Real-time respiratory antigen detection system and method

Abstract

The present invention relates to a system for antigen detection, comprising a first container adapted to collect aerosol from human breath and a second container suitable for a solvent, in which nanoparticles are dissolved and which are linked to antibodies, and in which a change in the optical properties of the solvent is detectable upon contact between the antibody and a compatible antigen. Here, a person can exhale into the first container, and the aerosol exhaled into the air sticks to a filter on one side of the first container. The first container is then non-cooperative in the second container, which contains a solvent containing dissolved nanoparticles and antibodies coupled thereto. The non-cooperative, in particular a back-and-forth stroking motion, causes the aerosol to be washed out into the solvent. If an antigen compatible with the antibody is present in the aerosol, the nanoparticles aggregate around the antigen, changing the optical properties of the solvent, which is illuminated by a light source and detectable by a light sensor. In a further aspect, the present invention relates to a method of using the system according to the present invention. The present invention further relates to diagnostic applications and methods, as well as corresponding medical uses and therapeutic methods for the prevention of pathologies associated with SARS coronavirus.

Description

[Technical field]

[0001] The present invention relates to a system for antigen detection, comprising a first container adapted to collect aerosol from the exhaled breath of a human subject and a second container suitable for a solvent, in which nanoparticles are dissolved and which are coupled to antibodies, and in which a change in the optical properties of the solvent is detectable upon contact between the antibody and the matching antigen. Here, a person can exhale into the first container, and the aerosol exhaled into the air sticks to a filter on one side of the first container. The first container is then incooperable in the second container, which contains a solvent containing dissolved nanoparticles and antibodies coupled thereto. The incooperability, in particular the back-and-forth stroke action, causes the aerosol to be washed out into the solvent. If an antigen compatible with the antibody is present in the aerosol, the nanoparticles aggregate around the antigen, changing the optical properties of the solvent, which is illuminated by a light source and detectable by a light sensor.

[0002] In a further aspect, the present invention relates to a method of using the system according to the present invention.

[0003] The invention further relates to diagnostic applications and methods, as well as corresponding medical uses and therapeutic methods for the prevention of pathologies associated with SARS coronavirus. [Background technology]

[0004] Current research has demonstrated that human pathogens, especially viruses, can be transmitted by direct and indirect contact, but mainly by droplets and aerosols, or particles in the inhalable size range (10 μm or less). Transmission via respirable particles (4 μm or less), which remain airborne for long periods and can be inhaled into the alveoli, has been particularly controversial. In the early 1940s, aerosol transmission was demonstrated between ferrets up to 2.75 m apart. More recent animal studies have supported the hypothesis of airborne transmission (i.e., by aerosols and / or large droplets) over much shorter distances, from 5 cm to about 1 m, and this transmission was species dependent. Results from animal studies in which influenza viruses were experimentally transmitted by aerosols provide indirect evidence that transmission of the virus within populations can occur via the aerosol route. However, the sometimes large differences between animal species indicate that the transferability of the results to humans is questionable.

[0005] Coronavirus (CoV)-mediated respiratory disease was first observed in 2002. Once infecting humans, the virus can spread rapidly via droplet transmission and close human-to-human contact, potentially resulting in epidemic or even pandemic scenarios. (1~3) .

[0006] Currently, the corona pandemic is of great clinical importance to many people, especially those at high health risk. The economic impact is another important factor.

[0007] As an example, the "coronavirus disease 2019 (COVID-19)" is caused by the pathogenic coronavirus SARS-CoV-2. Starting in December 2019, the virus spread worldwide within weeks, causing an international public health emergency. The global pandemic poses major challenges to healthcare systems, as well as restrictions on social life and weakening the global market economy. As no effective therapy is currently available and the disease leads to high morbidity and mortality, there is a strong need for not only therapeutic interventions for sick individuals, but also preventive measures to contain the outbreak. (1、2) .

[0008] Based on current knowledge, the primary route of transmission of SARS-CoV-2 is via droplets and aerosols expelled into the surrounding air by coughing, sneezing, breathing, or talking from an infected person to an infectious individual. Entry of SARS-CoV-2 into cells is mediated by binding of the viral spike protein (S protein) to the human angiotensin-converting enzyme 2 (ACE2) target receptor.

[0009] The possibility of spread and infection via the respiratory tract is currently causing great anxiety among the population.

[0010] The transfer of viruses into respirable particles that can be generated by coughing, sneezing, and breathing is especially problematic in enclosed rooms and large events with many people because low ventilation levels allow these particles to remain in the air for extended periods of time. If only we could know quickly and reliably whether a person is shedding the virus before entering a room and / or event, we could avoid infecting others and contain, or even prevent, the spread of the virus. (1、4) .

[0011] Commercially available virus detection test systems, such as SARS-CoV-2, can identify viruses in the nasopharynx and / or respiratory tract by swabbing. To assess the subject's current or past contact with a pathogen, specific antibodies in the mucous membranes of the nasopharynx and / or respiratory tract are also screened directly or indirectly, as described above. These known methods suffer from various problems, which are often simultaneous, such as some sample collection procedures being inconvenient for the subject, the need to prepare the analysis site, possible sample pretreatment / processing, time required to perform the analysis, the use of reagents, and the overall cost of one or more of the known methods described above. However, the presence of viral material in a swab from a mucous membrane does not necessarily indicate the infectiousness of the person, since the viral material could be particles that have been deposited there upon inhalation, especially inhalation of virus-rich air, or it could be virus that is no longer active, since it has already been destroyed by the local immune defense. This means, for example, that known methods may give false positive results, since the material detected by the swab may already be inactivated virus. (3、4) .

[0012] When an organism is infected with a pathogen, a particularly important variable is the number of infectious particles that the organism comes into contact with. However, less sensitive test systems require a higher viral load, increasing the risk of false negative results. Positive individuals may be in different stages of illness. Individuals with high viral loads are more infectious, but usually already have symptoms at this point and are more likely to stay at home. Infected individuals may have minor symptoms or even be asymptomatic, but may already be capable of infecting others. This does not include individuals with low but still infectious viral loads that may increase during the event. Furthermore, events and / or gatherings often last for a period of several hours. It is also well known that viral loads can increase exponentially every hour. Even if a test is negative, after only a short time it is no longer appropriate to speak about a person's viral status. Furthermore, gathering a group of people even with low viral shedding may be problematic, since on the one hand the virus may increase, and on the other hand the virus may remain in the air and accumulate there.

[0013] These issues limit the use of these methods and slow response times and therefore interventions such as isolation of positive subjects, which may contribute to the spread of infectious agents and infections. (5、6) .

[0014] Further approaches to provide sensors for detecting SARS-CoV-2 are also known in the prior art.

[0015] In (7), a nanomaterial-based sensor array with multiplexing capability to detect and monitor COVID-19 from exhaled breath is disclosed. The sensor used contains various gold nanoparticles linked to organic ligands. This creates a multi-species sensing layer that can expand or contract when exposed to volatile organic compounds (VOCs), thereby causing a change in electrical resistance. In these layers, inorganic nanomaterials are responsible for electrical conductivity, where the organic coating provides the adsorption sites for VOCs. VOCs diffuse into the sensing layer or rain down on the sensing surface and react with the organic segments or functional groups coated on the inorganic nanomaterials. The interaction results in a volume change (expansion / contraction) of the nanomaterial coating. As a result, contact between inorganic nanomaterials prevents a change with increasing or decreasing electrical conductivity (higher / lower), resulting in a change in the measured electrical conductivity even without a steric change in the sensing layer. The main idea behind the sensor array is based on the finding that viral agents and / or their microenvironment can release VOCs, which can reach the exhaled breath. The appearance of VOCs in exhaled breath may aid in the immediate detection of COVID-19, as this may occur early in the course of infection.

[0016] US Patent No. 5,999,333 discloses an apparatus for detecting biomarkers, in particular COVID-19 analytes. The apparatus includes a droplet collection structure for converting exhaled vapor into fluid droplets to form a fluid sample. The apparatus further includes a test system with a biomarker test zone for receiving the fluid sample and detecting the biomarker. The droplet collection structure includes a hydrophobic area for receiving the exhaled vapor and includes a hydrophilic channel for receiving the fluid droplets and transporting the fluid droplets towards the test system. The apparatus may also include a light emitter and a light detector, where the light emitter emits radiation towards the biomarker test zone and the light detector detects radiation from the biomarker test zone.

[0017] (8) describe an improved version of the Inflammacheck device for non-invasive detection of COVID-19 in exhaled breath at the point of care. An embedded electrochemical sensor allows collection of exhaled breath condensate. The sensor surface is coated with macromolecules to which the virus binds, thus generating a measurable signal indicating whether SARS-CoV-2 is present or not.

[0018] (9) presents a colorimetric sensor based on the interaction of gold nanoparticles induced by SARS-CoV-2. The extinction spectrum of a colloidal solution of a large number of virus-targeted gold nanoparticles functionalized with antibodies targeting the surface proteins of SARS-CoV-2 red-shifts within minutes when mixed with a solution containing virus particles. This colorimetric sensor was found to be able to detect very low viral loads compared to typical PCR tests.

[0019] New and emerging problems in the prior art that have not been solved so far are the long time between testing and people encounters and the low sensitivity of the test, and a further disadvantage of the prior art is that the testing systems are expensive and time consuming to use and are not suitable for repeated and / or mass testing just before events, at work or school. Another unsolved problem is the environmental and economic disadvantages of existing testing systems. Furthermore, the dyes used in the prior art testing systems can be bleached, leading to false negative results.

[0020] Therefore, the problem solved by the present invention is to develop a particularly sensitive, rapid and reusable system and method for detecting viruses and reliably identifying infected individuals.

[0021] There are no systems and methods for comprehensive, simultaneous high-throughput screening of human exhaled breath containing potentially infectious aerosols. There is a strong need for the general public, health care and education systems, as well as high economic interest, to have the ability to rapidly and reliably determine the infection risk of any individual in real time.

[0022] The present invention provides a simple, fast, versatile, sensitive and low-cost system that solves in one way the problems in the prior art. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 0325381 Summary of the Invention [Problem to be solved by the invention]

[0024] In light of the prior art, the technical problem underlying the present invention was to provide an alternative and simple system suitable for identifying human subjects shedding pathogens as aerosols. More specifically, the problem underlying the present invention is to provide a means for rapid and sensitive detection of pathogens using antibodies bound to non-dyed nanoparticles, while avoiding dye degradation and false results. Another problem underlying the present invention is to provide a means for high-throughput detection and quantification of pathogens in aerosols in the exhaled breath of human subjects.

[0025] A further problem to be solved by the present invention is to provide a fast-growing, optimized, low-maintenance and versatile testing system at lower cost while reducing the need for reagents and disposables. [Means for solving the problem]

[0026] The object is achieved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0027] Therefore, the present invention provides a system for antigen detection, comprising a first container adapted to collect aerosol from the breath of a human subject containing the aerosol, and a second container suitable for a solvent, in which nanoparticles are dissolved and which are linked to an antibody, and further, a change in the optical properties of the solvent is detectable upon contact between the antibody and a compatible antigen, The system includes a measurement cell, within which is a spectrometer having a light source and a light sensor; and The first container has a hole on a first side and a filter on a second side, and the second container is configured to incorporate the first container, where the filter of the first container is in contact with the solvent of the second container within the measurement cell; When the second container is assembled into the first container, the sealing between the shell surfaces of both containers forces the solvent enclosed in the second container through the filter of the first container; A viable back and forth stroking motion between the first container and the second container flushes the filter and dissolves the collected aerosol from the filter into the solvent; a light source configured to illuminate the solvent and a light sensor configured to detect a change in an optical property; and The system relates to a system for antigen detection comprising an alarm device adapted to emit an alarm signal upon detecting a change in an optical property.

[0028] Those skilled in the art will appreciate that the language above about the second container being suitable for the solvent preferably means that the solvent is present in the second container.

[0029] Therefore, in a further preferred embodiment, the present invention provides a system for antigen detection comprising a first container adapted to collect aerosol from the exhaled breath of a human subject containing the aerosol, and a second container in which a solvent is present, nanoparticles are dissolved in the solvent and the nanoparticles are linked to an antibody, and further comprising a system for antigen detection, the system comprising: The system includes a measurement cell, within which is a spectrometer having a light source and a light sensor; and The first container has a hole on a first side and a filter on a second side, and the second container is configured to incorporate the first container, where the filter of the first container is in contact with the solvent of the second container within the measurement cell; When the second container is assembled into the first container, the sealing between the shell surfaces of both containers forces the solvent enclosed in the second container through the filter of the first container; A viable back and forth stroking motion between the first container and the second container flushes the filter and dissolves the collected aerosol from the filter into the solvent; a light source configured to illuminate the solvent and a light sensor configured to detect a change in an optical property; and The system relates to a system for antigen detection comprising an alarm device adapted to emit an alarm signal upon detecting a change in an optical property.

[0030] In some embodiments, the solvent comprises Tris, Triton X100, Tween 20, sodium azide, and hydrochloric acid, where the pH of the solvent is between 6 and 8, preferably about pH 7.

[0031] In some embodiments, the solvent comprises 50 mM Tris (pH 7), 0.5% Triton X100, 1% Tween 20, 1% sodium azide, 0.01% hydrochloric acid, where the pH of the solvent is between 6 and 8, preferably about pH 7.

[0032] In some embodiments, the nanoparticles are also linked to an aptamer, where the aptamer can be an RNA aptamer or a DNA aptamer. The combination of features of the present invention creates surprising synergistic effects, such that the individual features interact with each other to provide the advantageous properties and associated overall success of the present invention.

[0033] The preferred system has proven to be particularly advantageous in a number of respects.

[0034] A particular advantage is that the preferred system allows for a much higher sensitivity in the detection of antigens, meaning that even minimal amounts of up to 10 antigens can be detected to determine the infectivity of exhaled breath.

[0035] Thus, it is particularly advantageous to further appreciate that the preferred system can provide a very short response time. Thus, the inventors have realized that the preferred system can detect antigens in aerosols very quickly. Advantageously, the result of the detection of the antigen can already be detected after about 20 seconds, after about 15 seconds, after about 10 seconds, in particular also after about 5 seconds. The particularly rapid measurement can be justified by the fact that nanoparticles to which antibodies are bound, dissolved in a solvent, aggregate particularly quickly, thus resulting in a change in optical properties, which can be measured particularly quickly by the preferred spectroscopic device.

[0036] Furthermore, the preferred system has the great advantage of allowing a particularly reliable detection of antigens in breath. Conventional tests, particularly rapid tests, such as rapid tests for the SARS-CoV-2 virus, tend to produce a certain percentage of false positive or false negative reactions. The preferred system achieves a significant improvement over the prior art, since the false positive rate of the prior art tests is significantly reduced.

[0037] Moreover, the efficient manageability of the preferred system is a major advantage. For example, the preferred system can be easily transported and easily operated due to its low mass and / or size. In a preferred embodiment, the system is configured as a single unit, and the components of the preferred system preferably interact and are operatively connected to each other. Preferably, the preferred system is located in, on or against a support frame. In a preferred embodiment, the preferred system has a total unit mass of up to about 5 kg, preferably up to about 2 kg, and most preferably up to about 0.5 kg. In a further preferred embodiment, the preferred system has a length, width and / or height, preferably as a unit, of up to about 1 m, preferably up to about 0.5 m, particularly preferably up to about 0.3 m, and most preferably up to about 0.1 m. Due to the low mass and / or small dimensions of the preferred system, the system according to the invention is advantageously suitable in a variety of applications.

[0038] For example, in the context of leisure activities, it would be possible to use the preferred system in a particularly simple manner to identify whether a person has a certain antigen load and therefore is diseased or not. In this way, it is possible to identify large numbers of people particularly quickly, efficiently and reliably in situations such as visiting discos, museums, restaurants, trains, etc.

[0039] It is also a particular advantage that the preferred system is particularly simple to deploy. Thus, due to the preferred components of the system according to the invention, no complex manufacturing steps are required to assemble the preferred system. Advantageously, components such as the first container, the second container, the filter, the solvent, the light source and the light sensor eliminate complex construction methods compared to the prior art. Advantageously, the above components can be manufactured and / or obtained at low cost, so that the preferred system proves to be particularly efficient in the context of mass production and / or mass application.

[0040] The system according to the invention can therefore be used to detect antigens in exhaled breath aerosols in a particularly efficient manner, meaning that people can be tested positive or negative for certain diseases. Due to its preferred design, the system according to the invention can be called a passive puff system or passive puff test.

[0041] The present invention describes a method for detecting viruses or other pathogens, particularly the SARS-CoV-2 virus, in the exhaled breath of a human subject, thus preventing or avoiding super-spreading events and vaccine breakthroughs altogether.

[0042] Currently, there are no rapid, real-time, versatile, cost-effective and non-invasive antigen detection systems available for detection of SARS-CoV-2 antigen, especially at detection of as few as 10 viral particles per sample collection.

[0043] In particular, a significant portion of the population has either never accepted existing measurement methods, such as nasal swab tests, or rejected them after initial acceptance. The testing system disclosed herein provides a simple and entirely harmless testing system and method that is easily accepted by the population.

[0044] Preferably, the person breathes, i.e. blows air, into the first container, the exhaled breath containing aerosols which enter the first filter during exhalation and subsequently strike and become deposited on the filter of the first container.

[0045] The first container is then incorporated into a second container, where the second container contains nanoparticles dissolved in a solvent and with antibodies bound to their surfaces. Preferably, the first container is incorporated into the second container by a reciprocating stroke motion, so that the second container flushes the filter of the first container, where the solvent is introduced into the first container and is in contact with the aerosol. If an antigen that is compatible with the antibodies of the nanoparticles is present in the aerosol, the binding of the antibody and the antigen to each other will cause aggregation of the nanoparticles, causing a change in optical properties that can be detected by a light sensor and a light source irradiating the solvent.

[0046] In some embodiments, the system comprises a calculation unit, where the calculation unit is configured to calculate a concentration of the antigen based on a change in an optical property of the solvent.

[0047] Advantageously, the calculation step for determining the concentration of the antigen can be carried out by the system. For this purpose, preferably, a computer program product such as software and / or algorithms capable of carrying out the corresponding calculation operations is installed in the calculation unit. Advantageously, the result, in particular the accurate result, for the concentration of the antigen can be determined by the system itself.

[0048] In some embodiments, the light source is a monochromatic laser comprising linearly polarized light, where the linearly polarized light is configured to excite the pathogen-nanoparticle aggregates at an extinction peak wavelength, which may be preferably between 600 nm and 700 nm, preferably between 610 nm and 690 nm, more preferably between 620 nm and 680 nm, even more preferably between 630 nm and 670 nm, even more preferably between 640 nm and 660 nm, and even more preferably between 645 nm and 655 nm.

