Diagnosis of respiratory diseases using exhaled breath and aerosol analysis

Abstract

To provide a method and device for analyzing non-volatile organic substances in exhaled breath. [Solution] A method and device for analyzing non-volatile organic compounds in exhaled breath and other aerosols is disclosed, using a variety of diagnostic tools that enable rapid, low-cost point-of-care assays for several diseases, including respiratory diseases such as COVID-19. The disclosed method and system selectively captures non-volatile organic compounds in exhaled breath and other aerosols in a packed bed column. The non-volatile organic compounds are eluted, and the sample is analyzed using a diagnostic instrument, including MALDI-TOFMS. The disclosed system and method provide diagnostic test results in less than approximately 20 minutes and enable autonomous operation with minimal human intervention.

Description

[Technical Field]

[0001] This disclosure relates to methods and devices for analyzing non-volatile organic compounds in exhaled breath and other aerosols using a variety of diagnostic tools that enable rapid, low-cost, and autonomous point-of-care assays for respiratory diseases. More specifically, but not limited to, this disclosure relates to methods and devices for analyzing non-volatile organic compounds in exhaled breath using mass spectrometry methods, including MALDI-TOFMS, for the detection of respiratory diseases such as COVID-19 and for the diagnosis of tuberculosis. [Background technology]

[0002] Coronavirus disease (COVID-19) is a disease caused by the newly emerged coronavirus SARS-CoV-2. This new coronavirus is a respiratory virus that spreads primarily through droplets produced when an infected person coughs or sneezes, or through saliva droplets or nasal secretions. The new coronavirus is highly contagious and has caused the ongoing COVID-19 pandemic. This suggests that the virus is spreading more rapidly than influenza. Rapid detection tools are needed to help mitigate the spread.

[0003] Furthermore, tuberculosis (TB) kills more than 4,000 people daily, surpassing HIV / AIDS as the leading cause of death (killer) globally (Patterson, B et al., 2018). The reported rate of incidence decline remains insufficient at 1.5% per year, making it unlikely that treatment alone would significantly reduce the disease burden. In HIV-saturated communities, genotyping studies of Mycobacterium tuberculosis (Mtb) have shown that recent infections, rather than relapses, account for the majority (54%) of tuberculosis cases. The physical processes of tuberculosis transmission remain poorly understood, and the application of new technologies to elucidate key events in the generation, release, and inhalation of infectious aerosols is slow. There are few empirical studies characterizing airborne infectious particles. Two major problems hindering investigations are the low concentrations of naturally generated Mtb particles and the complications of environmental and patient-derived bacterial and fungal contamination of airborne samples. Nevertheless, many attempts at airborne detection have been made. A 2004 proof-of-concept study in Uganda and subsequent feasibility studies sampled aerosols generated by coughing from pulmonary tuberculosis patients. Direct coughing into a sampling chamber equipped with two viable cascade impactors resulted in positive cultures from more than a quarter of participants, despite 1–6 days of chemotherapy. Follow-up studies using the same apparatus found that participants with high aerosol bacterial loads had higher rates of household infections, potentially leading to disease detection that could serve as a useful measure of clinically relevant infectivity. Thus, interruptions in transmission may have a rapid and measurable impact on tuberculosis incidence.

[0004] The best way to control tuberculosis transmission is to rapidly identify and treat active TB cases (Wood, RC et al., 2015). Diagnosis of pulmonary tuberculosis is typically made by microbiological, microscopic, or molecular analysis of the patient's sputum. In most developing countries, the “gold standard” test for tuberculosis infection is smear culture based on sputum samples. The sample is smeared onto a culture plate, a stain specific to Mtb is added, and the stained cells are counted using a microscope. If the cell concentration in the smear is higher than a set threshold, the sample is classified as positive. If the TB count is below this threshold, it is classified as negative. Diagnosis can take several hours. The need for sputum as a diagnostic sample is limited by the challenge of collecting sputum from patients and its complex composition. The viscosity of the material limits the sensitivity of the test, increases heterogeneity between samples, and increases the cost and effort associated with the test. Furthermore, sputum (requiring coughing) poses an occupational risk to healthcare workers. Sputum has several drawbacks as a sample medium. First, only about 50% of patients can provide a good sputum sample. For example, children around 8 years old often cannot provide a sample when requested because they usually have not developed the ability to "cough" sputum from the back of their throat. The elderly and sick may not have the strength to cough up phlegm. Others may simply not have phlegm in their throat. Therefore, diagnostic methods based on sputum analysis may not be able to provide a diagnosis for as many as 50% of patients who need one. Sputum is not useful as a diagnostic sample if it is collected 1-2 days after the patient has been treated with antibiotics. This is because the specimen no longer represents the disease deep in the lungs, and the number of living Mtb in the sputum decreases significantly within a few days of starting treatment. Urine and blood have been proposed as sample mediums for the diagnosis of tuberculosis infection. Blood is a highly invasive process, and the processing costs of blood samples, which are often HIV-positive, are high. This is because, in some parts of the world, many tuberculosis patients also have co-infection with HIV. Furthermore, patients infected with active tuberculosis may not have a large number of tuberculosis cells circulating in their blood.Although urine-based diagnostic methods have also been proposed, these tests are looking for biomarkers for diseases other than live Mycobacterium tuberculosis, and their widespread clinical use has not been validated.

[0005] Easier, safer, and more homogeneous samples to collect and handle would simplify TB diagnosis. Exhaled breath contains aerosols ("EBA") and vapors, which can be non-invasively collected and characterized to elucidate physiological and pathological processes in the lungs (Hunt, 2002). To capture exhaled breath for assay, it is passed through a condenser to produce a fluid accumulation called exhaled breath condensate ("EBC"). Although EBC is primarily derived from water vapor, it is dissolved in cytokines, lipids, surfactants, ions, oxidation products, and non-volatile compounds such as adenosine, histamine, acetylcholine, and serotonin. Furthermore, EBC traps potentially volatile water-soluble compounds such as ammonia, hydrogen peroxide, ethanol, and other volatile organic compounds. The pH of EBC can be easily measured. EBC contains aerosolized airway endothelial fluid and volatile compounds, non-invasively revealing biochemical and inflammatory activity ongoing in the lungs. The rapid increase in interest in EBCs stems from the recognition that, in lung diseases, EBCs possess measurable features that can be used to distinguish between infected and healthy individuals. These assays provide evidence of airway and lung redox deviations, acid-base status, and the degree and type of inflammation in acute and chronic asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome, occupational diseases, and cystic fibrosis. Characterized by uncertain and variable dilutions, EBCs may not be able to accurately assess the concentration of individual solutes in the intrinsic airway endothelial fluid. However, they can provide useful information when concentrations vary significantly between healthy and diseased states, or when based on the ratio of solutes present in the sample.

[0006] Patterson et al. (2018) used a custom-made respiratory aerosol sampling chamber (RASC), a novel device designed to optimize patient-derived exhaled aerosol sampling and isolate and accumulate breathable aerosols from a single patient. Environmental sampling detects Mtb present in the air within the chamber after an aging period. Thirty-five newly diagnosed GeneXpert (Cepheid, Inc., Sunnyvale, CA) sputum-positive tuberculosis patients were approximately 1.4 m 3 The samples were monitored during 1 hour of confinement in a RASC chamber with a volume of [volume not specified]. The GeneXpert gene assay is based on polymerase chain reaction (PCR) and may be used to analyze samples for tuberculosis diagnosis and to indicate whether tuberculosis samples have drug resistance genes. The GeneXpert PCR assay for TB accepts sputum samples and can yield positive or negative results in about 1 hour. The chamber incorporated aerodynamic particle size detection, viable and unviable sampling devices, real-time CO2 monitoring, and cough recording. Microbial cultures and droplet digital polymerase chain reaction (ddPCR) were used to detect Mtb in each bioaerosol collection device. Mtb was detected in 77% of aerosol samples, 42% of samples were positive by mycobacterial culture, and 92% were positive by ddPCR. A correlation was found between cough incidence and culturable bioaerosols. Mtb was detected in all viable cascade impactor stages and peaked at aerosol sizes of 2.0–3.5 μm. This is based on a median of 0.09 CFU per liter of exhaled breath positive for aerosol culture and an estimated median concentration of 4.5 x 10⁻¹⁰ exhaled particulate bioaerosols. 7 This suggests CFU / ml. Mtb was detected in bioaerosols exhaled by the majority of untreated TB patients using a RASC chamber. Molecular detection was found to be more sensitive than Mtb culture in solid medium.

