) and subsequent detection of viruses, especially after they are exhaled from an infected person in the aerosol phase. Recovering and measuring viruses encapsulated in the aerosol phase under different environmental conditions is crucial for building devices that can detect viral particles (e.g., CoV-2 virions) in a wide range of environments and conditions. [Example]

[0042] Without further explanation, it is believed that those skilled in the art can utilize the present invention to its fullest extent. Accordingly, the following examples are merely illustrative and should not be construed as limiting the present disclosure in any way. All numerical ranges described herein are understood to include all values ​​from the lower value to the higher value. For example, if a range is stated as 10 to 50, values ​​such as 12 to 30, 20 to 40, or 30 to 50 are intended to be expressly recited herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be expressly recited in this application.

[0043] Example 1 Biosensor

[0044] Electrochemical Biosensors. Described herein is a microimmunoelectrode (MIE) technology that utilizes square-wave voltammetry to measure the oxidation of tyrosine amino acids (at ∼0.65 V) in specific proteins (i.e., pathogen-displayed proteins). The oxidation of tyrosine releases electrons, which are detected as a current at a carbon electrode (Figure 8). The amount of current is directly proportional to the amount of analyte present. Antibodies covalently bound to the electrode surface recruit targets to the biosensor for measurement. In the case of SARS-CoV-2, nanobodies (produced in llamas, then sequenced and inexpensively cultivated in bacteria) are covalently bound to the biosensor surface, conferring biosensor specificity. In some embodiments, one or more nanobodies are bound to the biosensor instead of, or in addition to, the SARS-CoV-2 nanobody. In embodiments where two or more pathogens can be detected with a single biosensor, the signal (e.g., current) from the biosensor can be a multiplexed signal. Importantly, tyrosine oxidation is irreversible, meaning that protein binding to nanobodies on the electrode surface is measured only once. This contrasts with many electrochemical sensors, which measure the impedance of the electrode surface; essentially measuring the binding event rather than the actual protein. The biosensor of this disclosure uses inexpensive screen-printed carbon-based electrodes (SPiCE).

[0045] Amyloid beta (Aβ) microimmunoelectrodes (MIEs) in Alzheimer's disease research. As a prior example of the use of similar biosensors, an immuno-based voltammetric method was developed as a microimmunoelectrode (MIE) biosensor, which was used to measure human amyloid beta (Aβ) peptide concentrations in the brains of Alzheimer's disease model mice over a minute-by-minute timescale. The Aβ biosensor was surgically implanted in the mouse brain, allowing real-time measurements of brain interstitial fluid in awake, freely moving mice. While the designs of the Aβ and SARS-CoV-2 biosensors differ based on their intended applications (5 μm carbon fiber grafted onto glass and 1 mm screen-printed electrodes, respectively), the underlying principles of the biosensors are similar.

[0046] The Aβ biosensor was implanted into the brains of 1) APP / PS1 transgenic mice expressing human Aβ or 2) wild-type mice expressing only endogenous mouse Aβ. Importantly, mouse Aβ lacks the tyrosine amino acid found in human Aβ. According to the theory of how the biosensor functions, the tyrosine amino acid is required to generate an electrochemical signal on the biosensor, serving as a strong control for specificity in vivo. In APP / PS1 mice, the biosensor measured human Aβ every 60 seconds for 3 hours, exhibiting the minute-by-minute fluctuations expected based on continuous neuronal activity (Figure 9). In contrast, the signal in wild-type mice was negligible throughout the entire 3-hour measurement period. The Aβ biosensor is 8,000-fold more selective for human Aβ than any other tyrosine in the brain.

[0047] A series of biosensors have been developed for use in various mouse models of neurological diseases, including those detecting various types of Aβ peptides (Aβ 40 , Aβ 42 These included those targeting Aβ oligomers, tau, and α-synuclein. Another MIE was also developed for the neuromodulatory peptide metenkephalin. While standard Aβ oligomer ELISAs generally have sensitivity in the low pg / mL range, the biosensor has a sensitivity of 200 attograms / mL of oligomers, approximately 10,000 times higher.