[0049] The use of a monochromatic laser is advantageous in that it has a particularly high photon density and is therefore, in the context of the present invention, particularly suitable for irradiating with sufficient intensity the solvent and the aerosol dissolved therein by the introduction of the first container.

[0050] The aforementioned wavelengths have therefore proven to be particularly advantageous in that they are particularly sensitive and allow particularly accurate and high-resolution measurements.

[0051] In some embodiments, the light source comprises one or more LEDs, where the light source preferably emits unpolarized light configured to excite pathogen-nanoparticle aggregates at multiple extinction wavelengths.

[0052] In some embodiments, the wavelength may be between 400 nm and 800 nm, for example, 465 nm, 557 nm, 591 nm, 632 nm, 520 nm, 572 nm, 610 nm, 700 nm, and / or 730 nm, or any value in between.

[0053] In one embodiment, the light temperature of the LED light is about 2700K.

[0054] In one embodiment, the optical signal detected by the optical sensor is a reflected optical signal, where the reflection of the optical signal is preferably mediated by a reflective foil, which reflects the light on the other side of the measuring solution back to the optical sensor.

[0055] The use of at least two LEDs as light sources is particularly advantageous in that it makes it possible to assess the absorption spectrum of the measuring solution to be evaluated at different wavelengths, i.e. over two or more wavelengths, and to detect different sizes of GNPs and GNP aggregates for different pathogens.

[0056] In some embodiments, the optical sensor is preferably a spectral sensor, wherein preferably a channel of the spectral sensor is configured to detect Raman shifted light scattered by pathogen-nanoparticle aggregates excited by a laser or light source.

[0057] In some embodiments, the light sensor is horizontally and / or vertically spaced from the light source, where the detection opening of the light sensor, which is preferably configured to block light, is arranged to face in the same direction as the light source. The reflective foil of the housing wall of the detection unit reflects the light back to the sensor, so that the measurement signal of the light sensor is advantageously higher, and the absorption is higher due to the increased path length through the measurement liquid. In the context of the present invention, horizontally spaced means that the light sensor and the light source are arranged in the same plane. The plane here represents a reference plane oriented substantially parallel to the bottom of the system. In particular, horizontally spaced between the light sensor and the light source may also include radially spaced. Thus, vertically spaced between the light source and the light sensor is understood to mean that the light source and the light sensor are arranged in different planes. In particular, vertically spaced means that there is a height difference between the light sensor and the light detector, which are at different heights, taking the bottom of the system as a reference.

[0058] In some embodiments, the optical sensor is mounted along the illumination direction of the light source to collect a large amount of Mie scattered light caused by pathogen-nanoparticle aggregates, since these aggregates scatter the largest amount of light in the same direction as the incident light, advantageously resulting in a high measurement signal of the optical sensor.

[0059] In a further embodiment, a linear polarizing filter is placed in front of the light sensor to filter out the light and pass the depolarized light scattered by the pathogen-nanoparticle aggregates, which have a higher depolarization ratio than other contaminant particles that may be contained in the fluid, resulting in a better signal-to-noise ratio.

[0060] In some embodiments, the alarm signal is selected from the group comprising a visual signal and / or an audio signal.

[0061] Advantageously, the above mentioned signal types have been found to be particularly easy to implement, and furthermore, they are able to clearly inform the user that a (critical) amount of antigen load has been detected.

[0062] In some embodiments, the system comprises a communication unit.

[0063] Advantageously, the communication unit is able to transmit the measurement results to an external data processing unit, such as a smartphone, a tablet and / or a computer, etc. Thus, advantageously, multiple possibilities are provided for obtaining the measurement results.

[0064] In some embodiments, the nanoparticles comprise a material selected from the group including gold, silver, ruthenium, rhodium, palladium, osmium, iridium, and / or platinum, and / or the nanoparticles can be spheres in the range of 15 nm to 25 nm, or nanorods having a diameter of 10 nm to 25 nm and a length of 20 nm to 80 nm.

[0065] The above-mentioned materials for nanoparticles can be processed in an efficient and cost-effective manner for the detection of antigens, in particular for the binding of antibodies to nanoparticles. Likewise, these materials are particularly suitable for further processing, for example to make them suitable for binding to antibodies in the context of further processing steps. In particular, desired emission characteristics can be advantageously obtained.

[0066] The above dimensions are relevant in that they depolarize the illuminating light. In particular, the degree of depolarization depends on the shape of the particle. The essentially round shape of the nanoparticles causes very little depolarization of light, whereas the rather irregular shape of the pathogen-nanoparticle aggregates causes high depolarization of light.

[0067] In some embodiments, the filter may be a hydrophilic filter or a hypophilic filter, preferably a filter suitable for reversible attachment of antigens.

[0068] For filters as used herein, it is possible to choose from a variety of materials and topologies in the prior art. Such filters can be permeated with a solvent. The matrix should also be of a material suitable for reversibly trapping the captured antigen. In one embodiment, the method comprises contacting one or more filters with an aqueous solvent containing antigen-binding protein molecules (antibodies) bound to nanoparticles. In one embodiment, contacting the filter with the solvent results in the release of the antigen captured by the filter into the solvent.

[0069] In one embodiment, the filter is a hydrophilic filter, which has the advantage that the filter can be completely saturated with the solvent.

[0070] For example, the filter may be of the same material as FFP2 or FFP3 face masks known in the prior art. This filter has excellent properties for reliably retaining antigens. The human subject breathes into a first side of the first container, which is provided with a hole on the first side and is firmly connected to a filter material on the second side. In one embodiment, the hole on the first side of the first container is adapted for the human subject to cover and surround the opening with his mouth and breathe into it. In one embodiment, the second side of the first container is firmly connected to the filter. The breath hits the filter, which receives the dried antigen and the aerosol containing the antigen. In an important advantage, the filter receives and captures at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of aerosolized and dried antigens from the subject's breath. In one embodiment, the capture of antigens by the filter is reversible. The first container is introduced into the solvent of the second container on the second side connected to the filter. When inserted, the filter is completely covered by the solvent. Upon direct contact of the filter with the solvent, at least 50%, at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the captured antigen is released and dissolved in the solvent. In one embodiment, to release the antigen into the solvent, the filter is completely covered by the solvent.Releasing the antigen into the solvent includes squeezing, mixing, washing, and / or moving the filter up and down in the solvent. Releasing the antigen into the solvent also refers to distributing or mixing. In one embodiment, the filter is washed in the solvent. In one embodiment, the filter can be rinsed or washed by moving the first container up and down in the second container. In one embodiment, the filter is compressed in the solvent. In one embodiment, the antigen is released and distributed into the solvent by washing the filter in the solvent. In one embodiment, the antigen is released and distributed into the solvent by squeezing the filter in the solvent. In one embodiment, the antigen is released and distributed into the solvent by moving the filter up and down in the solvent. This is particularly advantageous for releasing and dissolving the antigen retained on the filter completely or nearly completely into the solvent.

[0071] In one embodiment, the filter is a fibrous filter. In one embodiment, the type of material of the fibrous filter can be cellulose, polymer, mineral wool, regenerated cellulose, polyamide, zein fiber, cellulose acetate, cotton, silk, wool, carbon, activated carbon, clay, synthetic resins and cellulose derivatives, ethyl cellulose, polyamide, and / or cellulose acetate propionate. In one embodiment, the fibrous filter comprises one or more layers of one type of material described herein. In one embodiment, the fibrous filter comprises two or more layers of two or more types of materials.

[0072] In one embodiment, the matrix of the filter has a thickness of 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or greater. In one embodiment, the maximum diameter of the matrix channels is suitable for retaining an antigen used to detect a particular pathogen. In one embodiment, the target particle size to be retained by the filter may be 10 μm or less, 5 μm or less, or 2 μm or less. In one embodiment, the target particle size to be retained by the filter may be 0.2 μm or less, or 0.1 μm or less. In one embodiment, the pore size may range from 0.01 μm to 200 μm. In one embodiment, the pore size may range from 0.02 μm to 100 μm. In one embodiment, the pore size may range from 0.02 μm to 50 μm. In one embodiment, the pore size may range from 0.05 μm to 20 μm. In one embodiment, the pore size may range from 0.075 μm to 1 μm. In one embodiment, the pore size may range from 0.1 μm to 0.75 μm In one embodiment, the pore size may range from 0.1 μm to 0.3 μm.

[0073] In one embodiment, the filter comprises a filter having a pore size of at least 0.1 μm and a filter thickness of less than 0.5 mm.

[0074] In one embodiment, the filter comprises a filter having a pore size of at least 0.1 μm and a filter thickness of less than 1 mm.

[0075] In one embodiment, the filter comprises a filter having a pore size of at least 0.5 μm and a filter thickness of less than 0.5 mm.

[0076] In one embodiment, the filter comprises a filter having a pore size of at least 0.5 μm and a filter thickness of less than 1 mm.

[0077] In one embodiment the filter material is an FFP2 filter material. In one embodiment the filter material is an FFP3 filter material.

[0078] In one embodiment, the filter is an inertial impaction filter comprising one or more nozzles and an impactor plate.

[0079] The combination of the filter, the first container, the second container and the solvent disclosed herein is surprisingly effective in capturing antigens from both dry breath and aerosol-containing breath and releasing them back into the solvent. The uptake of antigens by the filter is preferably complete or nearly complete. The release of antigens from the filter and dissolution of antigens into the solvent preferably occurs completely or nearly complete. This has a positive effect on the accuracy, reproducibility and specificity of the measurement. With this combination, the accuracy, speed, reproducibility and specificity of the measurement are particularly advantageous.

[0080] In some embodiments, the exhaled breath of a human subject may be collected by directly contacting the subject's mouth with the first container.

[0081] The advantage over the prior art is the independent and non-invasive introduction of air into the system by the human subject, which is particularly gentle to the human subject. Furthermore, the antigens to be measured are delivered directly from the human subject, without further processing of the sample, which may lead to loss of antigens and cause sample contamination and measurement delays. This further processing may bias the results, resulting in either false positive or false negative measurement results. Prior art measurement methods are also uncomfortable for the human subject and may cause injury. The system of the present invention offers the great advantage of injury-free measurements with very specific and highly accurate measurement results, since the antigens released by the human subject are completely detected and evaluated by the system.

[0082] In some embodiments, the solvent further comprises a salt, a detergent, an antifoaming agent, a stabilizer, a blocking agent, and / or water.

[0083] Various additives, concentrations, and their combinations suitable for the solvent are described herein as examples and are not intended to be limiting. Prior art and those skilled in the art are aware of various possible compositions of suitable solvents to provide optimal conditions for antibody-antigen recognition, binding, and color change. Those skilled in the art can certainly select suitable additives, combinations, and concentrations suitable for the disclosed inventive system and method from the solvents in the prior art.

[0084] In some embodiments, the solvent comprises one or more of maltodextrin, trehalose, PEG, a blocking agent (e.g., BSA), and / or sodium chloride. In exemplary embodiments, one or more of the solution components, e.g., maltodextrin, are lyophilized beads or pellets that are suspended in a liquid, e.g., water, saline, or a liquid biological sample. This may be provided as. For example, one or more of the solution components may be provided as beads that are suspended in a solution in a spectrophotometric cuvette or in a reaction chamber of an analyzer rotor upon addition of a liquid.

[0085] In one embodiment, the solvent comprises a salt. In some embodiments, the salt is NaCl, MgCl2, CaCl2, NaSCN, KCl, sodium phosphate, Tris-HCl. In one embodiment, the solvent comprises EDTA or EGTA. The salt is usually used in the form of a buffer in the solvent. For example, the solvent comprises a buffer, and phosphate buffered saline (PBS) or tris(hydroxymethyl)aminomethane (TRIS, e.g., TRIS-HCl) or imidazole (e.g., imidazole-HCl) is used. In one embodiment, the solvent comprises a detergent, such as SDS, Tween 20, or Triton X-100. In one embodiment, 1% or less SDS, 0.5% SDS, or 0.2% or less SDS is used. In one embodiment, 1% or less N-lauroyl sarcosine, 0.5% N-lauroyl sarcosine, or 0.2% or less N-lauroyl sarcosine is used. In one embodiment, 1% or less Tween 20, 0.5% Tween 20, 0.2% or less Tween 20 is used.

[0086] In one embodiment, the solvent includes an anti-foaming agent, such as an oil or wax.

[0087] In one embodiment, the solvent contains a stabilizer such as BSA or citric acid or sodium azide or glycine, preferably glycine and / or citric acid.

[0088] In embodiments, the pH of the solvent ranges between pH 6.5 and pH 7.5, preferably between pH 6.7 and pH 7.3, and more preferably close to pH 7.

[0089] In one embodiment, the solvent comprises 20 mM Tris / HCL, 150 mM sodium chloride, 0.1% BSA and / or Tween 20, where Tween 20 is preferably used at a concentration between 0.2% and 0.4%, said solvent is preferably for the detection of coronavirus antigens.

[0090] In one embodiment, the solvent comprises 20 mM PBS, 150 mM sodium chloride, 0.1% BSA and / or Tween 20, where Tween 20 is preferably used at a concentration between 0.2% and 0.4%, said solvent is preferably for the detection of coronavirus antigens.

[0091] In one embodiment, the solvent comprises 20 mM Tris / HCL, 150 mM sodium chloride, 0.2% NP40, and / or 0.1% BSA, wherein said solvent is preferably for detection of coronavirus antigens.

[0092] In one embodiment, the solvent comprises 20 mM PBS, 150 mM sodium chloride, Tween 20, and / or 0.1% BSA, wherein said solvent is preferably for the detection of coronavirus antigens.

[0093] Here, the solvent is particularly favorable for the stability of the bond between the nanoparticles and the antibody. Moreover, the antibody in the solvent has an optimal molecular structure suitable for specific recognition of the antigen, where the antigen is completely or almost completely dissolved in the solvent. The antigen desired to be detected is particularly well dissolved in the solvent and has an optimal molecular structure to be recognized and bound by the antibody. When the antibody binds to the antigen, aggregation is caused. The binding and aggregation leads to a change in the wavelength range of the light emitted by the nanoparticles on the antibody. The excitation light and the emission light can pass through the solvent without interference or loss. Surprisingly, the composition of the solvent does not cause any loss of the light radiation emitted by the laser and the nanoparticles.

[0094] In some embodiments, the antibody is suitable for contact with an antigen, where the antigen may be a protein, a peptide, a carbohydrate, a lipid or a nucleic acid, preferably a peptide or a protein, more preferably a protein, and the antigen is an antigen of a human pathogen, such as a viral antigen, a fungal antigen, or a bacterial antigen causing an infectious disease, such as COVID-19, respiratory syndrome, whooping cough, pneumonia, or tuberculosis.

[0095] In one embodiment, the antigen may be a nucleic acid, preferably a nucleic acid of a pathogen. In one embodiment, the antigen may be a toxin, preferably a toxin of a pathogen. In one embodiment, the antigen may be derived from a blood borne pathogen or a bacterium or a virus, where bacteria and viruses are preferred pathogens.

[0096] The antigen may be a protein, peptide, carbohydrate, lipid, or nucleic acid, preferably from a pathogen.

[0097] The invention described herein provides means and methods for detecting a soluble antigen, preferably an antigen of a pathogen. In some embodiments, the antigen is aerosolized. In some embodiments, the antigen is dissolved in a liquid. In some embodiments, the antigen is dry. For detecting pathogenic bacteria or fungi, the antigen is preferably expressed on the surface of the pathogenic bacteria or fungi or secreted by the pathogenic bacteria or fungi.

[0098] In some embodiments, the systems disclosed herein detect pathogenic bacteria or pathogenic fungi or pathogenic viruses, preferably antigens of pathogenic viruses.

[0099] In some embodiments, the systems disclosed herein detect antigens of pathogens that cause infectious diseases, preferably diseases of the respiratory tract, more preferably COVID-19, respiratory syndrome, whooping cough, pneumonia, and tuberculosis.

[0100] In some embodiments, the systems disclosed herein detect antigens of coronavirus, influenza virus, upper respiratory tract infection virus, pneumonia virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, preferably coronavirus.

[0101] In some embodiments, the human pathogen is selected from the group of bacteria, fungi, or viruses, such as coronaviruses, influenza viruses, Mycobacteriaceae, Streptococcusceae, preferably SARS coronavirus, respiratory syndrome virus, Streptococcus mutans, Mycobacterium tuberculosis, Streptococcus pneumoniae, and more preferably SARS-CoV-2.

[0102] In one embodiment, the system disclosed herein detects influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and / or bocavirus antigens, preferably coronavirus, more preferably SARS coronavirus, and even more preferably SARS-CoV-2. Examples of human pathogenic viruses that can be detected using the test system include:In some embodiments, the systems disclosed herein are capable of detecting and detecting viruses that are specific for the Adenoviridae, Alphaviruses, Astroviridae species, BK virus, Bunyaviridae species, Caliciviridae species, Colorado Tick Fever Virus (CTFV), Coxsackie B virus, Crimean-Congo hemorrhagic fever virus, Cytomegalovirus, Dengus virus, and the like. Viruses (DEN-1, DEN-2, DEN-3, and DEN-4)-Flaviviruses, Ebola virus (EBOV), multiple species of enteroviruses, enteroviruses, mainly Coxsackie A virus and enterovirus type 71 (EV71), Epstein-Barr virus (EBV), Gunnarito virus, Hendra virus, herpes simplex virus type 1 and herpes simplex virus type 2 (HSV-1 and HSV-2), herpes virus type 7 (HHV-7), human metapneumovirus human mycovirus (hMPV), human bocavirus (HBoV), herpesvirus type 6 (HHV-6) and human, human parainfluenza virus (HPIV), human T-lymphotropic virus type 1 (HTLV-1), Japanese encephalitis virus, Junin virus, Lassa fever virus, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Marburg virus, measles virus, Middle East respiratory syndrome-associated coronavirus, mumps virus, Nipah virus, norovirus, orthomyxovirus It detects antigens from multiple species of the Orthomyxoviridae family, Parvovirus B19, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, Rotavirus, Rubella virus, Sabia virus, SARS coronavirus, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Sin Nombre virus, Varicella zoster virus (VZV), Variola major or Variola minor virus, West Nile virus, Yellow fever virus, and Zika virus.