[0007] Mtb can be identified in EBA by culture, ddPCR, electron microscopy, immunoassay, and cell staining (e.g., oramine and dmn-Tre). Of these, PCR and immunoassay have the potential to be rapid and species-specific. PCR and other genomics-based techniques are specific at the strain level. Mass spectrometry has also been shown to be specific to the strain level of cultures obtained from bacterial infections. For example, Bruker Daltonics' (Germany) Biotyper has been shown to be able to identify up to 15,000 strains of bacteria that cause infection in humans. These techniques have been shown to be able to identify tuberculosis infection from EBA. Immunoassays for Mtb detection, such as those based on lipoarabinomannan, are also well known.

[0008] In the case of TB, people infected with TB are often diagnosed through passive case discovery when individuals visit a clinic. Active case discovery ("ACF") is generally considered to include other methods of contacting people suspected of having tuberculosis outside of primary healthcare systems. According to the WHO, ACF is "the systematic identification of people suspected of having active tuberculosis using rapidly applicable tests, screenings or other procedures." The goal of ACF is to treat infected individuals early to shorten the average duration of infection and thereby reduce the spread of the disease. In the case of tuberculosis, by the time a person seeks help at a clinic, they may have infected between approximately 10 and 115 people. ACF helps reduce or prevent serious tuberculosis infections. Diagnostic systems and methods such as sputum and blood analyses are not automated, not autonomously operated, or are not rapid. Many involve expensive assays consumed per analysis, so active case discovery is not generally useful, especially in developing countries and agro-regions. As mentioned above, EBA analysis appears to be a compelling diagnostic tool for tuberculosis detection, offering rapid analysis, portability, and low cost, as it eliminates the need for expensive assays and consumables. McDevitt et al. (2013) reported an EBA analyzer and method for influenza diagnosis. Large particles (>4 μm) are removed from exhaled breath using an impactor, followed by small particles (<4 μm) being removed with a wet film collector. The two sizes of collected particles were analyzed for influenza virus using a genomics-based method: reverse transcriptase polymerase chain reaction (rt-PCR). PCR technique uses biomolecular probes, combined with other biomolecules including enzymes, to amplify specific sequences of DNA if those sequences are present in the sample. Target sequences are thought to be specific to the identified disease. McDevitt et al. demonstrated that influenza can be diagnosed using EBA samples. The disclosed apparatus and method have several drawbacks from a practical standpoint. First, since exhaled aerosol samples are collected into individual samples with a volume of only a few milliliters, concentrating the samples requires considerable effort.Furthermore, the diagnostic device is not coupled to or integrated with a sample collector and is therefore unsuitable for use as an ACF tool. The ability to automate RNA assays to create autonomous diagnostic tools for tuberculosis analysis is unclear. There is no description of how to determine whether a sufficient amount of cough or exhaled aerosol was generated by a particular patient. Consequently, if a sample is found to be negative for influenza, this may be due to a false negative resulting from improper sample collection. It is well known that there is considerable variability among individuals regarding the amount of aerosolized lung fluid generated during various respiratory operations.

[0009] GeneXpert Ultra is a state-of-the-art genomics-based point-of-care diagnostic device that uses PCR technology. While it can be integrated with EBA sample collection methods to perform ACF for tuberculosis and other respiratory diseases, the sample collection time is too long to be practical. Patterson et al. have shown that 20–200 tuberculosis bacteria are typically generated by EBA and can be collected in a one-hour sampling period. A minimum of one hour of sampling is required to use GeneXpert Ultra as a diagnostic assay. GeneXpert can be integrated with systems that sample air to analyze air samples for airborne pathogens. The BDS system (Northup Grumman, Edgewood, MD) is used to screen U.S. Postal Service mail for anthrax-causing bacterial spores as it passes through distribution centers. A wet-wall cyclone combined with the GeneXpert PCR system autonomously samples air and reports the presence of pathogens. However, the GeneXpert Ultra assay is relatively expensive per test and takes approximately one hour to complete and produce results. Generally, PCR-based diagnostics are not suitable for tuberculosis screening in ACF applications because they require a long time for sampling and analysis, and the cost per test is relatively high.

[0010] The time associated with a diagnostic assay is a critical parameter for field tests or “point-of-care” tests. By definition, ACF is performed outside the healthcare system, and therefore ACF is an example of a field diagnostic assay. In the United States, point-of-care tests must provide a response within 20 minutes. Otherwise, the test is considered too slow and unacceptable for achieving short patient waiting times. In developing countries, particularly those with a history of tuberculosis outbreaks, a diagnosis can be made in about an hour using GeneXpert. As mentioned earlier, this assay is not yet widely deployed because it is expensive to implement on a “cost per test” basis. Due to its cost, it is not used to screen patients who appear healthy (asymptomatic) but may have tuberculosis, but rather to confirm a diagnosis that is strongly suspected based on other tests and factors.

[0011] Fennelly et al. (2004) described a TB analysis using a collection chamber containing cough aerosols and two Anderson Cascade Impactors in individuals known to be active patients. Individuals were asked to repeat a severe cough for 5 minutes each, twice. This approach is not suitable for automation as it took 30–60 days to culture the affected samples. A challenging aspect of EBA as a clinical sample is the relatively small sample volume of exhaled particulate matter that can be collected from breath. Furthermore, a significant portion of the collected mass is water. Molecules containing diagnostic information ("biomarkers") are only present in nanoliters or picograms. In addition, the aerosol collection method must be effective in capturing the majority of the biomass in exhaled breath. Exhaled breath includes air expelled from the lungs by various operations such as tidal breathing, deep breathing, coughing, and sneezing. Certain types of deep breathing operations, such as forced vital capacity (FVC), can be used to maximize vital capacity by inhaling as far as possible and exhaling as far (or deeply) as possible in order to measure the maximum amount of vital capacity. Forced expiratory volume (FEV1) measures the amount of air a person can force out. The amount of air exhaled can be measured during the first second (FEV1), second second (FEV2), and / or third second (FEV3) of forced breathing. Forced vital capacity (FVC) is the total amount of air exhaled during the FEV test. Forced expiratory volume and forced vital capacity are pulmonary function tests measured by spirometry. Forced expiratory volume is an important measure of lung function.

[0012] Although studies have shown that respiratory diseases can be detected from exhaled aerosols and exhaled condensates, modern clinical testing for infectious diseases or illnesses such as tuberculosis, influenza, and pneumonia continues to utilize sputum, blood, or nasal swabs. Exhaled breath analysis tools have not been commercialized due to a lack of methods and devices that efficiently collect and concentrate the trace amounts of samples present in exhaled breath. Furthermore, there are no criteria or methodologies for evaluating sufficient exhaled volume for specific diagnoses. The disclosed exemplary devices and methods overcome these limitations by collecting exhaled aerosols and exhaled condensates into relatively concentrated samples at high flow rates and with high efficiency. Furthermore, aerosol size sorting can be incorporated to increase the signal-to-noise ratio of specific samples before sample collection. The concentrated samples can then be analyzed in several ways, but preferably, a method that is highly sensitive, rapid, and highly specific to the analyte of interest is used. More preferably, the analysis is rapid and near real-time. Mass spectrometry, real-time PCR, and immunoassays are most likely to be highly sensitive, specific, and near real-time.