[0048] Example 2: Design of an exhaled aerosol collection device

[0049] The exhaled aerosol collection device (or box) has a lid with an inlet straw and two liquid inlet ports. Exhaled aerosols are collected in a condensation chamber, which contains the upper chamber of the box and consists of a tapered, sloped hydrophobic polyimide condensation surface supported by a scaffold. The hydrophobic condensation surface is formed using polyimide high-temperature masking tape. The collection device is stored in a -20°C freezer before testing to cool the condensation surface. If a freezer is not available, cold liquid, such as ice water, can be added to the lower chamber of the box through an inlet on the exterior of the collection device. When a person breathes into the device, the aerosols, along with any virus particles they may contain, impact and condense on the cold condensation surface. The tapered, sloped hydrophobic surface allows the exhaled breath condensate (EBC) to slide downward and settle in the bottom corner of the box, where a microimmunoelectrode (MIE) biosensor resides. After EBC collection, phosphate-buffered saline (PBS) solution is manually injected through the inlet port on the lid. PBS solution is added to wash any remaining EBC sample from the slope down to the biosensor at the bottom. Finally, after the EBCs have been analyzed by the biosensor, hypochlorous acid (HOCl) is injected through a second liquid injection port on the lid to disinfect the Breathalyzer and allow it to be safely disposed of.

[0050] Example 3 Microimmunoelectrode (MIE) biosensor

[0051] The electrochemical biosensor uses inexpensive screen-printed carbon-based electrodes (SPiCE, Catalog # SP-1401, BASi Research Products, West Lafayette, IN). The core technology for detecting SARS-CoV-2 virions in EBCs is based on microimmunoelectrode (MIE) technology. SPiCE is pre-processed in PBS (pH 7.4) and electrochemically activated using high-frequency cyclic voltammetry and chronoamperometry to enhance selectivity for tyrosine oxidation and increase the attachment of SARS-CoV-2-specific nanobodies. The nanobodies are derived from llamas and covalently bind to the electrode surface, directing the target to the MIE biosensor for measurement. During the prototype phase, EBC samples were diluted in cut-glass vials containing 1% bovine serum albumin (BSA) in PBS solution rather than analyzed directly in a breath analysis box. The SPiCE is connected to a commercially available potentiostat (PalmSens4, PalmSens BV, Houten, Netherlands). SPiCE is suspended in a sample vial so that the working electrode is completely submerged and prevents drying. Square-wave voltammetry (SWV) is performed to oxidize tyrosine in the spike protein and detect changes in current at the electrode surface. During SWV, the electrode potential is scanned from 0 to 1 V at a frequency of 15 Hz while measuring the current at the working electrode. When an electroactive species is oxidized, a peak in the oxidation current corresponding to the oxidation potential of that specific species is observed in the voltammogram. The presence of antibodies covalently bound to the electrode surface provides specificity for SARS-CoV-2 at a potential of 0.65 V. The tyrosine amino acid possesses a phenolic group that is easily oxidized at the electrode surface by voltammetry. Voltammetric studies of the electrochemical behavior of tyrosine in solution have revealed that tyrosine oxidation is either a 1:1 proton-coupled electron transfer process or a two-electron process. However, the protein backbone is involved in the electron transfer process, and oxidation of tyrosine residues in proteins using carbon-based electrodes has been reported to be in the two- to four-electron range.Therefore, when scanned at an electrode potential of 0.6 V, tyrosine residues on virus particles on the electrode surface are oxidized, releasing four electrons (per molecule) that MIE will detect as a current. Although the nanobody recognizes the spike protein, most of the virus is oxidized, which means that many other proteins or tyrosines on the virus surface also release electrons, causing signal amplification and likely contributing to the sensor's ultrahigh sensitivity.

[0052] Example 4 Materials and Methods

[0053] The materials and methods of this example are used in Examples 5-7.

[0054] Cells and viruses

[0055] Vero cells expressing human ACE2 and TMPRSS2 (Vero-hACE2-hTMPRSS2) were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.3), 100 U / mL penicillin-streptomycin, and 10 μg / mL puromycin. Vero cells expressing TMPRSS2 (Vero-hTMPRSS2) were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.3), 100 U / mL penicillin-streptomycin, and 5 μg / mL blasticidin.