[0103] In one embodiment, the system disclosed herein detects antigens of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and / or Moraxella catarrhalis. In one embodiment, the system disclosed herein detects antigens of B. anthracis spores, Bacillus anthracis trophozoites, and / or Vibrio cholera. Examples of human pathogenic bacteria that can be detected using the test system include:

[0104] In some embodiments, the systems disclosed herein are capable of detecting and / or detecting any of a wide variety of bacteria, including Acinetobacter baumannii, Arcanobacterium haemolyticum, Bacillus anthracis, Bacillus cereus, Bordetella pertussis, Burkholderia cepacia and other species of Burkholderia, Burkholderia pseudomallei, Chlamydophila pneumoniae, Clostridium botulinum, Corynebacterium diphtheriae, Coxiella burnetii, and / or other strains of the genus Burkholderia. burnetii, Cryptococcus neoformans, Ehrlichia chaffeensis, Fusobacterium species, Helicobacter pylori, Kingella kingae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria meningitidis, Nocardia asteroides and other Nocardia species, Pasteurella Rickettsia prowazekii, Rickettsia typhi, Prevotella species, RickettsiaIt detects antigens of Streptococcus typhi, Streptococcus agalactiae, multiple Streptococcus species, Streptococcus mutans, Vibrio cholerae, and Yersinia pseudotuberculosis.

[0105] In one embodiment, the system disclosed herein detects antigens of Aspergillus fumigatus, Cryptococcus neoformans, Cryptococcus gattii, endemic fungi, Histoplasma capsulatum, and / or Pneumocystis jirovecii. Examples of human pathogenic fungi that can be detected using the test system include: In one embodiment, the systems disclosed herein detect antigens of Blastomyces dermatitidis, Coccidioides immitis, Coccidioides posadasii, Geotrichum candidum, Histoplasma capsulatum, Pneumocystis jirovecii, and Sporothrix schenckii.

[0106] Examples of human pathogenic eukaryotes and nematodes that can be detected using the test system include: In one embodiment, the system disclosed herein detects antigens of Cryptosporidium species, Isospora belli, Plasmodium species, Trypanosoma cruzi, Giardia lamblia, Capillaria aerophila, and Angiostrongylus species.

[0107] In some embodiments, viral antigens can be selected from, but are not limited to, SARS-CoV-2 S protein, SARS-CoV-2 N protein, hPMV G protein, hPMV F protein, RSV F protein, RSV G protein, or influenza A / B proteins such as HA, M1, M2, and / or NA.

[0108] In one embodiment, to detect Foot and Mouth Disease virus, the VP1 structural polypeptide is targeted by a specific antibody or a specific aptamer. In one embodiment, to detect Hepatitis B virus, the E antigen is targeted by a specific antibody or a specific aptamer. In one embodiment, to detect Hepatitis C virus, the core antigen is targeted by a specific antibody or a specific aptamer. In one embodiment, to detect Ebola virus, the GP protein is targeted by a specific antibody or a specific aptamer. In one embodiment, to detect Severe Fever with Thrombocytopenia Syndrome virus, the nucleocapsid protein is targeted by a specific antibody or a specific aptamer. In one embodiment, to detect Zika virus, the NS1 protein is targeted by a specific antibody or a specific aptamer. In one embodiment, to detect Newcastle Disease virus, the entire virus is targeted by a specific antibody or a specific aptamer. Each pathogen can be detected using a single test or a variety of tests.

[0109] A single test can be used to detect two or more pathogens simultaneously, where accurate differentiation between various viruses is achieved.

[0110] The viral antigens are recognized and bound by antibodies linked to nanoparticles, where the solvent contains said antibodies linked to nanoparticles.

[0111] In one embodiment, the viral antigen is the S protein of SARS-CoV-2. In one embodiment, the viral antigen is the N protein of SARS-CoV-2.

[0112] The systems and methods disclosed herein also provide a means to identify the presence of influenza A and / or influenza B by detecting viral nucleoprotein antigens. Influenza viral proteins suitable for detection by the systems disclosed herein include proteins expressed within the viral structure, such as HA, NA, protein polymerase (PB1, PB2, PA), matrix protein (M1, M2), and nucleoprotein ("NP"). Immunodominant antigens of influenza include hemagglutinin (H) and neuraminidase (N). In influenza A, there are currently 15 known subtypes of hemagglutinin (H1-H15) and 9 subtypes of neuraminidase (N1-N9). In one embodiment, the influenza viral antigens can be HA, M1, M2, and / or NA antigens.

[0113] Human metapneumovirus (hPMV) and respiratory syncytial virus (RSV) are the most important causes of respiratory infections during infancy and early childhood. Both viruses encode similar surface proteins defined as surface glycoproteins (G) and fusion (F) proteins. In one embodiment, the viral antigen is the F or G protein of hPMV. In one embodiment, the viral antigen is the F or G protein of RSV.

[0114] In some embodiments, the bacterial antigen includes an antigen including, but not limited to, pertussis toxin, C-polysaccharide of Streptococcus pneumoniae, early secretory antigenic target, or culture filtrate protein.

[0115] Pathogenic bacteria can also cause respiratory infections. In one embodiment, the system disclosed herein detects antigens of pathogenic bacteria that cause respiratory infections, such as Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and / or Moraxella catarrhalis. In one embodiment, the bacterial antigen can be the C polysaccharide of Streptococcus pneumoniae. In one embodiment, the bacterial antigen can be streptolysin S, streptolysin O, toxin, exotoxin A, exotoxin C, and / or surface protein M of Streptococcus pyogenes.

[0116] As described herein, the antibodies used also recognize and bind to antigens of mycotoxins, particularly trichothecene (T2) mycotoxins, the diverse group of diacetoxyscirpenols, saxitoxin, or other dinoflagellate products, and / or microcystins (various types).

[0117] The present invention further provides a method for detecting an antigen in exhaled breath, including aerosols, of a human subject using the system disclosed herein, comprising the steps of: The detection of antigens is (a) collecting antigen from an aerosol of the human subject into a system disclosed herein by directly contacting the human subject's mouth with a first end of a first container having a filter at a second end; (b) attaching the antigen to a filter; wherein said attachment is reversible; (c) distributing the antigen in a solvent as described herein by completely covering the filter with the solvent; (d) contacting the antigen with the antibody; wherein contact causes a change in an optical property of the solvent, preferably a spectral sensor; (e) determining the change in said optical property by an optical sensor as described herein and transmitting it to a computing unit as described herein; Here, the change in optical properties; (f) calculating a first parameter, the concentration of the antigen, based on the change in the optical properties of the solvent; and and The detection of said antigen relates to methods that include identifying at least one type of human pathogen described herein.

[0118] The present invention provides a simple, non-invasive method of performing antigen detection, preferably SARS-CoV2 detection, using "breath aerosols" and other non-invasively collected samples. As used herein, "non-invasive" generally refers to devices or techniques that require minimal or no insertion into an orifice and do not cause significant discomfort to the patient. Non-invasive collection of respiratory specimens refers to collection methods that do not require the insertion of an object into the patient's nose, mouth, or throat. Non-invasive collection methods can usually be performed by individuals without special medical training, although minimal assistance may be required for children and infants. In one embodiment, the methods disclosed herein include non-invasive sample collection.

[0119] In one embodiment, the medium comprises a mixture of two or more different antibodies, where each different antibody is bound to a nanoparticle with a specific wavelength range of extinction and emission. In one embodiment, the medium comprises a mixture of two or more different antibodies that detect one or two or three or four or five or six or seven or eight different antigens, where the antigens are preferably from different pathogens. The system described herein allows for the detection of two or more different antigens, which is referred to as "multiplexed."

[0120] In some embodiments, the present disclosure provides multiplexed assays that allow for individual detection of multiple nanoparticles, for example of different wave spectra, using optical sensors that include one, two, three, four, five, six, seven, or eight channels that allow for multiplexed analysis.

[0121] In some embodiments, the first parameter is compared with the second parameter by the calculation unit, and the alarm device according to any one of claims 1 to 12 generates a visual and / or acoustic signal if the first parameter is greater than or equal to the second parameter, wherein the second parameter is a preset value.

[0122] The preset value is a second parameter used to compare with the first parameter. In the context of the present invention, this change includes a change in an optical property. The preset value is related to the minimum value for the detection of a change in an optical property, such as luminescence, that must be achieved to define the detection as a positive detection in the sense of the detection of an antigen. This comparison is a simple and fast mathematical check that informatively determines whether a visual and / or auditory signal is emitted, and this check can be performed by a simple calculation unit.

[0123] In one embodiment, a visual and / or audio signal is generated if the first parameter is greater than or equal to the second parameter.

[0124] In some embodiments, detecting the antigen comprises identifying a human pathogen, preferably a virus, a fungus, or a bacterium, more preferably a coronavirus, a Streptococcus spp., a Bordetella spp., a Mycobacteria spp., or an influenza virus, and even more preferably SARS-CoV-2.

[0125] In some embodiments, the pathogen causes an infectious disease in a human subject. Pathogens include microorganisms, viruses, and toxins that can cause disease. The antigen itself need not be infectious, but can indicate the presence of a pathogen, i.e., can be used to detect it.

[0126] For detecting pathogenic bacteria or fungi, the antigen is preferably expressed on the surface of the pathogenic bacteria or fungi or secreted by the pathogenic bacteria or fungi.

[0127] In one embodiment, the system disclosed herein detects antigens of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and / or bocavirus, preferably coronavirus, more preferably SARS coronavirus, and even more preferably SARS-CoV-2.

[0128] In one embodiment, the systems disclosed herein detect antigens of human pathogens in a genus-, species-, lineage-, and / or sublineage-specific manner.

[0129] In one embodiment, the systems disclosed herein detect antigens of SARS-CoV-2, where detection can be performed regardless of SARS-CoV-2 lineage, such as SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 lambda, or SARS-CoV-2 omicron.

[0130] In one embodiment, the systems disclosed herein detect antigens of SARS-CoV-2, where detection can be specific to one or more of the SARS-CoV-2 lineages, such as SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 lambda, or SARS-CoV-2 omicron.

[0131] In one embodiment, the systems disclosed herein detect antigens of SARS-CoV-2, where detection can be specific to one or more SARS-CoV-2 sublineages of the SARS-CoV-2 lineage, such as SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 lambda, or SARS-CoV-2 omicron.

[0132] Detection capacity and specificity can be tailored by selecting antigens specific to the genus, species, lineage, and / or sublineage of the human pathogen.

[0133] In the sense of the present invention, antigens of the Coronaviridae family are preferably detected. In one embodiment, antigen detection comprises identifying human pathogenic coronaviruses, including human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), preferably SARS-CoV-2.

[0134] A particular advantage of the present system is that it can detect more than one antigen, and that the detection of more than one antigen can be done simultaneously (also known as multiplex). Furthermore, it is advantageous to be able to assign antigens to specific pathogens. This is particularly useful for diagnosis, therapy, and classification of risk to infected human subjects and human subjects in close proximity to infected human subjects. Proximity is advantageous in allowing transmission of pathogens from an infected human subject to another human subject.

[0135] Surprisingly, multiplex measurements are as fast, sensitive and specific as single detection. Even those skilled in the art would not expect that the color detections of nanoparticles would not interfere with each other, for example, by scattering, loss of light energy, and transfer of resonance energy. Even more unexpectedly, the sensitivity or specificity of antigen-antibody binding is unchanged in multiplex detection compared to single detection.

[0136] Further example systems and methods are described in detail below.

[0137] Detailed Description of the Invention The present invention relates to a system for antigen detection comprising a first container configured to collect an aerosol from the exhaled breath of a human subject and a second container suitable for a solvent comprising nanoparticles linked to an antibody. In a further aspect of the invention, a change in the optical properties of the solvent is detectable upon contact between the antibody and a matching antigen, said change being associated with the presence of the antigen.

[0138] Unless otherwise explained, 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 disclosure belongs. The singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "Comprising" means "including." Thus, "comprising A or B" means "including A" or "including B" or "including A and B."

[0139] aerosol The term "aerosol" as used herein includes droplets, droplets, and droplet cores. Aerosol as used in the present invention is understood as a heterogeneous mixture of solid and / or liquid components produced by a human subject and contained in the exhaled breath of the human subject.

[0140] Aerosol particles, exhaled breath The term "aerosol particles" as used herein is also intended to include components of pathogens or pathogens exhaled from a human subject. Aerosol particles are also solid components within an aerosol. For the purposes of the present invention, aerosol particles travel in exhaled breath as liquid components, aerosol particles bound to the aerosol and / or in crystalline structure. The diameter of the aerosol particles is preferably between 0.01 μm and 500 μm, more preferably between 0.1 μm and 100 μm, and most preferably between 0.1 μm and 50 μm.

[0141] First container For the purposes of the present invention, a first container refers to a container having an internal cavity. In a preferred embodiment, the first container may have a substantially cylindrical, elliptical or spherical shape. Preferably, the first container is configured so that the user can exhale, in particular blow, air into the first container. For example, the user can lift the first container itself and exhale into it. Also, preferably, the first container may be guided to the user of the preferred system by another person. In a further embodiment, the first container may be attached to a holder during use.

[0142] A first side and a second side of the first container In a preferred embodiment, the first container comprises a first side and a second side. Preferably, the first side and the second side represent an area of ​​the first container. Preferably, the first side and the second side can describe to some extent an end of the first container. For example, in the preferred case of a cylindrical embodiment of the first container, the first side and the second side can form a bottom and a lid. Preferably, the first side of the container comprises a hole and the second side comprises a filter.

[0143] hole For the purposes of the present invention, a hole refers to an open area of ​​the first container, preferably arranged on the first side of the first container. The hole preferably allows exhaled air from the user to enter the cavity of the first container. The hole of the first container may preferably extend substantially along the first side of the container. In a further preferred embodiment, the hole may extend along a portion of the first side. In a further embodiment, the movable body is arranged in the cavity of the first container such that a cross-sectional change of the hole occurs substantially periodically as a result of exhalation, thus the user receives an acoustic feedback (similar to a whistle) when exhaling, which may have effects such as a pitch change.

[0144] Second container Preferably, the second container refers to a container having a cavity therein. In a preferred embodiment, the second container may have a substantially cylindrical, elliptical or spherical shape. Preferably, the container is configured such that the solvent enters the second container. Furthermore, the first container is preferably adapted to fit substantially snugly into the cavity of the second container. Likewise, it is preferred that there is a substantially air-tight contact between the first container and the second container. For this reason, it is preferred that the geometric configuration of the first container and the second container are substantially identical. Preferably, the diameter of the second container is slightly larger than the diameter of the first container and / or is substantially the same as the diameter of the cavity of the first container. In a further preferred embodiment, the second container may be a component or part of a support frame comprising the preferred components of the system of the present invention. Thus, when the first container is fed into the second container, the aerosol, preferably in the filter, comes into contact with the solvent of the first container (second container). Preferably, the second container is replaceable. Additionally, it is preferred that the second container has a body surface that comprises a substantially transparent material, thereby allowing light emitted from the light source to impinge on the nanoparticles located in the solvent.

[0145] Measurement Unit In the context of the present invention, the measuring unit preferably denotes a spatial region in which the optical properties of the solvent, in particular the change in the optical properties of the solvent after the first container is dispensed into the second container, are preferably measured. In a preferred embodiment, the measuring unit may be arranged in a carrier frame. The measuring unit thus preferably specifies in a volume in which a light source can illuminate the solvent and the optical properties can be detected by a light sensor, and in which the corresponding components of the preferred system can preferably be mounted in the measuring unit accordingly.

[0146] spectrometer The design and operation of a spectrometer is well known in the art. In the sense of the present invention, a spectrometer denotes a device comprising a light source and a light sensor. Thereto, it is preferred that a second container is arranged between the light source and the light sensor so that a measurement of the change in the optical properties by the light sensor can be performed. Furthermore, it is preferred to adapt and / or adjust the beam path, e.g. by arranging further optical components, such as lenses, mirrors, prisms and / or gratings, in the beam path between the light source and the light sensor. In particular, a diverging beam path can be advantageously compensated by further optical components.

[0147] Sealing In the sense of the present invention, a sealing preferably denotes an element and / or configuration that avoids undesired migration of the solvent into the first container, but preferably at the same time allows the desired migration through the filter, i.e. the first filter entering the first container, by introducing the first container into the second container. In this regard, a sealing may be, for example, but not limited to, a stuffing box, a piston ring, a bellows, a brush seal, a hydraulic seal, a pneumatic seal, and / or a translational seal, such as a carbon fin.

[0148] Reciprocating stroke motion The phrase reciprocating strokes preferably refers to a lifting motion of the first container into the second container. Here, in the context of the present invention, a stroke motion includes a substantially vertical up-and-down movement of the first container into the second container. Preferably, the lifting motion may be repetitive. The lifting motion, in particular the repeated lifting motion of the first container, may wash the aerosol previously deposited on the filter into the solvent.

[0149] Light source, light sensor In a further embodiment, the system further comprises exposing the conjugate to a light source in a wavelength range within the ultraviolet-visible-infrared spectrum. In yet a further aspect, the method comprises measuring an optical signal from the complex, where a change in the optical signal indicates the presence of the target analyte in the sample.

[0150] In another embodiment, the system includes exposing the conjugate to a light source in a wavelength range within the ultraviolet-visible-infrared spectrum and measuring an optical signal from the conjugate, where a change in the optical signal indicates the presence of a target analyte in the sample.

[0151] Alarm Device In the context of the present invention, an alarm device is a device capable of triggering an alarm signal when a change in the optical properties of the solvent is detected. The alarm signal can be, for example, an acoustic or optical signal.

[0152] optical properties Optical properties preferably refer to the properties and / or effects that appear when light emitted from a light source passes through a solvent. In particular, the presence of an antigen in the aerosol that is compatible with the antibody on the nanoparticles causes the nanoparticles to aggregate on the antigen, resulting in a change in optical properties. This also has an effect on the irradiating light. In particular, the scattering properties of the light change. In particular, the optical properties affect scattering. Scattering is the phenomenon in which part of the light is deflected from its direct path. However, other effects, such as reflection and absorption, may also be relevant in the context of the present invention.

[0153] Computational Unit The term "computing unit" preferably refers to any device that can be configured to perform computing operations. Preferably, the computing unit is a processor, processor chip, microprocessor, and / or microcontroller that is preferably configured to perform an evaluation of the changes in the measured optical properties. The computing unit may also preferably be a programmable circuit board that processes the changes in the optical properties. The computing unit may also preferably comprise a computer usable or computer readable medium such as a hard disk, a random access memory (RAM), a read only memory (ROM), a flash memory, etc.