[0013] There is a need for sample collection methods that can realize diagnostic assays that are faster, more reliable than sputum analysis, less invasive than blood analysis, can be combined with rapid diagnostic tools such as mass spectrometry ("MS"), and are characterized by being fast, sensitive, specific, and preferably having a low cost per test. Such systems can be used for proactive case detection (ACF) of tuberculosis and other lung and airway diseases. To be effective, ACF systems must be fast and inexpensive "per diagnosis". Low cost per test is a requirement for screening large numbers of individuals to prevent the transmission of tuberculosis and to find the small number of people who are actually infected with tuberculosis. Low-cost devices and methods are also needed for point-of-care diagnosis of influenza and other pathogenic viruses, as a patient who may have a "cold" may be infected with rhinovirus. In some cases, respiratory infections are caused by bacterial or fungal microorganisms and may be treatable with antibiotics. Otherwise, the microorganisms may be resistant to antibiotics, and diagnostic methods that can identify the resistance of microorganisms to antibiotics are desirable. A rapid EBA method is desired to differentiate between viral and bacterial infections in the airways while minimizing false negatives due to insufficient sample volume. Mass spectrometry, genomics including PCR, and immunoassays may offer high sensitivity and specificity. Mass spectrometry, particularly MALDI time-of-flight mass spectrometry (MALDI-TOFMS), has been demonstrated to be highly sensitive, specific, and near real-time, making it a suitable diagnostic tool for the analysis of EBA and EBC samples. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] Special Publication No. 2013-504074 [Patent Document 2] Japanese Patent Publication No. 2019-184288 [Patent Document 3] Japanese Patent Publication No. 2011-102747 [Patent Document 4] Japanese Patent Application Publication No. 10-227725 [Patent Document 5] Japanese Patent Application Publication No. 09-089863 [Overview of the project]

[0015] A breath sample collection system for diagnosing at least one respiratory disease using exhaled breath is disclosed, the breath sample collection system comprising: a breath collection element configured to accommodate an individual's face to collect exhaled breath having water, volatile organic components (VOCs), and non-volatile organic components; a sample capture element having a packed bed column for selectively capturing non-volatile organic components in the exhaled breath and being detachably and fluidly connected to the breath collection element; and a pump fluidly communicating with the sample capture element and configured to draw exhaled breath into the sample capture element. The non-volatile components in the exhaled breath may consist of exhaled aerosol particles having at least one of microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteome biomarkers characteristic of respiratory diseases. The disclosed system may further include a flow splitter positioned between the breath collection element and the sample capture element to split the exhaled breath flow such that a first portion of the exhaled breath flow is directed towards the sample capture element and a second portion of the exhaled breath flow is directed towards a HEPA filter. The system may further include a one-way valve located downstream of the sample capture element and positioned to be open under the flow exiting the sample capture element toward the pump, and otherwise positioned to be closed. The system may further include a large particle trap (first trap) for capturing larger particles of exhaled condensate to prevent them from reaching the packed sample capture element. The size of the large particles may be at least about 10 microns. The packed bed column may have solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. The packed bed column may have resin beads having octadecyl (C18) acrylate functional groups on their surface. The resin beads may have a nominal diameter between about 12 μm and about 20 μm. The resin beads may be packed between two porous polymer frit disks. The polymer frit disk located at the inlet end of the packed column may be characterized by an average pore size of at least 35 μm.The polymer frit disc positioned at the outlet end of the packed column may be characterized by an average pore size of about 10 μm. The weight of the backed bed may be about 25 mg. The pump may be a diaphragm pump. The nominal flow rate of the pump may be between about 200 ml / min and about 600 ml / min. The exhalation extraction element may have at least one of a CPR rescue mask, a CPAP mask, a ventilator mask, and a medical universal mouthpiece. The system may further have a second trap positioned between the sample capture element and the pump and configured to capture exhalation condensate (EBC) containing at least one of water vapor, volatile organic compounds, and non-volatile organic compounds passing through the packed bed. The second trap may be cooled below ambient temperature. The above solid particles may contain a functional group immobilized on the surface of the particles, and the functional group may have at least one of C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA. The above ion exchange phase may have at least one of diethylaminoethylcellulose, QAE Sephadex, Q Sepharose, and carboxymethylcellulose. The above polymer phase may have at least one of polystyrene-co-1,4-divinylbenzene, methacrylate, polyvinyl alcohol, starch, and agarose. The above antibody may have at least one of anti-human albumin, anti-influenza A virus NP, and anti-SARS-CoV-2 virus. The above antibody may be immobilized on protein A / G agarose beads. The capture element may be cooled to ambient temperature or below. The exhaled breath sample collection system may further include a humidifier located upstream of the inlet to the capture element in order to humidify the exhaled breath and increase the humidity in the packed bed column.

[0016] A breath sample collection system for diagnosing at least one respiratory disease using exhaled breath is disclosed, the breath sample collection system comprising: a breath collection element configured to accommodate an individual's face for collecting exhaled breath having water, volatile organic components (VOCs), and non-volatile organic components; at least one sample capture element, if the system comprises two or more capture elements, arranged parallel to each other, each element comprising a packed bed column for selectively capturing non-volatile organic components, and being removably and fluidly connected to the breath collection element; and at least one pump, fluidly communicating with the at least one sample capture element and configured to draw exhaled breath into the at least one sample capture element. The nominal flow rate of the at least one pump may be about 2.5 liters / minute. If, on one side, multiple sample capture elements are arranged parallel to each other, each of these capture elements is fluidly connected to its own pump.

[0017] A sample capture element for the diagnosis of respiratory diseases using exhaled breath is disclosed, the sample capture element having a packed bed column for selectively capturing non-volatile organic components in exhaled breath, the packed bed column having solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles, and functional groups immobilized on the surface of the particles, the functional group having at least one of C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethyl-aminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA. The solid particles may have a nominal diameter between about 12 μm and about 20 μm. The solid particles may be packed between two porous polymer frit disks. The polymer frit disc positioned at the inlet end of the packed column may be characterized by an average pore size of at least 35 μm. The polymer frit disc positioned at the outlet end of the packed column may be characterized by an average pore size of about 10 μm. The ion exchange phase may have at least one of diethylaminoethylcellulose, QAE Sephadex, Q Sepharose, and carboxymethylcellulose. The polymer phase may have at least one of polystyrene-co-1,4-divinylbenzene, methacrylate, polyvinyl alcohol, starch, and agarose. The antibody may have at least one of anti-human albumin and anti-influenza A virus NP. The antibody may be immobilized on protein A / G agarose beads.

[0018] A respiratory disease diagnostic system for diagnosing respiratory diseases using exhaled breath is disclosed, the respiratory disease diagnostic system comprising: an exhaled breath sample collection system; a sample extraction system; and a sample analysis system, wherein the exhaled breath sample collection system comprises: an exhaled breath collection element configured to accommodate an individual's face to collect exhaled breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds; a sample capture element having a packed bed column for selectively capturing non-volatile organic compounds; a sample capture element having fluid communication with the sample capture element and configured to draw exhaled breath into the sample capture element; the sample extraction system extracts non-volatile organic compounds from the packed bed column; and the sample analysis system comprises: a sample processing system for processing and concentrating the sample collected on a sample plate; and a diagnostic device for analyzing the sample. The diagnostic device may include at least one of PCR, ELISA, rt-PCR, mass spectrometer (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS. The diagnostic device may include MALDI-TOFMS. The extraction system may include means for flushing the packed bed column with a solvent to remove the solvent containing non-volatile organic matter from the packed bed. The solvent may have at least one of acetonitrile, methanol, acid, and isopropanol, with the remainder being water. The solvent may contain acetonitrile in water at a concentration of about 50% and about 70% by volume. The solvent may contain isopropanol in water at a concentration of about 50% and about 70% by volume. The solvent may contain methanol in water at a concentration of about 50% and about 70% by volume. The extraction system may include means for flushing the packed bed column with at least one of about 12.5% ​​acetic acid, about 5% TFA, about 5% formic acid, and about 10% HCl.