[0056] The original SARS-CoV-2 strain (2019-nCoV / USA-WA1 / 2020 strain) and the SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants were propagated in Vero-hTMPRSS2 cells. Infectious virus titers were determined by plaque assay on Vero-hACE2-hTMPRSS2 cells.

[0057] To inactivate SARS-CoV-2, culture supernatants containing infectious virus were treated with a 1:1000 dilution of β-propiolactone (BPL) for 18 hours. After inactivation with BPL for 1 hour at 37°C, SARS-CoV-2 inactivation was confirmed by plaque assay on Vero-hACE2-hTMPRSS2 cells, as previously reported. Inactivated samples and a positive control were included in the assay.

[0058] Aerosolization experiments in the laboratory

[0059] The experimental setup for the collection of EBC samples was CHεST (登録商標) (CH Technologies (USA), Inc) and exhaled aerosol analyzer. CHεST (登録商標)The device can simulate the particle generation and conditions associated with both exhalation and coughing / sneezing. It includes a Blaustein Atomizing Module (BLAM, CH Technologies (USA), Inc.), which simulates the size distribution of aerosols produced by various respiratory activities (e.g., exhalation and sneezing). The BLAM is a type of atomizer that generates aerosols using the jet atomization principle. The cycle period (i.e., aerosol generation time) and cycle interval (i.e., the interval between successive cycles) are defined by the user. To simulate a person breathing into the device, the BLAM was placed outside the jar. The cycle period and interval were set to 5 seconds each to simulate continuous exhalation. To estimate the flow rate of compressed air passing through the atomizer, the following calculation was performed: The aerosolization experiment mimicked 10–15 deep human breaths. The average resting human breathing rate is 7–8 LPM, and the forced expiratory volume (FEV) is typically 0.5 L. However, during deep breathing, the FEV can vary between 60 and 80% of the total lung capacity (6 L), depending on the person's gender and age. The FEV during deep breathing is estimated to be 4.8 L, so the total lung capacity during 10 to 15 deep breaths is in the range of 48 to 70 L. Therefore, compressed air (20 psi pressure setting) at a flow rate of 5.5 LPM is used for CHεST. (登録商標) The sample is collected over a 10-minute period. (登録商標) The BLAM atomizer is combined with a syringe pump for efficient infusion delivery. The inactivated virus solution (100 μL inactivated SARS-CoV-2 virus / 25 mL PBS) is delivered to the BLAM from the syringe pump at a flow rate of 0.9 mL / min. Thus, the inlet of the BLAM atomizer contains compressed air and the inactivated virus solution.

[0060] When the device is switched on, the atomizer generates an aerosol that travels to the breath analysis device. The BLAM simulates the aerosol generation and conditions associated with forced exhalation. A conical connector with an extended cylindrical section connects the atomizer outlet to the input straw of the breath analysis device. The 3D-printed conical connector narrows the path of the generated aerosol, thus resulting in greater impact on the breath analysis surface. The breath analysis device is placed in a -20°C freezer for at least 10 minutes before each test. The temperature difference between the aerosol stream generated by the atomizer (hot) and the cold surface of the collection device (cold) liquefies the impacting aerosol particles. The slope and hydrophobic nature of the cooled surface allow condensate to slide to the bottom of the collection device. After 10 minutes, the EBC sample collected on the surface of the breathalyzer is collected from the bottom, washed with 1 mL of PBS, and sent for analysis.

[0061] Electrode fabrication

[0062] The biosensor uses a screen-printed carbon electrode chip. To promote tyrosine oxidation and effective nanobody binding, the working electrode was pretreated in PBS with a 70 Hz triangular pulse voltage ranging from 0 to 3 V for 20 seconds, followed by holding at -0.8 V and 1.5 V for 5 and 10 seconds, respectively. The carboxyl groups on the electrode surface were activated using 0.4 M EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and 0.1 M NHS (N-hydroxysuccinimide) solution (Thermo Scientific, IL, USA) to generate semistable reactive amine NHS esters. The activated electrode was placed in a nanobody solution and incubated for 10 minutes at room temperature, followed by overnight at 4 °C. After the nanobody was bound to the electrode surface, the biosensor was incubated with 0.05% ethanolamine (to deactivate reactive amine sites) and 0.1% albumin (to block nonspecific protein binding sites).