[0154] The phrase "computational unit so configured" preferably means that a computer code and / or software capable of performing certain computational operations, such as determining the type and / or amount of antigen by changes in optical properties, is installed in the computational unit. The computer code and / or software may be written in any programming language or model-based development environment, such as C / C++, C#, Objective-C, Java, Basic / VisualBasic, MATLAB, Simulink, StateFlow, Lab View, or Assembler.

[0155] The computer code and / or software, which is preferably installed in the computing unit, can be considered as a technical feature since the direct physical effect of the system, for example by irradiating the solvent with a light source, is used. Therefore, the functional description of the computer code and / or software can be considered as a preferred defining embodiment of the invention. The specific computer code and / or software used is available to the person skilled in the art and can be installed accordingly using standard skills in the art.

[0156] In a preferred embodiment, reference data may also be available and stored in the computer unit, which preferably allows correlation of changes in the optical properties with the measurement process.

[0157] Thus, the reference data may relate to data that allows correlation of changes in optical properties with the antigen.

[0158] The reference data may be provided, for example, as a look-up table or as parameters for a mathematical relationship that allows correlation of physical quantities. For example, the reference data may be obtained by performing a suitable calibration experiment that measures the relationship between the change in optical property and the amount and / or type of antigen.

[0159] Typically, the reference data may be stored on a computer usable or computer readable medium in the control unit. Any format commonly used in the industry may be used. The reference data may be stored in a separate file and / or may be integrated into the computer code or software (e.g., source code) present in the computing unit.

[0160] Monochromatic Laser For the purposes of the present invention, a monochromatic laser preferably refers to a laser that emits monochromatic, ie, single-color, and thus substantially one wavelength or frequency, of light.

[0161] Pathogen-nanoparticle aggregates Pathogen-nanoparticle aggregates preferably refer to substantially solid collections of multiple nanoparticles, which preferably appear when antibodies on the nanoparticles are coupled with compatible antigens, resulting in multiple nanoparticles agglomerating into clusters that, when illuminated with a light source, change the optical properties of the medium, which can be measured.

[0162] Extinction peak wavelength The term extinction peak wavelength preferably refers to the light wavelength at which the maximum light intensity is absorbed and / or scattered.

[0163] Spectral Sensor A spectral sensor is preferably a sensor that is configured to split the incident light according to wavelengths, preferably with the aid of one or more detector elements, and is therefore preferably able to simultaneously detect the entire spectral distribution of light. In particular, a spectral sensor is able to detect scattered light. Thus, a spectral sensor allows detailed access to the spectral information in the radiant flux.

[0164] Spectral Sensor Channels The channels of the spectral sensor preferably refer to measurement channels of the spectral sensor that are preferably assigned to detector components to detect light of different ranges of wavelengths or frequencies.

[0165] Raman shift scattered light In the sense of the present invention, Raman shifted scattered light preferably means light that is scattered by the effect of Raman scattering and that is detectable by a light sensor.

[0166] To detect light scattered by Raman scattering, the polarizability must change during rotation or vibration of the molecules. In the spectrum of light scattered by a sample in a solvent due to the aggregation of nanoparticles (in the case of an antigen compatible with an antibody), other frequencies are observed in addition to the irradiated frequency (Rayleigh scattering). The frequency difference with the irradiated light corresponds to the energy of rotational, vibrational, phonon or spin-flip processes characteristic of the material. The reason lies in the interaction of light with matter, the so-called Raman effect, in which energy is transferred from light to matter (the "Stokes side" of the spectrum) or from matter to light (the "anti-Stokes side" of the spectrum). Since the wavelength of light, i.e. its color, depends on the energy of the light, this energy transfer causes a shift in the wavelength of the scattered light relative to the incident light, which is called the Raman shift. Raman scattering typically has a small scattering cross section, which is appropriate to increase the sensitivity of the preferred system.

[0167] Inline The term "in-line" preferably refers to an arrangement of the light source and the light sensor, preferably the spectral sensor, such that the light source and the light sensor form a horizontal line as a connecting line. The in-line arrangement of the light source and the light sensor allows a particularly accurate detection of changes in the optical properties.

[0168] Mie scattered light Mie scattered light is the basic term for light resulting from the Tyndall effect. In Mie scattering, light is scattered by nanoparticles with diameters comparable to or longer than the wavelength of the incident light. The Mie scattering signal is proportional to the square of the particle diameter of the scattering particle.

[0169] Linear Polarizing Filter A linear polarizing filter preferably refers to a component that can be used to polarize unpolarized light into linearly polarized light, thereby allowing light of only one polarization direction to pass through.

[0170] Depolarized Light In the sense of the present invention, preferably depolarized light means light that vibrates in any direction perpendicular to the direction of propagation.

[0171] Unpolarized Unpolarized light refers to light with a random, time-varying polarization. Natural light, like most other visible light sources, is produced independently by a large number of atoms or molecules whose emissions are uncorrelated. The term is somewhat imprecise, since at any given location and at any given time there is a constant plane of polarization. However, this means that the polarization changes so rapidly over time that it cannot be measured or related to the outcome of an experiment. Unpolarized light can be produced from an incoherent combination of vertical and horizontal linear polarization, or clockwise and counterclockwise circular polarization. Conversely, two linear polarization states of unpolarized light cannot form an interference pattern, even if they rotate relative to each other (Fresnel-Arago's third law). In particular, unpolarized light means that light vibrates in any direction perpendicular to the direction of propagation and / or that there is no uniform, fixed vibration direction relative to the direction of propagation.

[0172] reflective foil The term "reflective foil" refers to a material that reflects light because it has metallic optical interactions. That is, while some materials may cause light to be diffracted through, incident on, or scattered by an object, light is reflected back from the film in a very predictable manner. More precisely, the angle at which light is incident on the surface is the same relative to the normal angle (the vector perpendicular to the surface) at which the light is reflected from the surface.

[0173] Visual Signals By visual signal is preferably meant a signal that can be visually perceived by a human being, such as a display, a flashing lamp, a light signal emitted by an LED, a color change, and the like.

[0174] Acoustic signal An acoustic signal refers to sound waves that are preferably in the frequency range of approximately 10 Hz to approximately 20,000 Hz and thus perceptible to the human ear.

[0175] Communication Unit In the sense of the present invention, a communication unit preferably means a transmission unit which transmits a signal, which preferably transmits information about the change in optical properties and / or the antigen. The transmission of data and / or information via the communication unit is preferably carried out by directional or non-directional electromagnetic waves, whereby the frequency bands used can range from a few Hertz (low frequency) to several hundred Terahertz (visible light), depending on the application and technology used. According to the present invention, a wide variety of data transmission methods can be used, examples of which include, in the radio frequency range, Bluetooth, WLAN, ZigBee, Thread, RFID, NFC, Wibree or mobile phone wireless networks (especially campus networks) or WiMAX, but also, in the infrared or optical frequency range, IrDA and optical directional radio (FSO). Preferably, information can be transmitted by the communication unit to an external data processing unit.

[0176] In the sense of the present invention, an external data processing unit preferably refers to an external computing unit which can be configured to read out and preferably evaluate the data. For example, the external data processing unit may be a PC, a tablet computer and / or a mobile terminal such as a smartphone.

[0177] spread The term "spreading" includes diffusion, transport, and convection. In particular, dispersion may occur via air currents and air currents. In particular, for the purposes of the present invention, the term refers to the spread of viruses, pathogens, aerosol particles, aerosols, salt crystals, and / or salts dissolved in aerosols. Spreading can be interpreted as spreading of viruses, spreading of aerosol particles, spreading of aerosols, spreading of crystals, spreading of pathogens. Diffusion is the equalization of concentration differences in a liquid or gas by Brownian molecular motion. Convection is the movement of physical quantities in a flowing gas or liquid.

[0178] Direct spatial contact For the purposes of the present invention, the term "direct spatial contact" preferably means that two objects are in contact.

[0179] Continuous / Periodic / One-time The term "periodic" as used herein specifically describes a process that occurs periodically and repeatedly at equal intervals over a defined period of time. "Continuous" is also taken to mean a process that occurs continuously over a defined period of time. For purposes of the present invention, if an operation is triggered once and can be triggered repeatedly at unequal intervals over an indefinite period of time, the operation is also referred to as "one-off."

[0180] Medical Indication In one aspect of the invention, there is provided a system and method comprising an antibody or antibody fragment coupled to a nanoparticle according to the invention as described herein for use in detecting a pathogen of a condition associated with SARS-coronavirus, wherein the condition associated with SARS-coronavirus is preferably COVID-19 or a SARS-coronavirus associated respiratory disease.

[0181] As used herein, a "patient" or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, as well as other organisms.

[0182] As used herein, a patient with "symptoms of a respiratory tract viral infection" is a subject who suffers from one or more cold-like symptoms or flu-like illness, including, but not limited to, fever, cough, runny nose, sneezing, sore throat, difficulty breathing, headache, muscle pain, fatigue, rapid heartbeat, rapid breathing, nausea and vomiting, loss of taste and / or smell, and / or fatigue (feeling unwell).

[0183] In some embodiments, symptoms of infection with the SARS virus include fever, sore throat, cough, muscle aches, or fatigue, and in some embodiments, further include sputum production, headache, hemoptysis, and / or diarrhea. In some embodiments, symptoms of infection with a SARS coronavirus, such as SARS-CoV-2, include fever, sore throat, cough, loss of taste and / or smell, shortness of breath, and / or fatigue.

[0184] As used herein, the term "patient at risk of developing Severe Acute Respiratory Syndrome (SARS)" relates to a subject at an elevated (e.g., above average) risk of developing SARS, preferably different from any given person in the general population. In some embodiments, the patient has symptoms of SARS or symptoms of SARS coronavirus infection. In some embodiments, the patient does not have symptoms of SARS or symptoms of SARS coronavirus infection. In some embodiments, the subject has been in contact with people with SARS coronavirus infection or symptoms thereof. In some embodiments, a person at risk of developing SARS is one who has been tested for the presence of SARS coronavirus infection. In some embodiments, a person at risk of developing SARS is one who has tested positive for the presence of SARS coronavirus infection, preferably coronavirus infection.

[0185] In an embodiment, a patient at risk of developing SARS is an asymptomatic patient who does not (yet) show specific symptoms of SARS. An asymptomatic patient may be at risk of developing SARS because he or she has been in contact with a person infected with SARS coronavirus. For example, an asymptomatic patient may have been identified as at risk of developing SARS by a software application (app) installed on his or her smartphone or a corresponding (portable) device, indicating physical proximity or close physical proximity to an infected patient using a corresponding app on the respective mobile device / smartphone. Other methods of determining contact / physical proximity to an infected person are known to the skilled person and apply equally to the method of the present invention.

[0186] In some embodiments, the patient suffering from or at risk of developing Severe Acute Respiratory Syndrome (SARS) is suffering from a coronavirus infection.

[0187] Coronaviruses are a group of related viruses that cause disease in mammals and birds. The scientific name for coronaviruses is the family Orthocoronavirinae or Coronavirinae. Coronaviruses belong to the Coronavirinae family. This family is divided into the subfamily Coronavirinae and Torovirus, which are further divided into six genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus, Torovirus, and Bafinivirus. Viruses in the Alphacoronavirus and Betacoronavirus genera primarily infect mammals, whereas Gammacoronavirus infects avian species and members of the Deltacoronavirus genus have been found in both mammalian and avian hosts.

[0188] In humans, coronaviruses cause respiratory tract infections that can be mild, such as some cases of the common cold, or fatal, such as SARS, MERS, and COVID-19. Coronaviruses are enveloped viruses with a positive-sense, single-stranded RNA genome and a nucleocapsid with helical symmetry. The genome size of coronaviruses ranges from approximately 27 kilobases to approximately 34 kilobases, making them the largest of the known RNA viruses.

[0189] Various species of human coronaviruses are known, including, but not limited to, human coronavirus OC43 (HCoV-OC43) of the genus β-CoV, human coronavirus HKU1 (HCoV-HKU1) of the genus β-CoV, human coronavirus 229E (HCoV-229E) of the genus α-CoV, human coronavirus NL63 (HCoV-NL63) of the genus α-CoV, Middle East Respiratory Syndrome-related Coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0190] Coronaviruses vary widely in terms of risk factors. Some coronaviruses (e.g. MERS-CoV) are fatal in over 30% of infected individuals, while others are relatively harmless, like the common cold. Coronaviruses cause colds with major symptoms such as fever and sore throat, e.g. due to adenoid swelling, and occur mainly during the winter and early spring seasons. Coronaviruses can cause pneumonia (direct viral pneumonia or secondary bacterial pneumonia) and bronchitis (direct viral bronchitis or secondary bacterial bronchitis). Coronaviruses can also cause SARS.

[0191] In some embodiments, the patient is suffering from an infection, preferably an infection caused by SARS coronavirus (SARS-CoV). As used herein, SARS coronavirus refers to the coronavirus that causes Severe Acute Respiratory Syndrome (SARS), a viral respiratory disease of zoonotic origin that first surfaced in the early 2000s and was caused by the first identified strain of SARS coronavirus (SARS-CoV or SARS-CoV-1).

[0192] SARS is induced by droplet transmission, and viral replication can be found in the upper and lower respiratory tract or gastrointestinal mucosa. In parallel, the virus can also directly invade cells in various organs, such as the liver, kidney, heart, and brain. Another unique mechanism seems to be the direct invasion of the virus into T cells. Many SARS patients with COVID-19 have clinically low concentrations of lymphocytes in the blood, also known as lymphopenia. Clinically, patients present with respiratory symptoms such as dry cough and shortness of breath, fever, or diarrhea. However, symptoms related to acute liver, cardiac, or renal damage may also occur. In less severe cases of SARS, patients may show mild or no symptoms at all.

[0193] Embodiments of SARS coronavirus include, but are not limited to, any coronavirus that induces SARS or SARS-like pathology. Particular embodiments include, but are not limited to, SARS coronavirus (SARS-CoV-1), first discovered in 2003 (see above), Middle East Respiratory Syndrome (MERS-CoV), first discovered in 2012, and SARS-CoV-2, which causes COVID-19, the disease that caused the 2019-2020 coronavirus pandemic.

[0194] SARS-CoV-2 strains cause COVID-19, the disease that caused the ongoing 2019-2020 coronavirus pandemic. The disease was first identified in December 2019 in Wuhan, the capital of Hubei province in China, and has spread worldwide. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include muscle pain, diarrhea, sore throat, loss of taste and / or smell, and abdominal pain. Most cases cause mild symptoms, but some may progress to viral pneumonia and multiple organ failure.

[0195] Coronaviruses can be identified by molecular techniques, e.g., sequence-based analysis, e.g., PCR-based amplification of viral genetic material. Genome-wide phylogenetic analysis indicates that SARS-CoV-2 shares 79.5% and 50% sequence identity with SARS-CoV and MERS-CoV, respectively. However, 94.6% sequence identity is found between the seven conserved replicase domains in ORF1ab of SARS-CoV-2 and SARS-CoV, whereas less than 90% sequence identity is found between the replicase domains of SARS-CoV-2 and other -CoVs, suggesting that SARS-CoV-2 belongs to the beta-CoV lineage.

[0196] Similar to other CoVs, the SARS-CoV-2 virion, with a genome size of 29.9 kb, has a nucleocapsid composed of genomic RNA and phosphorylated nucleocapsid (N) protein. The nucleocapsid is embedded in a phospholipid bilayer and is covered by two different types of spike protein, namely the spike glycoprotein trimer (S) present in all CoVs, and the hemagglutinin esterase (HE) shared only by some CoVs. The membrane (M) protein and the envelope (E) protein are located in the S protein within the viral envelope. The SARS-CoV-2 genome has 5'- and 3'-terminal sequences typical of -CoVs (265 nucleotides at the 5'-end and 229 nucleotides at the 3'-end region) with the gene order 5'-replicase open reading frame (ORF) 1ab-S-envelope (E)-membrane (M)-N-30. The predicted S, ORF3a, E, M, and N genes of SARS-CoV-2 are 3822, 828, 228, 669, and 1260 nucleotides in length, respectively. Similar to SARS-CoV, SARS-CoV-2 has a predicted ORF8 gene (366 nucleotides in length) located between the M and N ORF genes.

[0197] Since the start of SARS-CoV-2 spread, SARS-CoV-2 has acquired an increasing number of polymorphic nucleotide positions in various reading frames of the viral genome (such as nsp2, nsp6, RDRP, S, ORF3A, ORF8, and N), based on which the virus can be divided into phylogenetic groups or lineages. Currently, intensive research is being carried out to determine whether and how certain mutations affect the properties of the virus, such as transmissibility, virulence, or immunogenicity (Alm, E., Broberg, EK, Connor, T., Hodcroft, EB, Komissarov, AB, Maurer-Stroh, S., Melidou, A., Neher, RA, O'Toole, A., Pereyaslov, D., et al. (2020). Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January-June 2020. Euro Surveill 25.).

[0198] The World Health Organization classifies virus variants as "variants of interest (VOI)" or "variants of concern (VOC)."

[0199] The term "variants of interest," or "VOI," refers to SARS-CoV-2 variants that (1) have a phenotypic change or a mutation that likely or definitely affects the phenotype, and (2) are causing multiple case clusters or cases in different countries in the context of community transmission, or (3) are classified as VOIs by the World Health Organization (2021b). COVID-19 Weekly Epidemiological Update (Supplement, February 25, 2021), Special Edition: Proposed Working Definitions of SARS-CoV-2 Variants of Interest and Concern (World Health Organization).

[0200] The term “variants of concern”, or “VOCs”, refers to virus variants with changes in epidemiology (especially increased transmissibility), clinical findings (especially increased virulence), or pathogenic characteristics that have been shown to adversely affect the effectiveness of countermeasures, diagnostic detection methods, vaccines or therapeutics ( World Health Organization, 2021b ).

[0201] The WHO currently classifies four SARS-CoV-2 variants, namely B.1.1.7, B.1.351, P.1, and B.1.617.2, as VOCs (World Health Organization (2021a); ​​COVID-19 Weekly Epidemiological Update - 11 May 2021 (WHO); Oh, D.-Y., Kroeger, S., Wedde, M., Hartkopf, F., Budt, M., Seifried, J., Radonic, A., Belarbi, E., Hoelzer, M., Boettcher, S., et al. (2021). SARS-CoV-2-Varianten: Evolution im Zeitraffer. Deutsches Aerzteblatt 118, A-460 / B-388.). According to the WHO complementary and simplified nomenclature system, they are also called alpha, beta, gamma, and delta variants by the Greek letters in the order of their discovery (World Health Organization, 2021c). The skilled person can find information about SARS-CoV-2 variants and their sequences in the relevant literature and databases such as NCBI, GISAID, or published in Germany by the Robert Koch Institute (www.rki.de / covid-19-varianten-nomenklatur).