[0019] A respiratory disease diagnostic system is disclosed for diagnosing respiratory diseases caused by viruses having at least one of SARS-CoV, MERS-CoV, and SARS-CoV-2 in exhaled breath, the respiratory disease diagnostic system comprising: an exhaled sample collection system; a sample extraction system; a sample processing system; and a diagnostic device, wherein the exhaled sample collection system comprises: an exhaled collection element configured to accommodate an individual's face to collect exhaled breath containing water, volatile organic components (VOCs), and non-volatile organic components; a sample capture element having a packed bed column for selectively capturing non-volatile organic components; a pump having fluid communication with the sample capture element and configured to draw exhaled breath into the sample capture element; the sample extraction system extracts non-volatile organic components from the packed bed column; the sample processing system has means for thermal acid digestion of non-volatile organic components having viral particles extracted from the sample extraction system to produce a peptide sample characteristic of the virus; and the diagnostic device analyzes the peptide sample. The extraction system described above may include means for flushing the packed bed column with at least one of approximately 12.5% ​​acetic acid, approximately 5% TFA, approximately 5% formic acid, and approximately 10% HCl. The packed bed column has solid particles having a functional group immobilized on the surface of the particles, the functional group may have at least one of carbohydrates such as glycan, heparin, heparan sulfate, and dextran.

[0020] A method for diagnosing a respiratory disease caused by a virus having at least one of SARS-CoV, MERS-CoV, and SARS-CoV-2 in exhaled breath is disclosed. This method for diagnosing a respiratory disease includes an exhaled breath sample collection step of collecting an exhaled breath sample from an individual, which comprises providing an exhaled breath collection element configured to receive the individual's face for collecting exhaled breath containing water, volatile organic components (VOCs), and non-volatile organic components, and drawing the exhaled breath into a sample capture element having a packed bed column to selectively capture non-volatile organic components using a pump; an exhaled breath sample collection step; in a sample extraction system, extracting non-volatile organic substances containing virus particles from the packed bed column using at least one of about 12.5% acetic acid, about 5% TFA, about 5% formic acid, and about 10% HCl; digesting the extracted non-volatile organic substances to generate a peptide sample characteristic of the virus; and analyzing the peptide sample using a diagnostic device. The analyzing step may include plating the peptide sample on a MALDI matrix-coated sample plate and analyzing the plated sample using MALDI-TOFMS. The packed bed column has solid particles having functional groups immobilized on the surface of the particles, and the functional groups may have at least one of glycan, heparin, heparan sulfate, and carbohydrates such as dextran.

[0021] A breath sample collection system for diagnosing at least one respiratory disease using exhaled breath is disclosed, the breath sample collection system comprising a mask configured to accommodate an individual's face for collecting exhaled breath having water, volatile organic components (VOCs), and non-volatile organic components, the mask including a stem and a port located below the stem; a HEPA filter removable and fluidly connected to the stem of the mask; a sample capture element having a packed bed column for selectively capturing non-volatile organic components in the exhaled breath, the sample capture element removable and fluidly connected to the port; and a pump fluidly communicating with the sample capture element and configured to draw exhaled breath into the sample capture element. The non-volatile components in the exhaled breath may consist of exhaled aerosol particles having at least one of microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteomics biomarkers characteristic of the respiratory disease. The packed bed column may consist of solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. The packed bed column may have resin beads having a C18 functional group on its surface. The resin beads may have a nominal diameter between about 12 μm and about 20 μm. The resin beads may be packed between two porous polymer frit disks. The system may further have a trap positioned between the sample capture element and the pump, configured to capture exhaled condensate (EBC) passing through the packed bed, containing at least one of water vapor, volatile organic components, and non-volatile organic components. The trap may be cooled below ambient temperature. The solid particles may have a functional group immobilized on the surface of the particles, the functional group may have at least one of C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA.

[0022] An exemplary sample collection system for collecting aerosol particles for the diagnosis of at least one respiratory disease is disclosed, the exemplary sample collection system having a sample capture element having a packed bed column for selectively capturing non-volatile organic components in the aerosol, and a pump in fluid communication with the sample capture element and configured to draw the aerosol into the sample capture element. The non-volatile components in the aerosol may have at least one of microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteomics biomarkers characteristic of respiratory diseases. The packed bed column may have solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. The packed bed column may have resin beads having a C18 functional group on the surface. The resin beads may have a nominal diameter between about 12 μm and about 20 μm.

[0023] An exemplary respiratory disease diagnostic system for the diagnosis of respiratory diseases caused by aerosolized viral and bacterial particles is disclosed, the exemplary respiratory disease diagnostic system comprising an exemplary sample collection system disclosed herein, a sample extraction system for extracting non-volatile organic matter from the packed bed column, and a diagnostic device for analyzing the extracted non-volatile organic matter sample. The extraction system may have means for flushing the packed bed column with at least one of about 12.5% ​​acetic acid, about 5% TFA, about 70% isopropanol, about 5% formic acid, and about 10% hydrochloric acid. The diagnostic device may have at least one of PCR, ELISA, rt-PCR, mass spectrometer (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS. The system may further have a sample processing system for processing and concentrating the sample collected on a sample plate. The sample processing system may have the steps of mixing the sample with a MALDI matrix and depositing the mixed sample and MALDI matrix onto a sample plate. The above sample processing system may further include the step of drying the sample plate after the mixed sample and the MALDI matrix have been applied to it. The above sample processing system may include means for hot acid digestion of a nonvolatile organic substance having virus particles extracted from the sample extraction system to produce a peptide sample characteristic of the virus; the step of mixing the peptide sample with the MALDI matrix; and the step of applying the mixed sample and the MALDI matrix to a sample plate. The above system may further include the step of drying the sample plate after the mixed sample and the MALDI matrix have been applied to it. The MALDI matrix may consist of α-cyano-4-hydroxycinnamic acid, acetonitrile, TFA, and water. The aerosolized virus particles may consist of at least one of SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0024] Other features and advantages of this disclosure are described in part in the following description and accompanying drawings, and the favorable aspects of this disclosure will become apparent to those skilled in the art through examination of the following detailed description, which is described and shown here and partially grasped in conjunction with the accompanying drawings, and will also be learned through the practice of this disclosure. The advantages of this disclosure may be realized and achieved by means and combinations specifically indicated in the accompanying claims. [Brief explanation of the drawing]

[0025] The aforementioned aspects of this disclosure and its many associated benefits will be more readily understood by referring to the following detailed description in conjunction with the attached drawings. [Figure 1] Figure 1 is a schematic diagram of an exemplary breath sample collection system having a packed bed column. [Figure 2] Figure 2 is a schematic diagram of an exemplary diagnostic system for respiratory diseases that includes a sample collection system. [Figure 3] Figure 3 is a schematic diagram of an exemplary diagnostic method using a system with a packed bed column. [Figure 4] Figure 4 shows the measurement of particle capture efficiency using an exemplary packed bed column. [Figure 5] Figures 5A–5E show the results for non-volatile organic molecules collected using an exemplary packed-bed column and analyzed using mass spectrometry. [Figure 6A] Figure 6A shows silver-stained images of proteins in a breath sample obtained by SDS-PAGE electrophoresis. [Figure 6B] Figure 6B shows the spectra collected using total ion chromatography (TIC) in LC-MS analysis of exhaled breath samples. [Figure 7] Figure 7 is a schematic diagram of an exemplary breath sample collection system having a packed bed column. [Figure 8A]Figures 8A and 8B show the results for aerosolized bacteria and viruses captured using an exemplary packed-bed column and analyzed using MALDI TOF-MS. [Figure 8B] Figures 8A and 8B show the results for aerosolized bacteria and viruses captured using an exemplary packed-bed column and analyzed using MALDI TOF-MS.

[0026] All reference numbers, identifiers, and callouts in the figure are incorporated herein by this reference as if they were fully described here. The absence of numbering in the figure elements is not intended as a waiver of any rights. Unnumbered references may also be identified by letters in the figure or appendices.

[0027] The following detailed description includes references to the accompanying drawings, which form part of the detailed description. The drawings illustrate specific embodiments in which the disclosed systems and methods may be carried out. These embodiments, which should be understood as “examples” or “options,” are described in sufficient detail to enable those skilled in the art to carry out the invention. Embodiments can be combined, other embodiments can be utilized, or structural or logical modifications can be made without departing from the scope of the invention. Therefore, the following detailed description should not be constrained, and the scope of the invention is defined by the accompanying claims and their legal equivalents.