[0063] Electrochemical measurement characterization

[0064] Prior to analysis, EBC samples were diluted in cut-glass vials containing 1% bovine serum albumin (BSA) in PBS solution. The biosensor was connected to a commercially available potentiostat (PalmSens4), and SWV was performed in the blank and sample solutions. SWV was scanned at an electrode potential of 0-1 V and a frequency of 15 Hz. Voltammograms were acquired in a three-electrode configuration using the handheld potentiostat (PalmSens) and the accompanying PSTrace 5.9 software.

[0065] Virus aerosol recovery rate (%)

[0066] In laboratory experiments, stock solutions of inactivated SARS-CoV-2 particles and PBS were first prepared, virus concentrations were measured from the stock solutions (using RT-qPCR), and the percentage of viral aerosol recovery from the exhaled aerosol sampler was determined.

[0067] Viral aerosol recovery was calculated as the ratio of the number of virus copies detected in the aerosolized sample divided by the total number of virus copies contained in the stock solution, and expressed as a percentage of the latter.

[0068] [ka]

[0069] The theoretical total RNA copy number depends on the stock RNA amount and the aerosolized sample volume entered into the collection device, while the sample RNA copy number depends on the sample RNA amount and the collected EBC volume. Because the EBC sample is diluted with 1 mL of phosphate buffer saline (PBS) solution, the dilution factor is also taken into account in the calculation.

[0070] Theoretical total RNA copy number = (stock viral load) * (aerosolized sample volume)

[0071] [ka]

[0072] Sample RNA copy number = (sample RNA volume) x (EBC volume) x (dilution factor)

[0073] Therefore, viral aerosol recovery (%) is estimated for different SARS-CoV-2 variants.

[0074] Predicted Exhalation Rate

[0075] The predicted exhalation rate is calculated from the limit of detection (LoD) of the MIE biosensor for various SARS-CoV-2 variants. The LoD is the oxidation current (I ox This refers to the minimum viral RNA concentration (copies / mL) required to produce a viral load (detectable). The typical viral load ranges from 70 to 30,000 copies / mL after 20 breaths, with an average of 2470 copies / mL. This range is used to estimate the number of viral copies per breath, and the minimum number of breaths required for detection can be calculated as follows:

[0076] The number of RNA copies per breath varies from 3.5 to 1500, with an average of 123.5.

[0077] The MIE biosensor LoD for the various mutants is shown in the table below. [Table 1]

[0078] Average EBC sample in lab experiments = 1.04 mL (including dilution with PBS)

[0079] Total RNA copy number in sample = (LoD) x (EBC sample volume)

[0080] [ka]

[0081] The minimum number of exhalations for biosensor detection varies between 0.04 and 9 for various SARS-CoV-2 variants. The boxplot in Figure 3A is constructed assuming two additional data points within ±33.33% of the mean viral load (2470 copies / ml).

[0082] Detection of SARS-CoV-2 from clinical samples

[0083] To evaluate the diagnostic device's performance in human patients, eight subjects (n = 6 COVID-positive and n = 2 COVID-negative) were tested using the clinical research device. In the clinical study, the design of the exhaled aerosol collection device was modified to include a slot at the bottom of the box that could fit an Eppendorf tube. Therefore, EBC samples could slide down the hydrophobic surface and be collected into the tube. The breathalyzer was placed in a -20°C freezer for at least 1 hour before EBC sample collection. Each subject breathed into the breathalyzer two, four, and eight times, and corresponding EBC samples were collected. In this manner, three samples were obtained from each subject. PBS was added through the injection port on the breathalyzer lid to flush any remaining EBC samples into the Eppendorf tube. After sample collection, the breathalyzer set was sterilized with HOCl and safely disposed of. The EBC samples were then analyzed for the presence of SARS-COV-2 in the laboratory using an MIE biosensor.