[0202] VOCs include the SARS-CoV-2 variants alpha, beta, gamma, delta, lambda, and omicron.

[0203] As used herein, "alpha variant", "B.1.1.7", "VOC202012 / 01", "GR / 501Y.V1", "20I / 501Y.V1" or "UK variant" is a SARS-CoV-2 variant defined by mutations in the spike protein (SEQ ID NO:2 and SEQ ID NO:13), including del69 / 70, del144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, and accounts for approximately 95% of new SARS-CoV-2 infections in England.

[0204] As used herein, "beta", "B.1.351", "501Y.V2", "VOC202012 / 02", "GH / 501Y.V2", "20H / 501Y.V2", or "South African variant" refers to a SARS-CoV-2 variant that contains mutations in the spike protein (SEQ ID NO:3 and SEQ ID NO:14) including L18F, D80A, D215G, LAL242-244del, R246I, K417N, E484K, N501Y, D614G, A701V, in particular three amino acid substitutions (K417N, E484K, and N501Y) in the receptor binding domain of the spike protein that are defined by non-synonymous mutations that can increase its affinity for the human ACE2 receptor (Tegally, H., Wilkinson, E., Giovanetti, M., Iranzadeh, A., Fonseca, V., Giandhari, J., et al., J. Immunol. 2014, 14:1311-1323, 1999). J., Doolabh, D., Pillay, S., San, E., Msomi, N., et al. (2020)).

[0205] As used herein, "Gamma", "P1", "B.1.1.28.1", "501Y.V3", "VOC202101 / 02", "GR / 501Y.V3", "20J / 501Y.V3", or "Brazil variant" is a SARS-CoV-2 variant defined by mutations in the spike protein (SEQ ID NO: 5), including L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F.

[0206] As used herein, "B1.617.2", "delta", "VOC202104 / 01", "G / 452R.V3", "21A / S:478K", ​​or "India variant" is a SARS-CoV-2 variant defined by mutations in the spike protein (SEQ ID NO: 4), including T19R, del157-158, L452R, T478K, D614G, P681R, D950N.

[0207] The "lambda variant", "C.37 variant" or "Peru variant" are defined by mutations in the spike protein (SEQ ID NO: 6) including D614G, T859N, F490S, L452Q or L452R, T76I, G75V, del246 / 252. Mutations L452Q and F490S are located within the spike protein receptor binding domain.

[0208] "Omicron". As used herein, SARS-CoV-2 variants of pangolin lineage B.1.1.529 include numerous sublineages (e.g., BA.1, BA.2, BA.2.12.1, BA.3, BA.4 / BA.5) that result from omicron variants. Compared to the original SARS-CoV-2 virus, there are approximately 50 amino acid changes, the majority of which (e.g., about 32) are located within the spike protein. For several of these amino acid changes (spike: K417N, S477N, E484A, N501Y, D614G, H655Y, P681H), phenotypic effects have been described (e.g., immune evasion) ( Andreano et al., 2021 , Greaney et al., 2021 , Starr et al., 2020 , Weisblum et al., 2020 , Zahradnik et al., 2021 ).

[0209] The present invention also covers additional SARS-CoV-2 variants that have not yet emerged.

[0210] The present invention can also be used to identify any of the above-mentioned variants of a pathogen, such as a viral variant, or to identify any species or subspecies. For example, SARS-CoV can be identified regardless of whether it is SARS-CoV or SARS-CoV-2. As another example, SARS-CoV-2 can be identified regardless of whether it is a SARS-CoV-2 variant alpha and / or beta and / or gamma and / or delta and / or lambda and / or omicron. In another example, SARS-CoV-2 variants alpha and / or beta and / or gamma and / or delta and / or lambda and / or omicron can be specifically identified.

[0211] Infectious diseases, viruses, pathogens, SARS-COV-2 The term "infectious disease" as used herein is also understood to mean, in the sense of the present invention, a disease in humans caused by contact and / or invasion of a pathogen into a cell, preferably caused by a virus. According to the skilled artisan, "virus" is also understood to mean an organic structure that spreads extracellularly by infection as a virion, but can only grow inside a suitable cell as a virus. According to the skilled artisan, the invasion of a virus into a cell can also be called infectious or potentially infectious.

[0212] pathogen As used herein, "pathogen" refers to a molecular structure or agent that is infectious, i.e., capable of causing disease, such as an infectious disease, in a particular organism. In some embodiments, the pathogen causes an infectious disease in a human subject. Pathogens include microorganisms, viruses, and toxins that can cause disease. The antigen itself need not be infectious, but can be indicative of the presence of a pathogen, i.e., can be used to detect it.

[0213] The taxonomic terms "genus," "species," "lineage," and "sublineage" are used as defined in the art.

[0214] The invention described herein provides means and methods for detecting a soluble antigen, preferably an antigen of a pathogen. In some embodiments, the antigen is aerosolized. In some embodiments, the antigen is dissolved in a liquid. In some embodiments, the antigen is dry. For detecting pathogenic bacteria or fungi, the antigen is preferably expressed on the surface of the pathogenic bacteria or fungi or secreted by the pathogenic bacteria or fungi.

[0215] Pathogenic fungi: Pulmonary fungal pathogens can cause life-threatening invasive disease. Pathogenic fungi that cause respiratory disease include Aspergillus, Cryptococcus, endemic fungi, and Pneumocystis. In one embodiment, the system disclosed herein detects antigens of Aspergillus fumigatus, Cryptococcus neoformans, Cryptococcus gattii, endemic fungi, Histoplasma capsulatum, and / or Pneumocystis jirovecii. Further examples are listed in Example 7.

[0216] As described herein, the antibodies used also recognize and bind to antigens of mycotoxins, particularly trichothecene (T2) mycotoxins, the diverse group of diacetoxyscirpenols, saxitoxin, or other dinoflagellate products, and / or microcystins (various types).

[0217] Pathogenic bacteria: As described herein, the antibodies used recognize and bind to antigens of pathogenic bacteria, including but not limited to B. anthracis spores, Bacillus anthracis trophozoites, Francisella tularensis, Yersinia pestis, Vibrio cholerae O139, Vibrio cholerae O1, Brucella spp, Chlamydia spp, Bacillus subtilis spores, Staphylococcal enterotoxin-B (SEB) and other superantigens, ricin, pertussis toxin, Salmonella spp, Shigella dysenteriae, and the like. The pathogenic bacteria that cause respiratory infections may be selected from the group consisting of Escherichia coli O157:H7, Ralstonia species, Listeria, or Clostridium botulinum. Pathogenic bacteria that cause respiratory infections include Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis. In one embodiment, the system disclosed herein detects antigens of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and / or Moraxella catarrhalis. Further examples are listed in Example 5.

[0218] Pathogenic virus: As described herein, the antibody used recognizes and binds to an antigen of a pathogenic virus, where the pathogenic virus may be selected from the group of, but is not limited to, coronavirus, influenza virus, upper respiratory tract infection virus, pneumonia virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, Venezuelan equine encephalitis virus, foot-and-mouth disease virus, dengue virus, orthopoxvirus, potyvirus. In one embodiment, the pathogenic virus is preferably a virus that causes a respiratory infection, such as pneumonia or an upper respiratory tract infection. Pathogenic viruses that cause respiratory infections include influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and bocavirus. In one embodiment, the system disclosed herein detects antigens of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, and / or bocavirus, preferably coronavirus, more preferably SARS coronavirus, and even more preferably SARS-CoV-2.

[0219] Several families of human pathogenic viruses are known, such as the Coronaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Rhaboviridae, Adenoviridae, Polyomaviridae, Papillomaviridae, Picornaviridae, which are listed here in a non-exhaustive manner, preferably Coronaviridae in the sense of the present invention. Among the human pathogenic coronaviruses, for example, but not limited to, human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), preferably SARS-CoV-2, are known. Table 1 provides examples of virus families that include pathogenic viruses and corresponding immunogens that can be used for detection.

[0220] [Table 1] Table 1: Examples of virus families Family Examples of viruses Immunogens Orthomyxoviridae influenza Paramyxoviridae Retroviridae Coronaviridae Arenaviridae Filoviridae Togaviridae Flaviviridae Bunyaviridae Rhabdoviridae Herpesviridae Family Bornaviridae Family Poxviridae Asfarviridae Arteriviridae Hepadnaviridae

[0221] In the case of influenza virus, the immunodominant antigens located on the surface of the virus are hemagglutinin (H) and neuraminidase (N). In influenza A, 15 subtypes of hemagglutinin (H1-H15) and 9 subtypes of neuraminidase (N1-N9) are currently known. Influenza virus proteins suitable for detection by the systems disclosed herein include proteins expressed within the viral structure, such as HA, NA, protein polymerases (PB1, PB2, PA), matrix proteins (M1, M2), and nucleoprotein ("NP"). Preferably, the conserved peptide sequences are conserved on at least two or more of M1, M2, HA, NA, or on one or more polymerase proteins, and preferably all, if not most, of the above.

[0222] Further examples are listed in Example 6.

[0223] Spike protein, corona protein Of particular interest from a diagnostic standpoint are the spike protein (S protein), due to its role in interaction with the human host receptor, and the highly conserved nucleocapsid protein (N protein), which has high immunogenicity.

[0224] Coronavirus spike proteins, also known as S proteins, are glycoprotein trimers, where each monomer of the trimeric S protein is approximately 180 kDa and contains two subunits, S1 and S2, which mediate attachment and membrane fusion, respectively. As explained in Xiuyuan Ou et al. (Nature Communications, 11, 1620, 2020), coronaviruses (CoVs) use the spike glycoprotein to bind to ACE2 and mediate membrane fusion and viral entry. In the S protein structure, the N- and C-terminal parts of S1 fold as two independent domains, the N-terminal domain (NTD) and the C-terminal domain (C domain). The RBD of mouse hepatitis virus (MHV) is located in NTD14, while most other CoVs, including SARS-CoV and MERS-CoV, use the C domain to bind to their receptors.

[0225] In one embodiment, the antibody conjugated to the nanoparticles recognizes the S protein and is used in the systems and methods of the invention to identify human subjects infected with SARS-CoV-2. In one embodiment, the antibody conjugated to the nanoparticles specifically recognizes a domain of the S protein, such as a human recombinant monoclonal antibody or antibody fragment that recognizes the receptor binding domain (RBD) of the S1 subunit (S1) of the recombinant spike glycoprotein of SARS-CoV-2.

[0226] Another protein that can be used as an antigen to detect SARS-CoV-2 in aerosols in human subjects is the nucleocapsid of the SARS-CoV-2 coronavirus.

[0227] The SARS-CoV-2 nucleocapsid protein (also SARS-CoV-2 N protein or N protein) is a highly immunogenic phosphoprotein and the most detectable protein in coronaviruses. The SARS-CoV-2 N protein is involved in the replication of the viral genome and in regulating cell signaling pathways. The SARS-CoV-2 nucleocapsid protein is of great interest for the development of SARS-CoV-2 diagnostic assays because the N protein is highly conserved in sequence and exhibits strong immunogenicity. The SARS-CoV-2 nucleocapsid protein is the major structural protein that binds to the RNA genome of the virus. In one embodiment, an antibody conjugated to the nanoparticles recognizes the N protein and is used in the systems and methods of the invention to identify human subjects infected with SARS-CoV-2. In one embodiment, the antibody conjugated to the nanoparticles, such as a human recombinant monoclonal antibody, a human recombinant polyclonal antibody, or an antibody fragment, specifically recognizes a domain of the N protein.

[0228] Thus, in some embodiments, the antibodies used may be specific for CoV-2, in other words, they bind to the spike or N protein of CoV-2 and not to those of other viruses. In some embodiments, the antibodies may bind to the spike or N protein of any given SARS coronavirus. In some embodiments, the antibodies may bind to the spike or N protein of coronaviruses that have sufficient similarity for the antibodies to bind, but have evolved or mutated from CoV-2 to form new viruses. In some embodiments, the antibodies bind to SARS coronaviruses (also referred to herein as "coronaviruses"), and more preferably to SARS-CoV-2. Thus, it is believed that the antibodies used bind to the RBD of the spike or N protein of SARS-CoV-2 as it exists in the subject, i.e., in the form it takes in the human subject before, during, or after infection of the host cell.

[0229] antibody, antigen As used herein, an "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes that specifically binds to, recognizes, or contacts an epitope of an antigen. Antibodies include monoclonal antibodies, polyclonal antibodies, or fragments of antibodies, and other forms known in the art. In some instances, the antibody is linked or conjugated to another molecule, such as a nanoparticle (e.g., a gold nanoparticle). As used herein, the term "polypeptide," "peptide," or "protein" refers to a multimer in which the monomers are amino acid residues (e.g., amino acid sequences) linked by amide bonds, including modified sequences such as glycoproteins. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer may be used. The term "polypeptide" or "protein" refers to both naturally occurring proteins and recombinantly or synthetically produced proteins.

[0230] Where the term "antibody" is used, reference may also be made to the term "antibody fragment." Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

[0231] Antibodies consist of heavy and light chains, each of which has a variable region called the variable heavy region (VH) and the variable light region (VL). Together, the VH and VL regions are responsible for binding to the antigen recognized by the antibody. Antibodies include intact immunoglobulins and their variants and portions known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are joined by a linker, whereas in dsFv, the chains are mutated to introduce a disulfide bond to stabilize the joining of the chains. The term also includes recombinant forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0232] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or a dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain (about 25 kD) and one "heavy" (H) chain (about 50 kD to about 70 kD). The N-terminus of each chain defines a variable region of about 100 to about 110 or more amino acids, which are primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains, respectively. Optionally, antibodies or immunological portions of antibodies can be chemically conjugated to or expressed as fusion proteins with other proteins.

[0233] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those of skill in the art, such as by generating hybrid antibody-forming cells from the fusion of a myeloma cell with an immune spleen cell. These fused cells and their progeny are called "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.

[0234] The term "conjugate" or "bioconjugate" refers to a compound in which one molecule, such as an antibody, is operatively coupled to another molecule, such as a nanoparticle, either directly or indirectly, by suitable means. In some embodiments, the molecule, preferably an antibody, may be directly covalently coupled to the nanoparticle, such as via a metal-thiol bond. In some embodiments, the molecule, preferably an antibody, may be coupled through the use of a "linker" molecule, so long as the linker does not substantially interfere with the function of the molecule, preferably an antibody. Preferably, the linker is biocompatible. Known molecular linkers include maleimide or succinimide groups, streptavidin, neutravidin, biotin, or similar compounds.

[0235] "Conjugate", "link", "bond" or "linked" refers to coupling of a first moiety to a second moiety, including but not limited to covalent binding of one molecule to another molecule (e.g., directly or through a linker molecule), non-covalent binding of one molecule to another molecule (e.g., electrostatic binding, electrostatic conjugation), non-covalent binding of one molecule to another molecule by hydrogen bonds, non-covalent binding of one molecule to another molecule by van der Waals forces, and all combinations of such couplings. Those skilled in the art are aware of methods of conjugating, linking, binding or linking, i.e., covalently binding, linking or non-covalently coupling a first moiety to a second moiety, and can apply them from the prior art in the sense of the present invention. As used herein, "direct linkage" is understood as coupling or conjugation of two molecules without an intervening linker molecule. In some embodiments, a direct linkage is formed when an atom of a first molecule is bound to an atom of a second molecule. In some embodiments, a direct link is formed when an atom of the antibody binds to an atom of the nanoparticle, hi some embodiments, the direct link is a covalent bond, such as a metal-thiol bond, preferably a gold-thiol bond.

[0236] As used herein, the term "colocalized" refers to occurring at the same or substantially the same location. In some instances, metal precipitates (e.g., metals in oxidation state 0) formed by the methods described herein are colocalized with a target molecule when they accumulate or aggregate within at least about 5 μm of the target molecule (e.g., within at least about 1 μm, within at least about 500 nm, within at least about 250 nm, within at least about 100 nm, within at least about 50 nm, within at least about 20 nm, within at least about 10 nm, within at least about 5 nm, within at least about 2 nm, within at least about 1 nm, or within at least about 0.5 nm of the target molecule).

[0237] The term "contact" as used herein refers to an arrangement that allows an association between two or more entities, in particular a direct physical association, e.g. in solid and / or liquid form (e.g. contact between a biological sample or a biological molecule, preferably an antigen attached to a filter or dissolved in a solvent, and an antibody).

[0238] The term "detect" or "detecting" as used herein is to be construed as determining whether a target molecule (e.g., an antigen) is present or absent in a sample, such as an aerosol exhaled by a human subject. In some instances, this may include quantification. "Detect" also refers to any method of determining whether something is present, such as determining whether a target molecule is present in a biological sample, such as an aerosol exhaled by a human subject. The term "detection" may include, for example, using visual, acoustic, and / or mechanical devices to determine whether a sample has a particular characteristic, such as whether an antigen is present in an aerosol exhaled by a human subject. In some embodiments, detection includes visual and / or acoustic observation that an antibody is bound to a target molecule, particularly an antigen, or observation that an antibody is not bound to a target molecule, particularly an antigen.

[0239] "Specifically binds" should be understood by those skilled in the art who clearly recognize the various experimental procedures that can be used to test binding and binding specificity. In many protein-protein interactions, some cross-reactivity or background binding may be unavoidable, but this does not diminish the "specificity" of the binding between an antibody and an epitope. The term "directed against" is also applicable when considering the term "specificity" in understanding the interaction between an antibody and an epitope.

[0240] "Antigen" refers to any target particle or target molecular structure that is recognized by an antibody. The antigen may be an antigen of a pathogen. The antigen may be a soluble antigen. In one embodiment, the antigen may be a nucleic acid, preferably a nucleic acid of a pathogen. In one embodiment, the antigen may be a toxin, preferably a toxin of a pathogen. In one embodiment, the antigen may be from a blood borne pathogen or a bacterium or a virus, etc., where bacteria and viruses are preferred pathogens.

[0241] The antigen may be a protein, peptide, carbohydrate, lipid, or nucleic acid, preferably from a pathogen.

[0242] The antigen may also be an antigen of a human pathogen, such as a viral, fungal or bacterial antigen causing an infectious disease, such as COVID-19, respiratory syndrome, whooping cough, pneumonia or tuberculosis.