[0028] In this disclosure, aerosol generally means a suspension of particles dispersed in air or gas. “Autonomous” diagnostic systems and methods mean that they produce diagnostic test results “without or with minimal intervention by a healthcare professional.” The U.S. FDA classifies medical devices based on the risks associated with them and by assessing the amount of regulation that reasonably ensures the safety and effectiveness of the devices. Devices are classified into one of three regulatory classes: Class I, Class II, or Class III. Class I includes the lowest-risk devices, and Class III includes the highest-risk devices. All classes of devices are subject to general regulations, which are the fundamental requirements of the Food, Drug, and Cosmetic (FD&C) Act, applicable to all medical devices. In vitro diagnostic products are reagents, instruments, and systems intended for use in diagnosing diseases or other conditions, including determining a health condition, in order to cure, mitigate, treat, or prevent diseases or their sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. The exemplary devices disclosed herein may operate autonomously, produce reliable results, and consequently be regulated as Class I devices. In some parts of the world where tuberculosis infections are a major burden, access to medically trained personnel is extremely limited. Autonomous diagnostic systems should be prioritized over non-autonomous ones.

[0029] In this disclosure, singular nouns (equivalent to the English terms "a" or "an") are used to include one or more, and the term "or" is used to refer to non-exclusive "or" unless otherwise specified. Furthermore, it should be understood that any expressions or terms used herein, unless otherwise defined, are for illustrative purposes only and not for limitation. Unless otherwise specified in this disclosure, the error range associated with the term "approximately" is ±10% of the disclosed value (e.g., dimensions, operating conditions). The error range associated with a value disclosed as a percentage is ±1% of the disclosed percentage. The word "substantially" used before certain words includes the meanings of "a substantial portion of the specified range" and "most of what is specified, but not all of it." [Modes for carrying out the invention]

[0030] Exhaled aerosol particles contain various non-volatile organic biomolecules such as metabolites, lipids, and proteins. Furthermore, these non-volatile molecules have a broad particle size distribution ranging from submicron size to approximately 10 micron size. There is a need for exhaled breath collection and disease diagnostic systems and methods that can efficiently capture various types of non-volatile molecules of various particle sizes from exhaled breath. Specific aspects of this invention are described below in considerable detail for the purpose of illustrating the configuration, principles, and operation of the disclosed methods and systems. However, various modifications can be made, and the scope of this invention is not limited to the exemplary embodiments described.

[0031] An exemplary diagnostic system 2000 (Figure 2) based on breath analysis ("EBA") may have a breath sample collection system 1000 arranged in fluid communication with a sample extraction system 2002 and an analysis system 2003.

[0032] An exemplary exhaled sample collection system 1000 (Figure 1) may have a sample capture element 1001 having a packed bed column for selectively capturing exhaled aerosols containing non-volatile organisms (including but not limited to bacteria and viruses) and molecules (including small molecules, lipids, and proteins) onto a highly efficient adsorption material. A trap 1003 is in fluid communication with the column 1001 using a tube 1002. The trap 1003 may be made of glass or plastic material. The trap 1003 may be cooled below ambient temperature using an ice bath or other suitable means. The trap 1003 may be used to collect water vapor, other volatile (check) and non-volatile molecules that may pass through the collection column as exhaled condensate (EBC). During exhaled analysis of a patient's respiration using a ventilator, the sample capture element 1001 is detachably connected to the capnography port of the ventilator's respirator tube and positioned immediately near the outlet or at the outlet from the patient's lungs. During respiratory analysis of a person breathing normally, element 1001 may be detachably connected to a mouthpiece (not shown) in which the patient is instructed to breathe, or it may perform the breathing operations previously disclosed herein. For example, the capture element 1001 may be detachably coupled downstream (at the outlet) of an exhalation collection element 1007 (Figure 1), e.g., an emergency CPR rescue mask (e.g., model EVR-CPR01, supplied by Dixie USA EMS Supply Co.), which the patient wears during exhalation analysis. A flow splitter 1008 may be positioned between the exhalation collection element 1007 and the capture element 1001 to split the exhalation flow so that a first portion of the exhalation is directed to the capture element 1001 and a second portion is directed to the HEPA filter 1009. The flow splitter 1008 may be integrated into the collection element 1007. Furthermore, a large particle trap 1012 may be placed upstream of the capture element 1001 to remove large particles (larger than approximately 10 μm) of exhaled condensate from the exhaled airflow before they enter the capture element 1001. A pump 1006 may be used to draw exhaled air into the packed bed column of the capture element 1001.An exemplary pump 1006 is a portable diaphragm pump (e.g., Parker Hannifin Corp., part number: D737-23-01). The flow rate from pump 1006 may be adjusted using a needle valve 1005 to achieve a desired flow rate. A check valve (one-way flow valve) 1011 may be placed between pump 1006 and the capture element 1001 and is configured to be in the open position only when pump 1006 is drawing exhaled air through the packed bed column. When there is no flow, the valve 1011 is in the closed position. A nominal flow rate of approximately 200 ml / min to 600 ml / min may be used. Furthermore, several capture elements 1001 may be used in parallel to increase the flow rate up to a maximum of 12 L / min. Furthermore, when one or more capture elements are in collection mode, one or more may be in elution mode, and some may be in standby mode. To determine whether the amount of exhaled sample was appropriate, a CO2 sensor and a particle counter (not shown) may be placed between the exhaled air collection element 1007 and the sample capture element 1001. CO2 monitoring and particle counting allow for approximation of the percentage of exhaled air. A HEPA filter may be placed downstream of the trap 1003. The capture element 1001 may be cooled using a cooling jacket or other means to lower its temperature below ambient temperature to improve the collection efficiency of non-volatile organic particles. The exhaled sample collection system may further include a humidifier 1010 located upstream of the inlet to the capture element to humidify the exhaled air and increase the humidity in the packed bed column.

[0033] The exhaled breath collection element 1007 may have a snug-fitting mask configured to accommodate an individual's face and may be detachably attached to the patient's / individual's face / head using straps or the like. The individual may sit in an optional containment booth to isolate the patient's EBA from the surrounding air of the laboratory or area. Element 1007 may be used to collect respiratory aerosol particles released from the patient's mouth and nose using the pump 1006 as described above, without allowing aerosol particles to accumulate on the walls of element 1007, and to guide them to the capture element 1001. Element 1007 may be disposable to limit the risk of the patient being contaminated or infected with pathogens released by a previous patient. Alternatively, element 1007 may be reusable, in which case it may be sterilized.

[0034] An exemplary packed bed column within the capture element 1001 may have Hamilton PRP-C18 resin beads supplied by Sigma Aldrich and other vendors. The bed may be held in place between two porous filter plates, such as frit discs. For example, a polyethylene disc with an average pore size greater than 35 μm may be placed upstream of the bed, and a polyethylene disc with an average pore size of 10 μm (Boca Scientific, Dedham, MA) may be placed downstream of the bed. The 35 μm frit disc allows for faster airflow, while the smaller 10 μm frit disc traps all the C18 resin well. In exemplary element 1001, the packed bed may have about 25 mg of C18 resin beads with a nominal diameter between about 12 μm and about 20 μm. Non-volatile organic components in exhaled breath are interacted with the C18 functional groups on the beads and are removedly captured. Water, volatile substances, and other hydrophilic molecules pass through the bed and may be trapped in the glass trap 1003.

[0035] In addition to the C18 functional group, other functional groups exhibiting affinity for non-volatile molecules may be used as adsorbents in columns immobilized on solid-phase beads such as resin beads. The solid-phase beads may be made from polymers and particles such as resins, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. The adsorbent material may have other functional groups, which are not limited to octadecyl, octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, and propylsulfonic acid, which are placed on the solid-phase beads. The functional group may also have at least one of the following: ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA.