[0084] Example 5 Biosensor

[0085] The biosensor described herein combines recent advances in EBC sampling with ultrasensitive electrochemical detection of SARS-CoV-2 variants using llama-derived nanobodies to develop a handheld, point-of-care exhaled aerosol analyzer with a microimmunoelectrode (MIE) biosensor for clinical diagnostics. The exhaled aerosol collector has a removable infusion straw through which patients breathe into the device (Figures 1 and 4). Virus-containing respiratory aerosols contained in warm exhaled breath strike a cold, hydrophobic surface and condense. The surface is washed with 1 mL of 1% bovine serum albumin (BSA) / phosphate-buffered saline (PBS) along a tapered slope, transporting the condensed aerosol to the bottom corner of the box where the MIE biosensor is located. The biosensor utilizes screen-printed carbon-based electrodes, and llama-derived nanobodies are covalently attached to the electrode surface to confer specificity for the SARS-CoV-2 spike protein (Figures 5A-5B). The biosensor detects the oxidation of tyrosine amino acids contained in the spike protein of SARS-COV-2 (Figure 1). The biosensor surface is pre-blocked with 1% BSA to prevent nonspecific binding. Importantly, tyrosine amino acids are reduced and cannot be re-oxidized. Therefore, tyrosine contained in nanobodies or BSA, which is oxidized during electrode preparation, does not emit a signal during the actual test. The MIE biosensor is connected to a commercially available potentiostat, where square-wave voltammetry is performed to oxidize tyrosine and measure the peak oxidation current corresponding to the presence of viral aerosols in a given sample. The test is disposable and provides results within one minute, an improvement over traditional viral diagnostics.

[0086] Example 6 Performance Parameters

[0087] The specificity of the MIE biosensor was demonstrated by measuring the peak tyrosine oxidation current (I) at various concentrations of SARS-CoV-2 and SARS-CoV-1 spike proteins. oxThe SARS-CoV-2 spike protein showed a strong signal up to 20 pg / mL of sample, saturating at approximately 20 ng / mL. Meanwhile, SARS-CoV-1 showed negligible signal (Figure 2A). Despite the fact that both spike proteins share over 70% genetic composition, the biosensor exhibited high specificity for SARS-CoV-2.

[0088] The limit of detection (LoD) of the MIE biosensor was determined by serially diluting the purified, inactivated SARS-CoV-2 stock solution and measuring the I at different virus concentrations (determined by RT-qPCR). ox The minimum viral RNA concentrations detected by the MIE biosensor were 32, 8, 6, and 21 RNA copies / mL for the USA / WAa1 / 2020 (WA1), beta (B.1.351), delta (B.1.617.2), and Omicron (BA.1) strains of SARS-CoV-2, respectively (Figure 2B). The LoDs of the biosensor were comparable to or superior to those of other comparable sensors. Furthermore, the LoDs for all variants were significantly lower than the typical viral RNA levels found in the breath of SARS-CoV-2 infected individuals, highlighting the potential of the MIE biosensor for ultrasensitive detection of viral aerosols in exhaled breath. Each oxidized tyrosine emits 2–4 electrons, which the MIE biosensor detects as a current. For the beta and delta variants, the sensor response plateaued at higher concentrations, likely due to a hook effect caused by excessive analyte concentration or nanobody restriction on the electrode surface.

[0089] To evaluate device performance, inactivated SARS-CoV-2 virions of three different mutants: WA1, Delta (B.1.617.2), and Omicron (BA.1) were aerosolized in laboratory experiments. Aerosols were created that mimicked the size distribution of exhaled air from the lower respiratory tract of the lungs, and the nebulized air volume corresponded to the volume exhaled by a person taking 10–15 "deep" breaths (Figures 4 and 5A–5B). Aerosolization tests using pure PBS solution served as a control for this method. Figure 2C shows the average I measured for aerosolized virus sampled with the exhaled aerosol collector. ox The value is the I ox The sensitivity of this device is shown normalized to the 1000 kJ / s value, demonstrating a sensitivity of 77.8% (n=45). The sensitivity of this method is comparable to other electrochemical detection techniques for SARS-CoV-2; however, this method focuses on directly detecting virus-containing aerosols and provides results within one minute.

[0090] RT-qPCR results showed that viral RNA in samples collected using an exhaled aerosol analyzer was 10 per sample. 1.3 ~10 3.7 The gene copy number was determined to be 100% (Figure 2C). These values ​​are consistent with viral loads reported using EBC-based methods and are approximately 3–4 orders of magnitude lower than those measured using nasal swabs. COVID-19 infection produces 200–600 viral particles per exhaled breath, supporting the feasibility of this method for detecting viral aerosols in exhaled breath.