[0243] Transmission The term "transmission" of a pathogen as used herein is taken to mean "contact transmission", "invasion", "transmission" and "infection", preferably the transmission of a virus to human cells. The skilled person is aware that using the device of the present invention to measure the spread and transmission of human pathogenic viruses and viral components outside the laboratory is an unacceptable hazard. In particular, the hazard may exist not only in the time situation immediately after the measurement, but also days and weeks after the measurement. Such measurements are also prohibited by the Biological Substances Ordinance. Aerosol particles can also be used to simulate the spread and transmission of viruses, particularly of coronaviruses, more preferably SARS-CoV-2.

[0244] sample The term "sample" refers to any liquid, semi-solid, or solid substance (or material) in which a target may be present. In particular, the sample may be a biological sample. Examples of biological samples include saliva and aerosols exhaled from a human subject. In some embodiments, the biological sample is obtained from a human subject.

[0245] Biological samples include any solid or liquid sample obtained from, excreted by, or exhaled by an organism, particularly samples from healthy or apparently healthy human subjects or human patients suffering from a condition or disease to be diagnosed or investigated, such as SARS-CoV-2. For example, the biological sample may be an aerosol or saliva produced by the human subject. In one embodiment, the sample is a quality control sample. In one embodiment, the sample is a test sample. For example, the test sample is a molecule (e.g., an antigen) obtained from a pathogen. In one embodiment, the subject is at risk for or has suffered from a particular condition or disease.

[0246] The present invention provides a simple, non-invasive method of performing antigen detection, preferably SARS-CoV2 detection, using "breath aerosols" and other non-invasively collected samples. As used herein, "non-invasive" generally refers to devices or techniques that require minimal or no insertion into an orifice and do not cause significant discomfort to the patient. Non-invasive collection of respiratory specimens refers to collection methods that do not require the insertion of an object into the patient's nose, mouth, or throat. Non-invasive collection methods are typically performed by individuals without special medical training, although minimal assistance may be required for children and infants. In one embodiment, the methods disclosed herein include non-invasive sample collection.

[0247] Multiple The sample may contain multiple targets, i.e., antigens, that can be specifically bound by antibodies. The easiest way to detect multiple targets is to use multiple antibodies bound to different nanoparticles with different wave spectra by increasing the number of detection channels. The above system exploits the diversity of antibodies to provide a means to detect multiple rare target particles or target antigens.

[0248] In some embodiments, multiple detectable nanoparticles that can be separately detected can be conjugated to different specific antibodies that specifically bind to different antigens to provide a multiplexed assay that can result in the detection of multiple pathogens in a sample.

[0249] In one embodiment, the medium comprises a mixture of two or more different antibodies, where each different antibody is bound to a nanoparticle with a specific wavelength range of extinction and emission. In one embodiment, the medium comprises a mixture of two or more different antibodies that detect one or two or three or four or five or six or seven or eight different antigens, where the antigens are preferably from different pathogens. The system described herein allows for the detection of two or more different antigens, which is referred to as "multiplexed."

[0250] In some embodiments, the present disclosure provides multiplexed assays that allow for individual detection of multiple nanoparticles, for example of different wave spectra, using optical sensors that include one, two, three, four, five, six, seven, or eight channels that allow for multiplexed analysis.

[0251] Nanoparticles, metal ions The term "nanoparticle" as used herein refers to a nanoscale particle having a size measured in nanometers, e.g., a nanoscopic particle having at least one size less than about 500 nm, or less than about 400 nm, or less than about 300 nm, or less than about 200 nm, or less than about 100 nm, or less than about 50 nm. Examples of nanoparticles include, but are not limited to, metal nanoparticles, magnetic nanoparticles, paramagnetic nanoparticles, superparamagnetic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers with covalently bound metal chelates, etc., nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In a preferred embodiment, the nanoparticles are metal nanoparticles, such as nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more of these metals. The nanoparticles may have a core, or a core and a shell, such as core-shell nanoparticles. In one embodiment, the nanoparticles are gold nanoparticles. In one embodiment, the nanoparticles are silver nanoparticles. In one embodiment, the nanoparticles are platinum nanoparticles.

[0252] In one embodiment, the nanoparticles range in size from 15 nm to 25 nm in diameter, and in one embodiment, the nanorods range in size from 15 nm to 25 nm in diameter and 30 nm to 60 nm in length.

[0253] A "metal ion" is a cation that requires reduction and electrons to convert to a metal (zero oxidation state). Metal ions include, but are not limited to, gold ions, gold ions, copper ions, nickel ions, platinum ions, palladium ions, cobalt ions, or iridium ions. Metal ions may also include transition metal ions, such as from titanium. The metal ions may be in the form of a solution of a metal salt, such as a metal nitrate, metal halide, metal acetate, or metal perchlorate (e.g., silver nitrate, silver acetate, silver fluoride, or silver perchlorate). In other examples, the metal salt may be a metal sulfite, metal phosphate, or metal carbonate.

[0254] In one embodiment, antibody-nanoparticle conjugates are used, which may be used in methods to detect target molecules such as proteins, lipids, carbohydrates, or nucleic acid molecules.

[0255] Antibody-Nanoparticle Conjugates As used herein, an antibody-nanoparticle conjugate includes one or more nanoparticles, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nanoparticles, or more nanoparticles, e.g., 2-10 nanoparticles or 2-5 nanoparticles, directly linked to an antibody by a metal-thiol bond between the nanoparticle and a thiol present on the antibody (e.g., an amino acid residue, such as a cysteine ​​residue, of the antibody).

[0256] In some embodiments, the nanoparticles used in the antibody-nanoparticle conjugates are metallic nanoparticles. In some embodiments, the nanoparticles are gold, palladium, platinum, silver, copper, nickel, cobalt, or iridium, ruthenium, rhodium, osmium, or iron, preferably gold. In other examples, the nanoparticles are alloys of two or more metals, such as two or more of the metals gold, palladium, platinum, silver, copper, nickel, cobalt, or iridium, preferably gold and silver. In some embodiments, the nanoparticles are core-shell nanoparticles having a metal core and a shell of another metal, preferably silver nanoparticles having a gold shell. In some embodiments, the nanoparticles have a metal core having about 10 to about 200 atoms, such as about 100 to 200, about 100 to about 150, about 11 to about 100, or about 11 to about 70 atoms.

[0257] In one embodiment, the nanoparticles are gold nanoparticles. Metallic nanoparticles and methods for making metallic nanoparticles are well known in the art. See, for example, Nanoparticles: From Theory to Application, Gunther Schmid, ed., Wiley-BCH, 2004.

[0258] In some embodiments, two or more nanoparticles conjugated to an antibody each have a diameter of about 0.5 nm to about 200 nm, e.g., about 1 nm to about 100 nm, about 2 nm to about 50 nm, about 2 nm to about 10 nm, or about 0.5 nm to about 50 nm. In certain instances, the nanoparticles have a diameter of about 5 nm or less, e.g., about 5 nm, about 4.5 nm, about 4 nm, about 3.5 nm, about 3 nm, about 2.5 nm, about 2 nm, about 1.5 nm, about 1 nm, or about 0.5 nm, or less. In other examples, the nanoparticles have a diameter of at least about 50 nm, e.g., about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, or greater.

[0259] The conjugates used include two or more nanoparticles associated with an antibody. The antibody may include monoclonal or polyclonal antibodies such as IgA, IgD, IgE, IgG, or IgM, antibody fragments including, but not limited to, proteolytic antibody fragments (such as F(ab')2 fragments, Fab' fragments, Fab'-SH fragments, and Fab fragments as known in the art), recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv")). In other examples, the antibody may include diabodies, genetically engineered antibodies, heteroconjugate antibodies (such as bispecific antibodies), and combinations thereof. In one embodiment, the antibody is a monoclonal antibody that recognizes the spike protein of a coronavirus, preferably the spike protein of SARS-CoV, more preferably the spike protein of SARS-CoV-2. In one embodiment, the antibody is a monoclonal antibody recognizing a coronavirus nucleocapsid protein, preferably a SARS-CoV nucleocapsid protein, more preferably a SARS-CoV-2 nucleocapsid protein. In one embodiment, the antibody is a polyclonal antibody recognizing a coronavirus spike protein, preferably a SARS-CoV spike protein, more preferably a SARS-CoV-2 spike protein. In one embodiment, the antibody is a polyclonal antibody recognizing a coronavirus nucleocapsid protein, preferably a SARS-CoV nucleocapsid protein, more preferably a SARS-CoV-2 nucleocapsid protein.

[0260] In one embodiment, the antibody-nanoparticle conjugate comprises a bond that directly links the antibody to the nanoparticle, such as a bond that is formed when an atom of a first molecule, the antibody, binds to an atom of a second molecule, the nanoparticle.

[0261] Aptamers are short oligomers that form specific bonds with target substances with high affinity by forming a stable tertiary structure, and can be mass-produced in a short time and at low cost using chemical synthesis techniques. Aptamers are also oligonucleotide chains of either RNA or DNA that have higher specificity and affinity for target proteins. Thus, aptamers are considered to be the nucleic acid version of antibodies. In addition, aptamers are very stable against environmental pH and temperature, and therefore their potential to be used in various fields such as environmental and medical diagnostics, for example, the detection of target substances and the development of sensors for pathogen detection, has been highly evaluated. Aptamers are generally created by selecting from a large random sequence pool using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. In addition to their excellent binding specificity and binding affinity, aptamers have many other advantageous properties that make them, as a whole, highly suitable molecules for functionalization of nanoparticles for active targeted delivery. Aptamer-functionalized nanoparticles have already demonstrated their effectiveness in targeted delivery of anticancer drugs in numerous preclinical studies and animal experiments, but have not yet reached clinical trials or clinical applications. Those skilled in the art will know how to make aptamers and will select suitable methods in the art for conjugating aptamers to nanoparticles. An example is disclosed herein (Example 3).

[0262] In one embodiment, an aptamer is conjugated to a gold nanoparticle, where the aptamer has excellent in vivo stability and can specifically bind to an antibody or any other target substance used in the detection of pathogens and diagnosis of infectious diseases.

[0263] The skilled artisan will be aware of methods for producing antibody-nanoparticle conjugates suitable for the systems and methods described herein. Suitable methods for producing antibody-nanoparticle conjugates are provided in the prior art.

[0264] Gold nanoparticles under various coating conditions Coating conditions suitable for the systems and methods described herein to produce antibody-nanoparticle conjugates are known and have been described in the prior art, for example, Pramanik A, Gao Y, Patibandla S, Mitra D, McCandless MG, Fassero LA, Gates K, Tandon R, Chandra Ray P. The rapid diagnosis and effective inhibition of coronavirus using spike antibody attached gold nanoparticles. Nanoscale Adv. 2021 Jan 18;3(6):1588-1596.

[0265] Certain specific examples of coating GNPs with antibodies are described below but should not be construed as limiting, other methods are possible to achieve the goal of optimally functional GNPs.

[0266] Synthesis of citrate-coated gold nanoparticles Citrate-coated gold nanoparticles (GNPs) were synthesized using HAuCl4·3H2O and sodium citrate. For this purpose, 0.01% HAuCl4·3H2O solution and 1 wt% sodium citrate were used. Transmission electron microscopy (TEM) as well as colorimetric and dynamic light scattering (DLS) data were used to characterize the newly synthesized nanoparticles. The color of the newly fabricated GNPs can be pink. GNPs exhibit unique optical properties due to surface plasmon resonance (SPR). It is well documented that the SPR band of citrate-coated gold nanoparticles is due to a phase change resulting from an increased rate of electron-surface collisions compared to larger particles. The absorption spectrum of citrate-coated gold nanoparticles with λmax is identified to be around 520 nm. For SPR of gold nanoparticles, the extinction coefficient ε(15)520nm=3.6×10 8 cm -1 M -1Since it gives a significantly higher absorption, it can be used for sensing COVID-19 antigens or virus using colorimetric studies with the naked eye.

[0267] X-ray diffraction (XRD) spectrum from citrate-coated gold nanoparticles. The particle concentration was calculated based on the maximum absorption wavelength of the gold colloid (ε(15) 519 nm = 3.6 × 10 8 cm -1 M -1 ) was measured by UV-Vis spectroscopy using the molar extinction coefficient at 100 nm.

[0268] Synthesis of PEG-coated gold nanoparticles To make GNPs biocompatible, they can be coated with HS-PEG-COOH. For this purpose, carboxy-PEG12-thiol (HS-PEG12-COOH) can be used. 10 mg of HS-PEG12-COOH can be dissolved in 5 mL of water and the citrate-coated GNPs can be added using a syringe with vigorous stirring. The mixture can be sonicated for 30 minutes, after which the final product can be separated by centrifugation. After coating with PEG, the size of the GNPs increases to about 18±4 nm. The color of the newly made PEG-coated GNPs can be pink. The absorption spectrum of the PEG-coated gold nanoparticles with λmax is confirmed to be about 521 nm, which can indicate the optical spectrum for the GNPs.

[0269] Synthesis of anti-spike antibody-coated gold nanoparticles Anti-spike antibodies can be attached to gold nanoparticles. EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) / NHS (N-hydroxysuccinimide) chemistry can be used. 0.2 molar (M) EDC and 0.05 M N-hydroxysulfosuccinimide sodium salt (1:3 (volume / volume) ratio) can be added to a solution containing GNPs and anti-spike antibodies, and the mixture can be sonicated for 30 minutes. GNPs with attached anti-spike antibodies can be separated from antibody-free GNPs using centrifugation at 6000 rpm for 15 minutes, followed by resuspension in buffer. After the antibodies are attached, the size of the GNPs can increase to about 27±6 nm. The number of antibodies attached to the GNPs can be determined by synthesizing GNPs conjugated with anti-spike antibodies attached with Cy3 dye. After separating the GNPs conjugated with antibodies attached to Cy3 dyes from unconjugated molecules, 10 μM potassium cyanide was added to oxidize the GNPs and the number of antibodies attached to the GNPs was calculated from the fluorescence data at 10 ng mL -1 The antibody attached to the GNPs can be estimated as:

[0270] The color of the newly created antibody GNPs may be pink. Figure 2D shows that the absorption spectrum of the antibody-attached gold nanoparticles with λmax is confirmed to be about 522 nm, which may indicate the optical spectrum for the GNPs.

[0271] solvent The term "solvent" as used herein refers to a solution, e.g., an aqueous solution, suitable for dissolving an antigen and specifically binding an antibody to said antigen. The solvent provides optimal reaction conditions, including pH, for the antibody to be bound and remain bound to the nanoparticles and for the antibody to specifically recognize and optimally bind to the antigen. The solvent includes nanoparticles bound to the antibody in water and one or more components including salts, detergents, blocking agents, reducing agents, antifoaming agents, stabilizers, and / or carbohydrates.

[0272] Various additives, concentrations, and their combinations suitable for the solvent are described herein as examples and are not intended to be limiting. Prior art and those skilled in the art are aware of various possible compositions of suitable solvents to provide optimal conditions for antibody-antigen recognition, binding, and color change. Those skilled in the art can certainly select suitable additives, combinations, and concentrations suitable for the disclosed inventive system and method from the solvents in the prior art.

[0273] The term "reducing agent" refers to an element or compound that reduces another molecule or compound. When another molecule or compound is reduced, the reducing agent becomes oxidized and is an electron donor. Specific examples of reducing agents include, but are not limited to, dithiothreitol (DTT), beta-mercaptoethanol, and sodium thiosulfate.

[0274] A "carbohydrate" can also be a polysaccharide. In one embodiment, the solvent comprises a polysaccharide at a final concentration of about 0.5% (w / v) to about 25% (w / v). In one embodiment, the solution comprises a polysaccharide at a final concentration of about 1% (w / v) to about 20% (w / v). In one embodiment, the solution comprises a polysaccharide at a final concentration of about 2% (w / v) to about 15% (w / v). In one embodiment, the solution comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or more (including all values ​​in between). In some embodiments, the sensitivity of the methods described herein is improved by using a polysaccharide, such as sucrose, trehalose, maltodextrin, sorbitol, mannitol, or ficoll. In one embodiment, the polysaccharide is Ficoll. In yet another exemplary embodiment, the polysaccharide is dextran.

[0275] As used herein, a "blocking agent" is understood to prevent non-specific binding of an antibody to an antigen. In one embodiment, the solvent comprises a blocking agent at a final concentration of about 0.1% (w / v) to about 20% (w / v). In another embodiment, the solution comprises a blocking agent at a final concentration of about 0.5% (w / v) to about 10% (w / v). In yet another embodiment, the solution comprises a blocking agent at a final concentration of about 1% (w / v) to about 5% (w / v). In exemplary embodiments, the solution comprises a blocking agent at a final concentration of about 1%, about 2%, about 3%, about 4%, or about 5% (including all values ​​therebetween). In various embodiments described herein, the addition of a blocking agent to the solution can improve the sensitivity of the assay compared to an assay performed in the absence of a blocking agent. In some embodiments, the blocking agent is selected from bovine serum albumin, casein, gelatin, ovalbumin, and gamma globulin. In an exemplary embodiment, the blocking agent is bovine serum albumin (BSA).

[0276] In some embodiments, the solvent comprises one or more of maltodextrin, trehalose, PEG, a blocking agent (e.g., BSA), and / or sodium chloride. In exemplary embodiments, one or more of the solution components, e.g., maltodextrin, are lyophilized beads or pellets that are suspended in a liquid, e.g., water, saline, or a liquid biological sample. This may be provided as. For example, one or more of the solution components may be provided as beads in a spectrophotometric cuvette or in a reaction chamber of an analyzer rotor that are suspended in a solution upon addition of a liquid.

[0277] Furthermore, salts such as chaotropic salts may have an effect on the stability of the gold conjugate colloid. In some embodiments, the solvent comprises a salt. A salt is known to those skilled in the art as a chemical compound consisting of a positively charged ion, called a cation, and a negatively charged ion, called an anion, between which there exists an ionic bond. A cation in the sense of the present invention may be, inter alia, a sodium, potassium, calcium, magnesium, or lithium ion, preferably a sodium ion. An anion in the sense of the present invention may be, inter alia, a chloride, an iodide, a sulfate, a nitrate, an acetate, a succinate, or a citrate ion, preferably a chloride ion. In some embodiments, the salt is NaCl, MgCl2, CaCl2, NaSCN, KCl, sodium phosphate, Tris-HCl. In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In one embodiment, the solvent comprises EDTA or EGTA. The salt is usually used in the form of a buffer in the solvent. For example, the solvent comprises a buffer, and phosphate buffered saline (PBS) or HEPES or tris(hydroxymethyl)aminomethane (TRIS, e.g., TRIS-HCl) or imidazole (e.g., imidazole-HCl) is used. Methods of adding phosphate buffer, sodium chloride, etc. are known (JP 3-504499 A). Buffer systems suitable for optimal antibody-antigen recognition and binding are known in the prior art (e.g., Antibody Compositions and Buffer Systems Therefor, WO 2014 / 191560 A).