[0036] An exemplary diagnostic system 2000 (Figure 2) may have a breath sample collection system 2001 arranged in fluid communication with a sample extraction system 2002 and an analysis system 2003. The sample collection system 2001 may have the exemplary sample collection system 1000 (Figure 2) described earlier. The sample extraction system 2002 may be used to extract captured non-volatile organic matter from the packed bed column of system 1000 and may be arranged inline or offline in system 2000. If system 2002 is arranged offline, at the end of breath sample collection, the capture element 1001 may be removed from system 1000 and eluted in an organic solvent in the extraction system 2002 to remove the non-volatile organic matter from the packed bed column. The exemplary organic solvent includes, but is not limited to, about 50-70% acetonitrile in water for extracting non-volatile organic matter (highly polar non-volatile organic molecules, proteins, etc.) captured from the packed bed column. Extraction may be repeated using the same or other solvents to extract less polar lipid molecules from the packed bed, the solvent being, but not limited to, 50–70% isopropanol in water. Other organic solvents include about 50% to about 70% methanol in water and about 50% methanol in about 50% chloroform. If system 2002 is placed inline, at least one of a CO2 sensor and a particle counter may be placed upstream of extraction system 2002. System 2002 may have a solvent container, a pump for transferring the solvent from the solvent container to the packed bed column, and a container for collecting the solvent containing the non-volatile biomarker into another container or cup. Alternatively, system 2002 may have an injector for injecting the solvent into the packed bed column and collecting the extract containing the non-volatile organic and biomarker into a suitable cup or container, or another laboratory tube of small volume. The captured sample in the solvent may be further processed and analyzed in analysis system 2003.

[0037] The analysis system 2003 may include a sample processing system 2004 and at least one diagnostic device 2005. The sample processing system 2004 may have elements necessary to perform one or more of the following steps.

[0038] (a) The step of placing the sample in at least one of a cup, vial, and sample plate. For example, the Series 110A Spot Sampler (aerosol device) uses a 32-well plate with a circular well shape (well volume 75 μL) or a teardrop well shape (well volume 120 μL), which is heated to evaporate the solvent and excess liquid / liquid and concentrate the sample. (b) A step of concentrating the sample by placing it in a cup and exposing it to a vacuum source or freeze-drying apparatus to evaporate the solvent. and; (c) High-temperature digestion step of proteins and virus particles.

[0039] The sample may be centrifuged to remove chemical contaminants. Many diagnostic devices may be adapted for use in the Analytical System 2003, which include, but are not limited to, devices that perform genomics-based assays (PCR, rt-PCR, whole-genome sequencing, etc.), biomarker recognition assays (ELISA, etc.), and spectral analyses such as mass spectrometry (MS). Among these diagnostic devices, MS is preferred in terms of analytical speed. Suitable MS techniques for biomarker identification are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) time-of-flight MS (TOFMS). ESI may be combined with a high-resolution mass spectrometer. MALDI-TOFMS devices are compact and lightweight, consume less than 100 watts of power, and can perform sample analysis in less than 15 minutes. MALDI-TOFMS is a suitable diagnostic device for point-of-care diagnostics suitable for ACF. The sample must be inserted into the vacuum chamber of the MS and dried before being exposed to laser pulses from an ultraviolet laser. This interaction between the sample and the laser generates large, beneficial biological ion clusters, which are characteristic of biological materials. If a concentrated sample containing only trace amounts of water, or only trace amounts of an organic solvent such as acetonitrile, methanol, or isopropanol at 50%–70% in the water, is provided by Sample Processing System 2004, sample analysis using MS will take less than 5 minutes (including sample preparation). This is because the time required to evaporate the water from the sample is short.

[0040] MALDI-TOFMS is often used to identify live / active agents, which include, but are not limited to, anthrax spores (multiple strains), Staphylococcus aureus enterotoxin (SEA), Staphylococcus aureus enterotoxin B (SEB), lysine, abrin, Ebola Zaire strain, aflatoxin, saxitoxin, conotoxin, Enterobacteriaceae T2 (T2), HT-2 toxin (HT2), cobra toxin, B. globigii spores, B. cereus spores, B. thuringiensis Al Hakam spores, B. anthracis Sterne spores, Y. enterocolitica, E. coli, MS2 virus, T2 virus, adenovirus, and NGA (non-volatile), bradykinin, oxytocin, substance P, angiotensin, diazepam, cocaine, heroin, and fentanyl. Furthermore, the exemplary systems and methods disclosed herein may be used to achieve accurate detection and identification of SARS-CoV-2 from human breath samples.

[0041] In matrix-assisted laser desorption / ionization (MALDI), target particles (analytes) are coated with a matrix chemical that preferentially absorbs light (often ultraviolet wavelengths) from a laser. Without the matrix, biological molecules decompose by thermal decomposition when exposed to the laser beam in a mass spectrometer. The matrix chemical also transfers charge to vaporized molecules, generating ions and accelerating the flight tube through an electric field. Microbiology and proteomics have become major application areas of mass spectrometry. Examples include bacterial identification, chemical structure discovery, and protein function derivation. MALDI-MS is also used for lipid profiling of algae. In MALDI-MS, a liquid typically consisting of an acid such as trifluoroacetic acid (TFA) and a MALDI matrix chemical such as α-cyano-4-hydroxycinnamic acid is dissolved in a solvent and added to the sample. Solvents include acetonitrile, water, ethanol, and acetone. TFA is usually added to minimize the effect of salt impurities on the sample's mass spectrum. Water allows for the dissolution of hydrophilic proteins, while acetonitrile allows for the dissolution of hydrophobic proteins. The MALDI matrix solution is spotted onto the sample on a MALDI plate to obtain a uniform and homogeneous layer of MALDI matrix material on the sample. The solvent evaporates, leaving only the recrystallized matrix, and the sample spreads throughout the matrix crystals. The acid partially degrades the cell membranes of the sample, making the proteins available for ionization and analysis by MS. Other MALDI matrix materials include 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), α-cyano-4-hydroxycinnamic acid (α-cyano or α-matrix), and 2,5-dihydroxybenzoic acid (DHB), as described in U.S. Patent No. 8,409,870.

[0042] Furthermore, the volatile organic compounds collected in trap 1003 (Figure 1) may be heated using a heater and then flushed to a diagnostic device performing GC-MS, GC-IMS, volatile ion chromatography, or other types of analytical methods suitable for the analysis of volatile organic compounds.

[0043] The detection of viruses (e.g., SARS-CoV-2) focuses on the detection of viral proteins, which is a challenging task. An exemplary method for virus detection may have a glycan-based capture matrix (beads) for extracting the target virus from a background matrix (e.g., other non-viral biomolecules, contaminants). Aliquots of a sample that would contain the virus are collected, for example, using a sample collection system 1000, which may also contain other background contaminants, which may be coated onto beads carrying the capture probe. At least one of glycans, heparins, and carbohydrates may be used as a capture material or probe, bound to resin beads or some other type of beads. An optional washing step may be used to remove non-target viral contaminants. The concentrated and purified virus may be eluted from the beads into a sealed heating chamber containing an organic acid, which may include formic acid or acetic acid, using a suitable solvent, and heated to about 120°C for about 10 minutes to digest the protein toxin into specific peptide fragments. This high-temperature acidic protein digestion protocol cleaves the protein at aspartic acid residues, producing a highly reproducible peptide pattern. The capture and digestion processes described here may be achieved with antibodies and enzymes, respectively. Using this example of MALDI-TOFMS sample preparation, sensitivity to ricin biotoxins exceeding 100 ng / mL (with an S / N ratio of approximately 50:1) was achieved with clean buffer. With an S / N ratio of 3:1, a limit of detection (LOD) of less than 10 ng / mL would be achieved. For a 1 μL sample used in a MALDI-TOFMS analytical system, an LOD of approximately 10 ng / mL corresponds to approximately 10 pg(10) on the probe. -12This corresponds to the total mass of g), which is equivalent to approximately 20,000 virus particles. An exemplary microfluidic sample processing system for carrying out the method disclosed above may be configured to analyze samples collected from air or from other sources such as nasal swabs. The glycan-based capture column and other microfluidic components are reusable. A large liquid reservoir containing buffers, weak acids, and alcohols may be used to provide sufficient volume to measure hundreds of samples in one channel of the system. Multiple systems may be run in parallel to process multiple samples simultaneously. This system is cost-effective because it does not require fragile and expensive biomolecular reagents.