[0091] Example 7 Clinical Evaluation

[0092] To demonstrate the system's performance in human patients, the device was used in a clinical trial. EBC samples from eight subjects were analyzed for CoV-2 EBC (six COVID-positive and two COVID-negative, as determined by RT-qPCR of nasopharyngeal swabs). Assuming a viral load range of 70 to 30,000 copies / mL (mean = 2470 copies / mL) in 20 exhaled breaths (20), the minimum exhaled breath for MIE biosensor detection was predicted to range from 0.05 to 9 for various SARS-CoV-2 variants. This is illustrated in Figure 3A. Therefore, the standard sample collection protocol for the clinical trial required each subject to take two, four, and eight breaths for various EBCs, with an approximate 3-minute interval between successive samples (n = 24 samples). Initial results (Figure 3B) show that the sensitivity of this method is 77.9% (n=8 subjects) and 100% specificity, since the analyte signal in negative patients is below the LoD. ox The values ​​also indicate that two exhalations are sufficient for detection using the MIE biosensor. The biosensor was tested in vitro using known inactive virus particles derived from the BA.1 variant, but clinical studies were conducted in the summer and fall of 2022, when the BQ.1 variant was dominant. However, human patients were not sequenced to identify which variants were present.

[0093] overview

[0094] In summary, we present a portable point-of-care testing platform integrating a novel exhaled aerosol sampler and a nanobody-based MIE biosensor. It can provide results within 1 minute at a cost of less than US$10 per test. The sampling technique is noninvasive, and the detection method is rapid and simple, requiring no highly trained personnel. Furthermore, satisfactory results were obtained with only 20 seconds of sampling (two exhaled breaths), compared with the 5–30 minutes required for sampling in conventional EBC-based studies. Finally, the MIE biosensor exhibits high sensitivity for SARS-CoV-2 detection, with a low LoD compared to similar devices.

[0095] Tyrosine oxidation peak current (I ox The absolute value of I depends on the amount of surface-bound nanobody and analyte concentration, along with external factors (e.g., ambient relative humidity and temperature). Due to variations in individual electrode responses, the gene copy number and I obtained from RT-qPCR may differ significantly. ox Direct comparison of values ​​was not possible. The impact of the hook effect at higher viral loads requires further investigation.

[0096] The platform can be readily adapted for the detection of various CoV-2 variants as well as other respiratory pathogens of interest.

[0097] conclusion

[0098] Airborne transmission via virus-containing aerosols is a major route of respiratory disease (including SARS-CoV-2) transmission. Direct, noninvasive screening of patient respiratory viral aerosols has long been a technical challenge. A point-of-care testing platform for direct detection of CoV-2 aerosols from just two patient exhalations is provided herein. It combines a handheld exhaled aerosol sampler with a llama-derived CoV-2 spike protein-specific nanobody bound to an ultrasensitive micro-immunoelectrode biosensor, which detects oxidation of the tyrosine amino acid contained in the CoV-2 spike protein. Results were within 20% of those obtained using standard detection methods. This platform has the potential to be adapted for multiplexed detection of various respiratory viruses, providing a rapid, noninvasive alternative to conventional viral diagnostics.

[0099] The definitions and methods set forth herein are provided to better define the present disclosure and to guide those skilled in the art in the present disclosure. Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the relevant art.

[0100] To facilitate understanding of the embodiments described herein, numerous terms are defined below. Terms defined herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, terms such as "a," "an," and "the" are not intended to refer to a single individual, but rather include general types of which particular examples may be used for purposes of illustration. While the terminology herein is used to describe particular embodiments of the disclosure, its use does not delimit the scope of the disclosure, except as set forth in the claims.

[0101] In some embodiments, numerical values ​​expressing quantities of ingredients, properties (e.g., molecular weight), reaction conditions, and the like, used to describe and claim particular embodiments of the present disclosure, are sometimes understood to be modified by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine that value. In some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters are construed in light of the number of significant digits reported and ordinary rounding approaches applied. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. The numerical values ​​set forth in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation in their respective testing measurements. The recitation of ranges of values ​​herein is merely intended as a shorthand method of referring to each individual value falling within the range. Unless otherwise specified herein, each value is incorporated into the specification as if it were individually set forth herein.