[0278] A "detergent" is a surfactant or mixture of surfactants. Detergents include long-chain alkyl hydrogen sulfates or sodium salts of long-chain benzene sulfonic acids. Detergents have an effect on antigen-antibody interactions, reducing background signals and promoting specific antibody binding. Certain suitable detergents, and typical concentrations of such detergents, are described herein, but one skilled in the art will recognize that other detergents and concentrations can be used. One skilled in the art will select detergents and concentrations available in the prior art that are suitable for the disclosed inventive methods and systems (MJ CRUMPTON and RME PARKHOUSE, FEBS LETTERS, Vol. 22, No. 2, May 1972, pp. 210-212).

[0279] Detergents used in the solvent for antigen-antibody binding include, for example, sodium dodecyl sulfate (SDS, ranging between 0.01% (weight / volume) and 4% (weight / volume)), Triton X-100 (ranging between 0.01% (weight / volume) and 4% (weight / volume)), Tween 20 (ranging between 0.1% (weight / volume) and 5% (weight / volume)), Tween 80 (ranging between 0.1% (weight / volume) and 45% (weight / volume)), Nonidet P40 (ranging between 0.1% (weight / volume) and 5% (weight / volume)), Brji 58 (range between 0.1% (wt / vol) and 5% (wt / vol)), deoxycholic acid sodium salt (DOC, range between 0.1% (wt / vol) and 4% (wt / vol)), cetyltrimethylammonium bromide (CTAB, range between 0.1% (wt / vol) and 4% (wt / vol)), cholic acid (range between 0.1% (wt / vol) and 4% (wt / vol)), dioctyl sulfosuccinate (range between 0.1% (wt / vol) and 3% (wt / vol)), or N-lauroyl sarcosine (range between 0.01% (wt / vol) and 2% (wt / vol)). The solvent may contain detergent at a final concentration (weight / volume) of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, and all values ​​therebetween. In one embodiment, 1% or less SDS is used. In one embodiment, 0.5% or less SDS is used. In one embodiment, 0.2% or less SDS is used. In one embodiment, 0.01% or less SDS is used. In one embodiment, 1% or less N-lauroyl sarcosine is used. In one embodiment, 0.5% or less N-lauroyl sarcosine is used. In one embodiment, 0.1% or less N-lauroyl sarcosine is used. In one embodiment, 0.01% or less N-lauroyl sarcosine is used. In one embodiment, 1% or less Tween 20 is used. In one embodiment, 0.5% or less Tween 20 is used. In one embodiment, 0.1% or less Tween 20 is used.In one embodiment, 0.01% or less Tween 20 is used.

[0280] "Defoamer" or "antifoaming agent" refers to a chemical additive that reduces and prevents the formation of foam. Antifoaming agents may be selected from, but are not limited to, mineral oil, vegetable oil, white oil, or any other oil that is insoluble in the foaming medium, wax, ethylene bisstearamide (EBS), paraffin wax, ester wax, fatty alcohol wax, powder defoamers, water-based defoamers, silicone-based defoamers. For example, antifoaming agents are added in certain types of detergents to reduce foaming that may weaken the detergent's action. In one embodiment, a solvent is included.

[0281] "Stabilizer" refers to a chemical compound that is added to a metastable system to prevent transition to a lower energy state. In the solvents described herein, the stabilizer helps to prevent antibody degradation and minimize protein aggregation. For example, the stabilizers N-acetylcysteine, or BSA, or citric acid, or sodium azide, or glycine (also glycerol), or urea, or imidazole are added to the solvent containing the antibody.

[0282] The amount of glycine added may be any concentration as long as the glycine concentration is in the range of 10 mg / mL to 30 mg / mL. Examples of the form of glycine added include glycine, glycine hydrochloride, and other pharma- ceutically acceptable salts of glycine. In one embodiment, the solvent contains glycine at 20 mg / mL to 25 mg / mL, more preferably 22 mg / mL to 23 mg / mL.

[0283] The amount of citric acid added may be any concentration as long as the citric acid concentration is in the range of 0.1 mmol / L to 50 mmol / L. Examples of the form of citric acid added include citric acid, pharma- ceutically acceptable salts of citric acid such as sodium citrate, etc. In one embodiment, the solvent contains citric acid at 0.5 mmol / L to 20 mmol / L, preferably 1 mmol / L to 10 mmol / L.

[0284] The amount of N-acetylcysteine ​​added may be any concentration as long as the N-acetylcysteine ​​is in the range of 0.1 mM to 50 mM. Examples of the form of N-acetylcysteine ​​added include N-acetylcysteine ​​and pharma- ceutically acceptable salts of N-acetylcysteine ​​such as N-acetylcysteine ​​salts. In one embodiment, the solvent contains N-acetylcysteine ​​at 0.5 mM to 20 mM, preferably 1 mM to 10 mM.

[0285] The amount of BSA added can be any concentration, so long as the BSA concentration is in the range of 0.1 mg / ml to 20 mg / ml. In one embodiment, the solvent contains BSA at 0.5 mg / ml to 10 mg / ml, preferably 1 mg / ml to 5 mg / ml.

[0286] The amount of sodium azide added may be any concentration as long as the concentration of sodium azide is in the range of 0.005% (w / v) to 0.2% (w / v). Examples of the form of sodium azide added include sodium azide and pharma- ceutically acceptable salts of sodium azide. In one embodiment, the solvent contains sodium azide at 0.01% (w / v) to 0.1% (w / v), preferably 0.02% (w / v) to 0.05% (w / v).

[0287] The amount of urea added may be any concentration as long as the urea concentration is in the range of 0.1% (w / v) to 20% (w / v). Examples of the form of urea added include urea and pharma- ceutically acceptable salts of urea. In one embodiment, the solvent contains urea at 1% (w / v) to 10% (w / v), preferably 2.5% (w / v) to 7.5% (w / v).

[0288] The solvent has a pH between 4 and 8, for example, pH 4 or more, pH 5 or more, pH 6 or more, pH 7 or more, or pH 8 or more, and all values ​​in between. In one embodiment, the pH of the solvent ranges between pH 6.5 and pH 7.5, preferably between pH 6.8 and pH 7.3, more preferably between pH 6.9 and pH 7.1.

[0289] In one embodiment, the solvent may contain 100 mM HEPES (pH 7.5), 50 mM NaCl, 0.5% (wt / vol) sucrose, 70 mM urea, 3 mM N-acetylcysteine, 1 mM EDTA, and 0.1% (wt / vol) Tween 20.

[0290] In one embodiment, the solvent may contain 200 mM HEPES (pH 7.5), 100 mM NaCl, 1% (w / v) sucrose, 140 mM urea, 6 mM N-acetylcysteine, 2 mM EDTA, and 0.1% (w / v) Tween 20.

[0291] The solvent may comprise Tris, Triton X100, Tween 20, sodium azide, and hydrochloric acid, where the pH of the solvent is between 6 and 8, preferably about pH 7. In one example, the solvent comprises 50 mM Tris (pH 7), 0.5% Triton X100, 1% Tween 20, 1% sodium azide, 0.01% hydrochloric acid, where the pH of the solvent is between 6 and 8, preferably about pH 7.

[0292] Filters As used herein, "filter" refers to any material that provides a porous or fibrous matrix to which antigens can reversibly bind or reversibly attach, filtering based on physical absorption within the matrix channels, e.g., size, solubility, or charge. The matrix may have a thickness of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or greater. In one embodiment, the filter is a size filter. The filter is attached directly to the first container. In one embodiment, the filter has hydrophilic properties. In one embodiment, the filter has hydrophilic properties.

[0293] In one embodiment, the filter is a charged filter. Charged filters include a porous matrix that filters molecules and / or solutions based on electrokinetic adsorption, for example, based on the charge on the matrix. A variety of positively charged ions, preferably metal ions, are suitable for use in such filters. In some embodiments, the charged filter includes at least one layer of diatomaceous earth and / or positively charged ions, preferably metal ions.

[0294] In one embodiment, the filter is a size filter. Size filters (also retention filters) comprise a material with a porous matrix, where the matrix channels retain any antigens with a size larger than the maximum diameter of the matrix channels and are suitable for retaining antigens on the material, preferably antigens from an aerosol of a human subject. Any antigens with a size smaller than the maximum diameter of the matrix channels can pass through the matrix channels. In one embodiment, the maximum diameter of the matrix channels is suitable for retaining antigens used to detect specific pathogens. Another type of filter is a membrane filter that retains particles such as antigens on its surface. "Retention" refers to the ability of the filter to retain particles of a given size.

[0295] In the context of the present invention, the matrix of the selected filter is capable of binding and retaining antigens during the process of collecting antigens from an aerosol of a human subject and releasing said antigens during a subsequent distribution process, maintaining them in a form in which antibodies can recognize said antigens.

[0296] Such filters can be permeable by the solvent. The matrix should also be of a material suitable for reversibly trapping the captured antigen. In one embodiment, the method includes contacting one or more filters with an aqueous solvent containing antigen-binding protein molecules (antibodies) bound to nanoparticles. In one embodiment, contacting the filter with the solvent results in the release of the antigen captured by the filter into the solvent.

[0297] In one embodiment, to release the antigen into the solvent, the filter is completely covered by the solvent. Releasing the antigen into the solvent includes squeezing, mixing, rinsing, and / or moving the filter up and down in the solvent. Releasing the antigen into the solvent also refers to distributing or mixing. In one embodiment, the filter is rinsed in the solvent. In one embodiment, the filter is compressed in the solvent. In one embodiment, rinsing the filter in the solvent releases the antigen and distributes it into the solvent. In one embodiment, squeezing the filter in the solvent releases the antigen and distributes it into the solvent. In one embodiment, moving the filter up and down in the solvent releases the antigen and distributes it into the solvent.

[0298] While certain suitable materials, and general properties of such materials, are described herein, one of ordinary skill in the art will recognize that other materials may be used and will be able to select filters available in the prior art that are suitable for the methods and systems of the disclosed invention.

[0299] In one embodiment the filter material is an FFP2 filter material. In one embodiment the filter material is an FFP3 filter material.

[0300] Materials used for filters may be selected from cellulose, quartz, polymers, and glass. In one embodiment, the filter material is cellulose, preferably unmodified. Filters are often referred to in terms of retention of target particle sizes, such as very fine particles from a few micrometers (e.g., 10 μm or less, 5 μm or less, or 2 μm or less) to the sub-micrometer size range (e.g., sizes up to 0.1 μm, even 0.2 μm). Pore sizes may range from 0.01 μm to 100 μm, or from 0.02 μm to 50 μm, or from 0.05 μm to 20 μm, or from 0.075 μm to 1 μm, or from 0.1 μm to 0.75 μm, or from 0.1 μm to 0.3 μm. Fiber materials for the filters may be selected from, but are not limited to, cellulose, quartz, polymers, and glass, asbestos, potassium titanate, aluminum silicate, mineral wool, regenerated cellulose, polyvinyl chloride, polyvinylidene chloride, polyacrylonitrile, polyethylene, polypropylene, rubber, polymers of terephthalic acid and ethylene glycol, polyamide, casein fibers, zein fibers, cellulose acetate, viscose rayon, hemp, jute, linen, cotton, silk, wool, mohair, paper, and metallic fibers. Particulate materials such as diatomaceous earth, fuller's earth, silicon, magnesia, silica, talc, silica gel, alumina, quartz, carbon, activated charcoal, clay, synthetic resins, and cellulose derivatives, e.g., polyethylene, polystyrene, polypropylene, urea-formaldehyde, phenol-formaldehyde, polytetrafluoroethylene, polytrifluorochloroethylene, polymers of terephthalic acid and ethylene glycol, polyacrylonitrile, ethyl cellulose, polyamide, and cellulose acetate propionate, as well as metal particles such as aluminum, iron, copper, nickel, chromium, and titanium, and metal alloys of all kinds, such as Monel, brass, stainless steel, bronze, Inconel, cupro-nickel, Hastelloy, beryllium, and copper, can also be used. Combinations of fibers and particles, such as diatomaceous earth and glass fibers, are also useful.

[0301] In one embodiment, the filter comprises a filter having a pore size of at least 0.1 μm and a filter thickness of less than 0.5 mm.

[0302] In one embodiment, the filter comprises a filter having a pore size of at least 0.1 μm and a filter thickness of less than 1 mm.

[0303] In one embodiment, the filter comprises a filter having a pore size of at least 0.5 μm and a filter thickness of less than 0.5 mm.

[0304] In one embodiment, the filter comprises a filter having a pore size of at least 0.5 μm and a filter thickness of less than 1 mm.

[0305] drawing The invention will now be illustrated by way of example only through the drawings disclosed herein. The drawings provided depict certain non-limiting embodiments and are not intended to limit the scope of the invention. [Brief description of the drawings]

[0306] [Figure 1] Illustrated diagram of a passive blow inspection system: external view and cross-sectional view. [Diagram 2] Illustrative illustration of a passive blow inspection system: cross section. [Diagram 3] Illustrative illustration of a passive blow inspection system: cross-section of the optical unit. [Figure 4] Illustrated passive blow inspection system: external view and cross-sectional view. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0307] Detailed Description of the Drawings Figure 1: Schematic of a passive blow inspection system: external view and cross section. External view (left) and cross section (right) of an evaluation unit 7 and a collection tube (first container) 1. The collection tube, i.e. first container 1, features a single filter 6. The evaluation unit incorporates a light source 2, a lens arrangement 3, a light sensor 4, a disposable fluid container 5, and a user interface 8.

[0308] Figure 2: Illustrative illustration of a passive blow test system: cross-section. Collection principle: Left: When an individual breathes through a disposable collection tube 1 at the site with a filter 6, aerosol particles in the breath are deposited on the filter 6. At the second site 9 of the collection tube, there may be a feature to control the amount of exhaled air, e.g. a whistle, a respirometer, or a balloon. Right: The collection tube is docked in a fluid container. The sealing between the body surface of the collection tube and the fluid container, i.e. the second container 5, forces the fluid through the filter. The reciprocating stroke action between the collection tube, i.e. the first container 1, and the fluid container, i.e. the second container 5, flushes the filter and dissolves the collected aerosols from the filter into the solvent.

[0309] Figure 3: Schematic of a passive blow inspection system: cross-section of the optical unit. Light from a light source 2 is magnified by optical lens 3a and illuminates the fluid in a transparent container 5. Scattered and unabsorbed light is focused onto a light sensor 4 by optical lens 3b.

[0310] Figure 4: Illustrative view and cross section of the passive blow test system. View (left) and cross section (right) of the evaluation unit 12 and the mouthpiece 1, the collection tube (first container) 3. The collection tube, i.e. the first container 3, features a filter 14 in one place. The first container 3 is pressed through a lid 2 with a sealing 4 into a transparent fluid container 15 containing the measurement liquid 5. The evaluation unit incorporates a microcontroller board 6 with a light source 8, a light sensor 7 with a lens arrangement, a disposable fluid container 15, a user interface 10 with an illuminated signal 11, and a battery 13. A light reflecting unit 9 reflects the light from the light source through the measurement liquid 5 to the light sensor 7. EXAMPLES

[0311] The present invention will be demonstrated through the examples disclosed herein. The examples provided are representative of specific embodiments and are not intended to limit the scope of the present invention. These examples should be considered as providing non-limiting explanations and technical assistance for the implementation of the present invention.

[0312] The following examples illustrate the individual components, fluids, and structures of a passive blow inspection system, as well as the pathogens that can also be inspected and detected by the inspection system.

[0313] Example 1: System configuration and structure - Passive blow inspection When an individual breathes through a disposable collection tube at the site with the filter, aerosol particles in the breath are deposited on the filter. At a second site of the collection tube, there may be a feature to control the amount of exhaled air, such as a whistle, a respirometer, or a balloon. After this, the collection tube is docked in the fluid container. The sealing between the body surface of the collection tube and the fluid container forces the fluid through the filter. The back and forth stroke action between the collection tube and the fluid container flushes the filter and dissolves the collected aerosol from the filter into the solvent. If a specific antigen is present in the aerosol, the measurement fluid changes its optical properties, which can be detected by the optical unit.

[0314] Example 2: Multiplexed Assays Containing Various Functionalized Nanoparticles The procedure is as described in Example 1. The measurement fluid is multiplexed and contains a mixture of gold and silver nanoparticles of different sizes between 10 and 100 nm, each equipped with different antibodies that bind to the following pathogens: SARS_Cov-19 Omicron variant (BA.2, BA.4, BA.5, BA 2.75, BQ1, Influenza A, and Influenza B). Antibodies that do not bind to pathogens not to be detected are selected. Agglutination, and therefore optical properties, occurs only for nanoparticles in the presence of a particular pathogen. A characteristic absorption spectrum of the measurement fluid is recorded by switching on up to 18 LEDs in succession with different peak wavelengths between 400 and 800 nm. The evaluation unit determines which of the different nanoparticles are agglutinated by comparing the variations in the properties of each nanoparticle and its agglutination. Several pathogens can be detected simultaneously.

[0315] Example 3: Measurement solution containing GNPs functionalized with aptamers The test solution contains GNPs functionalized with DNA aptamers targeting the COVID-19 nucleocapsid protein. The Selex procedure was used to select aptamers that can bind to different sites on the protein and form ternary complexes with the nucleocapsid protein, e.g., aptamer pair (Np-A48 / Np-A58), forming aggregates.

[0316] Example 4: Fluids are made as multiplexes and the device is able to distinguish between SARS-Cov19, influenza A, and influenza B Oligonucleotide aptamers are used to simultaneously and specifically detect SARS-CoV-2, influenza H1N1, and influenza H5N1. The RBD protein for SARS-CoV-2, the N protein for SARS-CoV, the whole virus for influenza virus H1N1, and the HA1 protein for influenza virus H5N1 are each targeted by a specific aptamer in a single test or in multiple tests.

[0317] The test systems disclosed herein can be used to detect antigens of SARS-CoV-2, where detection can be performed regardless of SARS-CoV-2 lineage, such as SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 lambda, or SARS-CoV-2 omicron.

[0318] The test systems disclosed herein can be used to detect antigens of SARS-CoV-2, where detection can be specific to one or more of the SARS-CoV-2 lineages, such as SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 lambda, or SARS-CoV-2 omicron.