[0044] Hot acid digestion reproducibly cleaves proteins at aspartic acid residues, creating known peptide sequences with known masses. These peptide mass distributions are characteristic of the precursor protein. Therefore, digestion provides excellent specificity when the target protein is far removed from the background material. Furthermore, the peptide mass distribution is directly determined by the genome, taking post-translational modifications into account. As soon as a new virus is isolated, its sequence can be rapidly determined. The RNA sequence of the SARS-CoV-2 virus can be used to accurately predict protein sequences using state-of-the-art bioinformatics tools (ExPASy Bioinformatics Portal). These proteins can be "digested" in silico (computer-aided) using bioinformatics tools to create a theoretical peptide map. Thus, peptides resulting from SARS-CoV-2 digestion can be predicted and compared with experimental data to generate a specific MALDITOFMS signature for the organism. Reports suggest that the main proteins of SARS-CoV are characterized by a nucleocapsid protein of approximately 46 kDa and a spike protein of 139 kDa. Reasonable amounts of other proteins are the E, M, and N proteins.

[0045] The detection specificity of target viruses requires some background removal, especially when the background contains other proteins. In the presence of large amounts of exogenous proteins, the peptide map may be dominated by non-target peptides. As previously mentioned, affinity capture probes for viral toxins based on glycan-modified agarose beads allow for easy cleanup of toxins, even in the presence of excess background proteins and other biomolecules. When analyzing exhaled breath for viral targets such as SARS-CoV-2, other human proteins in the exhaled breath (Figure 6 and Example 3) may interfere with the detection specificity. To ensure the highest specificity, affinity-based sample cleanup is necessary. Viral detection may require bead materials that offer more selective affinity compared to the glycan-modified beads described above. For example, while dextran-based adsorbents may be used for the purification of viruses, including coronaviruses, the affinity of this resin for target viruses may not be satisfactory. Instead, carbohydrates may be used for the purification of viruses and proteins, including target viruses such as SARS-CoV and SARS-CoV-2. Furthermore, heparin and heparan sulfate may be used as binders to attach to resin beads. Heparin covalently bound to Sepharose beads (GE Healthcare Life Sciences, Heparin Sepharose 6 Fast Flow Affinity Resin, product number 17099801) may be used as an alternative to glycan capture beads. This resin may enable a bead-based capture affinity capture system for collecting viral particles from exhaled breath. In an exemplary diagnostic system, an exhaled sample may be drawn through a capture bed in a sample collection system 1000 to collect particles from the exhaled breath. The resin beads (bed) may be washed to remove background material.The virus particles adsorbed onto the beads may then be eluted using a high-concentration acid solution, for example, at least one of approximately 12.5% ​​acetic acid, approximately 5% TFA, approximately 5% formic acid, and approximately 10% HCl, and sent to a high-temperature acid digestion chamber to generate characteristic peptides. The peptide sample may be mixed with a MALDI matrix and deposited as a substrate suitable for MALDI TOFMS analysis. The sample may also be deposited on a suitable substrate or disk pre-coated with a MALDI matrix.

[0046] Figure 3 is a schematic diagram of an exemplary diagnostic method 3000 using an exemplary system 2000. An autonomous point-of-care diagnosis based on exhalation may be performed using the exemplary method 3000. In step 3001, the individual (i.e., patient) may be instructed to sit down. The chair may be placed in a containment booth as needed. In step 3002, a sample exhalation collection element 1007 may be detachably attached to the individual's head. The individual is then instructed to breathe or perform one or more predetermined operations 3003, which may include a predetermined number of repetitions. Non-volatile organic matter in the breath is captured using system 1000, extracted using system 2002 in step 3004, and eluted using a suitable solvent. During sample collection, human exhalation passes through a column at a predetermined flow rate drawn by a suction pump. Non-volatile molecules in exhaled breath interact with the functionalized beads of capture element 1001 (e.g., C18 functional groups immobilized on resin beads), so these molecules are captured in the column bed of element 1001, while hydrophilic molecules, mostly composed of water and aqueous electrolytes in the breath, pass through the column. Non-volatile organic molecules in human exhaled breath exhibit a strong affinity for alkyl chains via intermolecular forces such as hydrogen bonding and non-covalent interactions. Elution of non-volatile molecules from the column bed may be achieved using organic solvents, which include, but are not limited to, acetonitrile, methanol, and isopropanol as described above. In step 3005, the sample may be further processed using component 2004. The type of sample processing depends on the type of diagnostic instrument and the non-volatile analyte particles of interest. As described above, the viral sample may be treated in a hot acid digestion chamber to produce characteristic peptides. The peptide sample may be mixed with a MALDI matrix and deposited as a substrate suitable for MALDI TOFMS analysis. The sample may be deposited on a suitable substrate or disk pre-coated with a MALDI matrix. Next, in step 3006, the sample is analyzed by a diagnostic device.When the diagnostic device is MALDI-TOF MS, sample processing may also include steps of plating the sample on a MALDI-TOF MS sample disk, heating the disk to concentrate the sample, and drying the disk. The sample disk is analyzed using MALDI-TOF MS. The TOFMS detector may be modified to incorporate an ion gate and a reflectron to enable analysis and sequencing of COVID-19 type virus peptides fragmented during MALDI-TOF / MS. The resulting spectra are compared with spectra of samples known to be positive for a particular respiratory infection, spectra from a known database, and spectra of samples from patients known to be healthy, and a diagnosis of the patient is made. The results may then be communicated to a clinician or the patient.

[0047] When the exhalation collection element 1007 is attached to the patient and sample extraction is initiated, the exemplary system and method is preferably autonomous (except for asking the patient to leave the chair after performing the necessary operations) and generates a diagnostic test result as is. In the case of virus particles such as SARS-CoV-2, the particle diameter is about 0.1 micron and the sensitivity can be between about 10 3 and 10 4 virus particles.

[0048] Reports suggest that analysis of nasal and throat swabs from influenza patients and COVID-19 patients yields virus counts between about 10 3 and 10 10 virus particles. Little is known about the number of virus particles in a patient's exhaled breath. Other reports suggest that influenza patients exhale more than 10 4 particles in about 30 minutes of breathing. If the SARS-CoV-2 output is similar to that of influenza, 10 3 to 10 4If the particle output and particle collection efficiency exceed 99.9%, the exemplary methods and systems disclosed herein should be sufficient to identify target viral particles in exhaled breath. The detection time using the exemplary systems and methods, including the steps of sample extraction (breathing operation), sample collection, sample processing (digestion), and analysis using MALDI TOF-MS, would be between 10 and 20 minutes. This detection time is significantly faster than that of existing detection systems.

[0049] An exemplary sample processing component may have a thermoacidization module or cartridge for autonomously extracting a sample from a packed bed column 1001, performing sample cleanup, performing thermoacidization, and providing a sample ready for plating onto a MALDI-TOFS sample substrate or disk. The cartridge may be designed to be reusable by adding a function to flush the cartridge after each use.