[0102] In some embodiments, when used in the context of describing a particular embodiment (particularly in the context of certain claims below), the terms "a," "an," "the," and similar expressions are interpreted as including both the singular and the plural, unless otherwise specified. In some embodiments, when used herein (including the claims), "or" is used to mean "and / or," unless clearly indicated to refer to only one alternative or to mutually exclusive alternatives.

[0103] The terms "contain," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs (e.g., "comprises," "comprising," "has," "having," "includes," and "including") is also open-ended. For example, any method that "contains," "has," or "includes" one or more steps is not limited to only those one or more steps and may include other steps that are not listed. Similarly, any composition or device that "contains," "has," or "includes" one or more features is not limited to only those one or more features and may include other features that are not listed.

[0104] All methods described herein may be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided in connection with specific embodiments herein is intended solely to more clearly describe the disclosure and does not limit the scope of the disclosure, which is defined by the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0105] Groups of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements described herein. One or more members of a group may be included in or deleted from a group for reasons of convenience or patentability. When such additions or deletions occur, the modified group shall be deemed to be included herein, thereby satisfying the description requirement of all Markush groups used in the appended claims.

[0106] All of the compositions and / or methods disclosed and claimed herein may be made and / or executed without undue experimentation in light of the present disclosure. While the compositions and methods described herein have been described in terms of the embodiments contained herein, those skilled in the art will recognize that changes can be made in the compositions and / or methods in the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined in the appended claims.

[0107] This written description uses examples to disclose the disclosure, including the best mode, and to enable any person skilled in the art to practice the disclosure, including making and using any device or system, and practicing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if the structural elements do not differ from the literal language of the claims, or if they include equivalent structural elements that do not differ insubstantially from the literal language of the claims.

Claims

1. A biosensor comprising an activated electrode containing a nanobody attached thereto.

2. the activated electrode comprises amine reactive groups on the electrode surface; The biosensor of claim 1 .

3. the activated electrode comprises amine-reactive NHS ester groups on the electrode surface; The biosensor of claim 1 .

4. The nanobody is attached to the activated electrode via an amide bond. The biosensor of claim 1 .

5. The nanobody binds to the target protein. The biosensor of claim 1 .

6. The electrode is a carbon-based electrode; The biosensor of claim 1 .

7. the nanobody is derived from the Camelidae family, The biosensor of claim 1 .

8. Nanobodies confer specificity and / or recruit proteins to electrodes; The biosensor of claim 1 .

9. designed to perform voltammetry, The biosensor of claim 1 .

10. It is designed to measure the oxidation of tyrosine amino acids in proteins. The biosensor of claim 1 .

11. Designed to analyze environmental air samples and detect airborne pathogens, The biosensor of claim 1 .

12. It is designed to detect multiple pathogens simultaneously in a single test. The biosensor of claim 1 .

13. designed to detect airborne pathogens selected from the group consisting of viruses, bacteria, parasites, fungi, molds, their variants, strains, and combinations thereof; The biosensor of claim 1 .

14. It is designed to detect the repeat binding domain (RBD) of the SARS-CoV-2 spike protein. The biosensor of claim 1 .

15. designed to detect at least one SARS-CoV-2 variant; The biosensor of claim 1 .

16. 1. A method for analyzing an aerosolized sample in an exhaled breath or environmental air sample to detect an aerosolized pathogen, comprising: receiving an environmental air sample with a biosensor; and Detecting airborne pathogens A method comprising: the biosensor comprises an activated electrode containing a nanobody attached thereto; method.

17. 1. An airborne pathogen detection device for analyzing an aerosolized sample in an exhaled breath or environmental air sample to detect aerosolized pathogens, comprising: a biosensor comprising an activated electrode containing a nanobody attached thereto; and Sample Collection Device Including, the device.

18. the sample collection device is an exhaled aerosol collection device; 16. The airborne detection device of claim 15.

19. A single test is designed to detect multiple pathogens simultaneously.

16. The airborne detection device of claim 15.

20. designed to detect airborne pathogens selected from the group consisting of viruses, bacteria, parasites, fungi, molds, their variants, strains, and combinations thereof; 16. The airborne detection device of claim 15.

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