[0319] The test systems disclosed herein can be used to detect antigens of SARS-CoV-2, where detection can be specific to one or more SARS-CoV-2 sublineages of the SARS-CoV-2 lineage, such as SARS-CoV-2 alpha, SARS-CoV-2 beta, SARS-CoV-2 gamma, SARS-CoV-2 delta, SARS-CoV-2 lambda, or SARS-CoV-2 omicron.

[0320] Example 5: Pathogenic Bacteria In another example, pathogenic bacteria that affect the respiratory tract, upper respiratory tract, and lower respiratory tract, more specifically pathogenic bacteria to humans, can be tested for and detected by the test system disclosed herein. The pathogen antigens that are the subject of the test to identify the pathogen are preferably present in exhaled breath, aerosols, droplets, and / or saliva and are accessible to the test system. An exemplary list of specific bacterial families, genera, or species that can be screened and detected using the test system is shown in Table 2.

[0321] Typically, these bacteria are identified and diagnosed by laboratory criteria including, for example, culture on blood agar plates, Gram staining, electron microscopy, enzyme-linked immunosorbent assay (ELISA), immunofluorescence and polymerase chain reaction, serology, cell culture, pathogen-specific antibodies, blood counts, biopsy specimens, sputum tests, blood tests, and / or urine tests. Obtaining results from the above tests in the prior art typically takes at least 10 minutes and often several hours or days.

[0322] [Table 2] Table 2: Pathogenic bacteria Infectious agent common name Acinetobacter baumannii Acinetobacter infection Arcanobacterium haemolyticum Arcanobacterium haemolyticum infection Bacillus anthracis anthrax Bacillus cereus Bacillus cereus infection Bordetella pertussis Whooping cough (whooping cough) Burkholderia cepacia and other Burkholderia species Burkholderia infection Burkholderia pseudomallei Melioidosis (Whitmore's Disease) Chlamydophila pneumoniae Chlamydophila pneumoniae infection (Taiwan Acute Respiratory Agent, or TWAR) Clostridium botulinum Botulism (and infant botulism) Corynebacterium diphtheriae diphtheria Coxiella burnetii Q fever Cryptococcus neoformans Cryptococcosis Ehrlichia chaffeensis Human monocytic ehrlichiosis Several species of Fusobacterium Fusobacterium infection Haemophilus influenzae Haemophilus influenzae infection Helicobacter pylori Helicobacter pylori infection Kingera Kingae Kingella kingae infection Legionella pneumophila Legionnaires' disease (Legionnaires' disease) Mycobacterium tuberculosis tuberculosis Mycobacterium ulcerans Buruli ulcer Mycoplasma pneumoniae Mycoplasma pneumonia Neisseria meningitidis meningococcal disease Nocardia asteroides and other Nocardia species Nocardiosis Multiple species of Pasteurella Pasteurellosis Several species of Prevotella Prevotella infection Rickettsia prowazekii Epidemic typhus Rickettsia typhi Murine typhus (typhus fever) Streptococcus agalactiae Group B streptococcal infection Streptococcus pneumoniae pneumococcal infection Multiple species of Streptococcus scarlet fever Streptococcus mutans Dental caries Streptococcus pyogenes Group A streptococcal infection Vibrio cholerae cholera Yersinia pseudotuberculosis Yersinia pseudotuberculosis infection

[0323] Example 6: Pathogenic viruses In further examples, pathogenic viruses that also affect the respiratory tract, upper respiratory tract, and lower respiratory tract, more specifically pathogenic viruses to humans, can be tested for and detected using the test system disclosed herein. The pathogen antigens that are the subject of the test to identify the pathogen are preferably present in exhaled breath, aerosols, droplets, and / or saliva and are accessible to the test system. An exemplary list of specific virus families, genera, or species (infectious agents) that can be screened and detected using the test system is shown in Table 3.

[0324] Typically, these viruses are identified and diagnosed by laboratory criteria including, for example, electron microscopy, enzyme-linked immunosorbent assay (ELISA), immunofluorescence and polymerase chain reaction, serology, cell culture, pathogen-specific antibodies, blood counts, biopsy specimens, blood tests, and / or urine tests. Obtaining results from the above tests in the prior art usually takes at least 10 minutes and often several hours or days.

[0325] [Table 3-1] [Table 3-2] Table 3: Pathogenic viruses Infectious agent common name Adenoviridae Adenovirus infections Alphaviruses Chikungunya Multiple species of Astroviridae Astrovirus infection BK virus BK virus infection Multiple species of Bunyaviridae Hemorrhagic fever with renal syndrome (HFRS) Multiple species of Caliciviridae Calicivirus infections (Norovirus and Sapovirus) Colorado Tick Fever Virus (CTFV) Colorado Tick Fever (CTF) Coxsackie B virus Coxsackie B virus infection Crimean-Congo hemorrhagic fever virus Crimean-Congo hemorrhagic fever (CCHF) Cytomegalovirus Cytomegalovirus infection Dengue Virus (DEN-1, DEN-2, DEN-3, and DEN-4) - Flavivirus Dengue fever Ebola virus (EBOV) Ebola Multiple enteroviruses Enterovirus Infections Enteroviruses, mainly Coxsackie A virus and enterovirus type 71 (EV71) Hand, foot and mouth disease (HFMD) Epstein-Barr virus (EBV) Epstein-Barr virus infectious mononucleosis (Mono) Ganaritovirus Venezuelan hemorrhagic fever Hendra virus Hendra virus infection Herpes simplex virus type 1 and herpes simplex virus type 2 (HSV-1 and HSV-2) Herpes simplex Herpesvirus type 7 (HHV-7) Human metapneumovirus (hMPV) Human metapneumovirus infection Human bocavirus (HBoV) Human bocavirus infection Herpesvirus type 6 (HHV-6) and Human Roseola infantum (6th disease) Human parainfluenza virus (HPIV) Human parainfluenza virus infections Human T-lymphotropic virus type 1 (HTLV-1) Human T-lymphotropic virus type 1 infection Japanese encephalitis virus Japanese encephalitis Junin virus Argentine hemorrhagic fever Lassa virus Lassa fever Lymphocytic choriomeningitis virus (LCMV) Lymphocytic choriomeningitis Machupo virus Bolivian hemorrhagic fever Marburg virus Marburg hemorrhagic fever (MHF) Measles virus measles Middle East Respiratory Syndrome-related Coronavirus Middle East Respiratory Syndrome (MERS) Mumps virus Mumps Nipah virus Nipah virus infection Norovirus Multiple species of the Orthomyxoviridae family Influenza (flu) Parvovirus B19 Erythema infectiosum (5th disease) Rabies virus rabies Respiratory syncytial virus (RSV) Respiratory syncytial virus infection Rhinovirus Rhinovirus infections Rotavirus Rotavirus infection Rubella virus rubella Sabia virus Brazilian hemorrhagic fever SARS coronavirus Severe acute respiratory syndrome (SARS) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Coronavirus disease 2019 (COVID-19) Sin Nombre virus Hantavirus Pulmonary Syndrome (HPS) Varicella zoster virus (VZV) Shingles (herpes zoster) Variola major or Variola minor virus smallpox (variola) West Nile virus West Nile fever Yellow fever virus yellow fever Zika virus Zika fever

[0326] For example, the VP1 structural polypeptide in the case of Foot and Mouth Disease Virus, the E antigen in the case of Hepatitis B Virus, the core antigen in the case of Hepatitis C Virus, the GP protein in the case of Ebola Virus, the nucleocapsid protein in the case of Severe Fever with Thrombocytopenia Syndrome Virus, the NS1 protein in the case of Zika Virus, and the whole virus in the case of Newcastle Disease Virus are targeted by specific aptamers in a single test or in various tests.

[0327] Example 7: Pathogenic Fungi Additionally, pathogenic fungi that also affect the respiratory tract, upper respiratory tract, and lower respiratory tract, more specifically pathogenic fungi to humans, can be tested for and detected using the test system disclosed herein. The pathogen antigens that are the subject of the test to identify the pathogen are preferably present in exhaled breath, aerosols, droplets, and / or saliva and are accessible to the test system. An exemplary list of specific fungal families, fungal genera, or fungal species that can be screened and detected using the test system is provided in Table 4.

[0328] Typically, these fungi are identified and diagnosed by, for example, KOH preparations, cytology, or histology, fungus-containing samples taken from sputum (by bronchoalveolar lavage), blood, or infected organs, and / or antigen detection. Obtaining results from the above tests in the prior art usually takes at least 10 minutes, and often several hours or days.

[0329] [Table 4] Table 4: Pathogenic fungi Infectious agent common name Blastomyces dermatitidis Blastomycosis Coccidioides immitis or Coccidioides posadacii Valley Fever Geotrichum candidum Geotrichosis Histoplasma capsulatum Histoplasmosis Pneumocystis jirovecii Pneumocystis pneumonia (PCP) Sporothrix schenckii Sporotrichosis

[0330] Example 8: Pathogenic eukaryotes, nematodes In different embodiments, eukaryotes and nematodes that also affect the respiratory tract, upper respiratory tract, and lower respiratory tract, more specifically eukaryotes and nematodes that are pathogenic to humans, can be tested for and detected using the test system disclosed herein. The pathogen antigens that are the subject of the test to identify the pathogen are preferably present in exhaled breath, aerosols, droplets, and / or saliva and are accessible to the test system. An exemplary list of specific eukaryotes, nematode families, eukaryotes, nematode genera, or eukaryotes, nematode species that can be screened and detected using the test system is shown in Table 5.

[0331] Typically, these eukaryotes and nematodes are identified and diagnosed by, for example, microscopic demonstration (bright field, differential interference contrast (DIC), and epifluorescence), acid-fast staining, Giemsa staining, stool tests, biopsy or consistency tests (Enterotest), antigen detection, blood tests, lumbar puncture, brain imaging, and / or serology. Obtaining results from the above tests in the prior art typically takes at least 10 minutes and often several hours or days.

[0332] [Table 5] Table 5: Pathogenic eukaryotes, nematodes Pathogen Infectious agent Common name Eukaryotes; Parasitic alveolata Multiple species of Cryptosporidium Cryptosporidiosis Isospora berry Isosporosis Multiple species of Plasmodium malaria Eukaryotes; Parasitic Euglena algae Trypanosoma cruzi Chagas Disease (American Trypanosomiasis) Eukaryotes; parasitic netamonada or metamonada Giardia lamblia Giardiasis Capillaria aerophila Capillariasis Parasitic nematode Angiostrongylus Angiostrongyliasis References (1) Guglielmi, G. The Explosion of New Coronavirus Tests That Could Help to End the pandemic. Nature 2020, 583 7817, 506 - 509, DOI: 10.1038 / d41586 - 020 - 02140 - 8 (2) Bhatraju, P. K.; Ghassemieh, B. J.; Nichols, M.; Kim, R.; Jerome, K. R.; Nalla, A. K.; Greninger, A. L.; Pipavath, S.; Wurfel, M. M.;Evans, L.; Kritek, P. A.; West, T. E.; Luks, A.; Gerbino, A.; Dale, C. R.; Goldman, J. D.; O’Mahony, S.; Mikacenic, C. COVID - 19 in Critically Ill Patients in the Seattle Region Case Series. N. Engl. J. Med. 2020, 382, 2012 - 2022. (3) Udugama, B.; Kadhiresan, P.; Kozlowski, H. N.; Malekjahani, A.; Osborne, M.; Li, V. Y. C.; Chen, H.; Mubareka, S.; Gubbay, J. B.; Chan, W. C. W. Diagnosing COVID - 19: The Disease and Tools for Detection. ACS Nano 2020, 14, 3822 - 3835. (4) Lassauniere, R.; Frische, A.; Harboe , ZB ; Nielsen , AC ; Fomsgaard, A.; Krogfield, CA; Jorgensen, CS Evaluation of Nine Commercial SARS-CoV-2 Immunoassays. withRxiv 2020, 1, DOI:10.1101 / 2020.04.09.20056325 (5) Borges , JT , Nakada , LYK , Maniero , MG et al. SARS-CoV-2: a systematic review of indoor air sampling for virus detection. Environ Sci Pollut Res 28, 40460–40473 (2021). (6) Bhalla, N.; Pan, Y.; Yang, Z.; Payam, AF Opportunities and Challenges for Biosensors and Nanoscale Analytical Tools for Pandemics: COVID-1 ACS Nano2020, 14, 7783. (7) Shan , B. , Broza , YY , Li , W. , Wang , Y. , Wu , S. , Liu , Z. , ... & Haick , H. (2020). Multiplexed nanomaterial-based sensor array for detection of COVID-19 in exhaled breath. ACS nano, 14(9), 12125-12132. (8) Maniscalco, M., Ambrosino, P., Ciullo, A., Fuschillo, S., Valente, V., Gaudiosi, C., … & Motta, A. (2021). A rapid antigen detection test to diagnose SARS-CoV-2 infection using exhaled breath condensate by a modified inflammacheck(R) device. Sensors, 21(17), 5710. (9) Ventura, B. D., Cennamo, M., Minopoli, A., Campanile, R., Censi, S. B., Terracciano, D., ... & Velotta, R. (2020). Colorimetric test for fast detection of SARS-CoV-2 in nasal and throat swabs. ACS sensors, 5(10), 3043-3048.

Claims

1. 1. A system for antigen detection, comprising a first container (1) adapted to collect aerosol from the exhaled breath of a human subject containing the aerosol, and a second container (5) suitable for a solvent, in which nanoparticles are dissolved and which are linked to antibodies, and in which a change in the optical properties of the solvent is detectable upon contact between the antibody and a compatible antigen, The system comprises a measurement cell in which there is a spectrometer with a light source (2) and a light sensor (4); The first container has a hole on a first side and a filter (6) on a second side, and The second container (5) is configured to incorporate the first container (1), wherein the filter (6) of the first container (1) is in contact with the solvent of the second container (5) in the measuring cell; When the first container (1) is assembled into the second container (5), the sealing between the body surfaces of both containers forces the solvent contained in the second container (5) through the filter (6) of the first container (1), A viable reciprocating stroke between the first container (1) and the second container (5) flushes the filter and dissolves the collected aerosol from the filter into the solvent; the light source (2) irradiates the solvent and the optical sensor (4) is arranged to detect a change in an optical property; and A system for antigen detection, the system comprising an alarm device adapted to emit an alarm signal upon detecting a change in the optical property.

2. The system of claim 1 , wherein the system comprises a calculation unit, wherein the calculation unit is configured to calculate a concentration of the antigen based on a change in an optical property of the solvent.

3. the light source (2) is a monochromatic laser containing linearly polarized light, wherein the linearly polarized light is configured to excite pathogen-nanoparticle aggregates at an extinction peak wavelength; 2. The system of claim 1, wherein the wavelength may be preferably between 600 nm and 700 nm, preferably between 610 nm and 690 nm, more preferably between 620 nm and 680 nm, even more preferably between 630 nm and 670 nm, even more preferably between 640 nm and 660 nm, even more preferably between 645 nm and 655 nm.

4. The system of claim 1 , wherein the alarm signal is selected from the group comprising a visual signal and / or an acoustic signal.

5. the nanoparticles comprise a material selected from the group comprising gold, silver, ruthenium, rhodium, palladium, osmium, iridium, and / or platinum; and / or The system of claim 1 , wherein the nanoparticles can be spheres ranging from 15 nm to 25 nm, or nanorods having a diameter of 15 nm to 25 nm and a length of 30 nm to 60 nm.

6. 2. The system according to claim 1, wherein the filter (6) can be a hydrophilic filter and / or a low affinity filter, preferably a filter suitable for reversible attachment of antigens.

7. 10. The system of claim 1, wherein the exhaled breath of the human subject can be collected by direct contact of the subject's mouth with the first container.

8. The system of claim 1 , wherein the solvent further comprises a salt, a detergent, an antifoaming agent, a stabilizer, a blocking agent, and / or water.

9. 2. The system of claim 1, wherein the antibody is suitable for contact with the antigen, wherein the antigen may be a protein, peptide, carbohydrate, lipid or nucleic acid, preferably a peptide or protein, more preferably a protein, and wherein the antigen is an antigen of a human pathogen, such as a viral, fungal or bacterial antigen causing an infectious disease, such as COVID-19, respiratory syndrome, whooping cough, pneumonia or tuberculosis.

10. 10. The system of claim 9, wherein the human pathogen is selected from the group of bacteria, fungi, or viruses such as coronaviruses, influenza viruses, Mycobacteriaceae, Streptococcusceae, preferably SARS coronavirus, respiratory syndrome virus, Streptococcus mutans, Mycobacterium tuberculosis, Streptococcus pneumoniae, more preferably SARS-CoV-2.

11. 10. The system of claim 9, wherein the viral antigen may be selected from, but is not limited to, SARS-CoV-2 S protein, SARS-CoV-2 N protein, hPMV G protein, hPMV F protein, RSV F protein, RSV G protein, or influenza A / B proteins such as HA, M1, M2, and / or NA.

12. 10. The system of claim 9, wherein the bacterial antigen comprises an antigen including, but not limited to, pertussis toxin, Streptococcus pneumoniae C-polysaccharide, early secretory antigenic target, or culture filtrate protein.

13. A method for the detection of antigens in the exhaled breath of a human subject, including aerosols, using a system according to any one of claims 1 to 12, comprising: The detection of the antigen comprises: (a) collecting antigens from an aerosol of the human subject into the system of any one of claims 1 to 12 by directly contacting the human subject's mouth with a first end of a first container (1) having a filter (6) at its second end; (b) attaching the antigen to the filter (6); wherein said attachment is reversible; (c) distributing the antigen in the solvent according to claims 1 to 12 by completely covering the filter (6) with the solvent; (d) contacting the antigen with the antibody; wherein said contacting causes a change in an optical property of said solvent, preferably a spectral sensor; (e) determining the change in the optical property by the optical sensor (4) according to claim 3 and transferring it to a calculation unit according to claim 2; wherein said change in optical properties; (f) calculating a first parameter, the concentration of the antigen, based on the change in the optical properties of the solvent; and and 11. The method of claim 10, wherein detecting the antigen comprises identifying at least one type of the human pathogen.

14. 14. The method of claim 13, wherein the first parameter is compared with a second parameter by the calculation unit, and the alarm device according to any one of claims 1 to 12 generates a visual and / or acoustic signal if the first parameter is greater than or equal to the second parameter, wherein the second parameter is a preset value.

15. 14. The method of claim 13, wherein detecting the antigen comprises identifying a human pathogen, preferably a virus, a fungus, or a bacterium, more preferably a coronavirus, a Streptococcus family, a Bordetella family, a Mycobacteria family, or an influenza virus, even more preferably SARS-CoV-2.

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