[0050] Other exemplary sample collection systems 7000 (Figure 7) are disclosed. An exemplary sample capture element 7001 may have a packed bed column containing C18-bound resin beads. These resin beads have C18 functional groups immobilized on their surface. The capture element 7001 may be attached to or detachably connected to a first-aid CPR rescue mask 7007 with minor modifications. The stem 7008 of the mask 7007, which normally connects to a resuscitation bag, may be modified to be detachably connected to a HEPA filter 7009. The HEPA filter prevents contamination of exhaled breath by contaminants from the ambient air. An oxygen inlet 7010 to the mask, which is normally located below the stem and configured to be close to the chin of a person when the person wears the mask, may be modified to be detachably connected to the capture element 7001. The element 7001 may be detachably inserted into the mask 7007 through the inlet 7010, or may be detachably connected to or inserted into the mask 7007 to form a substantially leak-free mating with the mask 7007. The mask 7007 may include an elastic band or string that can be looped behind the subject's head to ensure a tight fit of the mask to the patient's face. The mask 7007, configured as described above, prevents direct contact between the mouth of element 7001 and the column inlet, minimizing or eliminating saliva contamination of the column inlet, and also maximizing the collection of non-volatile organic particles from exhaled breath. A trap 7003 immersed in ice water may be installed after (downstream of) the capture element 7001. The flow rate (air suction rate) using the pump 7006 is controlled using the needle valve 7005 and can be suctioned at approximately 600 mL / min. A nominal flow rate of approximately 200 mL / min to 600 mL / min may be used. An optional HEPA filter 7011 may be installed between the trap 7003 and the needle valve 7005. Other fluid components, such as a check valve (see Figure 1), may be installed in the system 7000 to prevent backflow of element 7001 into the column bed. CO2 in the exhaled breath passes through the column bed of element 7001. A CO2 sensor may be placed between the outlet of the respiratory capture element 7001 and the trap 7003 to determine whether the amount of exhaled sample and / or the respiratory operation is appropriate.CO2 monitoring allows for estimating the percentage of air expelled. A particle counter may also be installed between the outlet of element 7001 and trap 7003 to detect the size and number of particles exiting the column bed, and may also be used to detect column bed saturation and breakthrough of non-volatile organic molecules from the column bed. The exemplary system 7000 may also include a bypass line (not shown) for the capture element 7001 to allow for standardization of the respiration rate before routing to the column bed of element 7001. The CO2 sensor and particle counter may also be fluidically connected to the bypass line. The capacity of the functionally immobilized solid beads in the column bed of the capture element 7001 for capturing non-volatile organic molecules may be between approximately 0.05 mg (non-volatile organic) / mg beads and approximately 0.5 mg / mg. The capacity of the C18-bonded resin beads in the column bed of the exemplary capture element may be approximately 0.1 mg / mg. In other words, a column bed equipped with 25 mg of C18 beads would have the ability to capture or adsorb approximately 2.5 mg of non-volatile organic molecules.

[0051] In addition to the C18 functional group, other functional groups exhibiting affinity for non-volatile molecules may be used as adsorbents in columns immobilized on solid-phase beads such as resin beads. The solid-phase beads may be manufactured from polymers and particles such as resins, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. The adsorbent material may contain other functional groups arranged on the solid-phase beads, including, but not limited to, octadecyl, octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, and propylsulfonic acid. The functional groups may also have at least one of the following: ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA.

[0052] The exemplary systems and methods described herein are not necessarily limited to their diagnostic capabilities for respiratory infections. For example, lung cancer may also release biomarkers into the peripheral pulmonary fluid, and these biomarkers would be readily detectable by the disclosed systems and methods. Furthermore, because blood is in close contact with the alveolar lining of the lungs, biomarkers from infections or cancers in other parts of the body (other than the lungs) may migrate beyond the alveolar lining into the peripheral pulmonary fluid, which is detectable by EBA analysis. Consequently, the scope of this invention is not limited to the detection and diagnosis of respiratory diseases. The exemplary systems and methods may be used to capture aerosol chemical particles such as ricin and analyze the particles to prevent the threat of chemical attack.

[0053] [example] [Example 1. Particle capture efficiency using exemplary packed bed column 1001] Approximately 200 μL of HPLC-grade water was aerosolized in a 2-liter chamber using a portable Aeroneb Go nebulizer (Philips, Amsterdam, Netherlands) that produced particles between approximately 0.3 μm and 5 μm in size. Column inlet particle size was measured. Particle counts were recorded using a portable laser particle counter (Met One Instruments, Grants Pass, OR) under four test conditions: a bare column without a particle bed, a column with a pore size of approximately 0.2 μm × 25 mm ID syringe filter (VWR International, Radnor, PA), column 1001 with 30 mg of C18 resin beads (C18 column), and column 1001 with C18 beads (30-minute incubation) after a 30-minute run time. The particle counter was placed downstream of the column. The particle count for 0.3 μm particles was approximately 37,000 without a C18 packed column, and decreased to approximately 480 with a C18 packed column (Figure 4), demonstrating a capture efficiency of over 99%. Similar capture efficiencies were observed for other particle sizes investigated. More importantly, high capture efficiency was maintained even after running collection for approximately 30 minutes. For a 0.3 micron sized bin (the particle size with the highest permeability of the filter), the laboratory background count was 146,000 particles / L. With the collection column attached, the count decreased to 14 particles / L, which represents a collection efficiency of approximately 99.99% (Figure 4), equivalent to or better than that of a HEPA filter.

[0054] [Example 2. Analysis of non-volatile organic molecules collected using exemplary packed bed column 1001 and MS] Based on size and chemical properties, non-volatile organic molecules can be classified into three major categories: polar small molecules, non-polar small molecules (lipids), and macromolecules (peptides and proteins). To demonstrate the collection efficiency of these three types of molecules using exemplary column 1001 containing a bed of C18 beads, representative molecules from each category were selected and characterized using high-resolution mass spectrometry for accurate mass measurements. 10 nM methadone (Sigma-Aldrich, St. Louis, Missouri) was selected to represent small polar molecules, 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (Matreya LLC, State College, Pennsylvania) was selected to represent lipids, and insulin from porcine pancreas (Sigma-Aldrich) was selected to represent peptides and proteins. Approximately 200 μL of each chemical, prepared in HPLC-grade water, was aerosolized using a portable AeronebGo nebulizer (Philips) and placed in a 50 mL conical tube placed on a heating block maintained at approximately 50°C. Column 1001, containing approximately 30 mg of C18 beads, was placed at the bottom of a 50 mL conical tube, and the flow rate of pump 1006 was set to approximately 200 mL / min. In each case, the aerosolized chemical was collected on the bed of column 1001 for approximately 5 minutes. After the collection of methadone and insulin, the collection column was washed four times with 400 μL of water. After rapid centrifugation (optional step), methadone and insulin were eluted using 400 μL of 70% acetonitrile. In the case of phosphorylcholine, the column was washed three times with approximately 400 μL of 70% acetonitrile, and elution (adsorption extraction) was performed using 400 μL of 70% isopropanol. In each case, the washing solution leaving the bed was reserved for analysis. The methadone and insulin collected (extracted) from the bed of column 1001 were lyophilized and resuspended in 200 μL of 70% acetonitrile containing 1% acetic acid. Phosphorylcholine collected from bed 1001 was lyophilized and resuspended in 200 μL of 50% isopropanol with 1% acetic acid and 25% acetonitrile.Mass spectrometry data for methadone, phosphorylcholine, and insulin were collected using an OrbitrapLTQ mass spectrometer (Thermo Fisher Scientific) via direct injection in cation mode. The direct injection flow rate was set to 3 μL / min, and data acquisition was recorded for 10 minutes at 200 m / z with a resolution of approximately 60,000. Target molecules were identified by precise mass measurements. As shown in Figure 5, the mass spectrometry signal for each representative molecule was detected only in the elution solution containing molecules extracted from the C18 bed, and not in the washing solution. Thus, exemplary column 1001 with a bed of C18 beads is effective for collecting small polar molecules, small nonpolar molecules (lipids), and macromolecules (peptides and proteins), enabling highly efficient sample collection of these types of non-volatile organic molecules.

[0055] [Example 3. Analysis of breath samples collected from human subjects] In the exemplary system 7000 described earlier, the column of element 7001 contained approximately 25 mg of C18 beads. Exhaled breath samples were collected from four healthy human subjects with different respiratory volumes at the same flow rate. Subject 1 produced a respiratory volume of 144 L, Subject 2 produced 40 L, and Subjects 3 and 4 produced 81 L. After exhaled breath sample collection, the collection column was removed and eluted with 400 μL of 70% acetonitrile to collect small polar molecules and proteins. Protein samples were lyophilized to remove the solvent and resuspended in 100 μL of 0.1% formic acid. Next, to collect nonpolar molecules (lipids), the column was eluted with 400 μL of 70% isopropanol.

[0056] Samples were analyzed using SDS-PAGE electrophoresis and bottom-up proteomics. Electrophoresis was performed using a Criterion Tris-HCl Gel system (Bio-Rad Laboratories, Hercules, CA) with approximately 25 μL of collected sample per subject. After SDS-PAGE electrophoresis, the SDS-PAGE gel was treated with silver staining (Thermo Fisher Scientific) to visualize proteins. Bovine serum albumin (BSA) was used as an internal positive control. During bottom-up proteomics, approximately 50 μL of total collected sample per subject was processed as described here. Briefly, approximately 50 μL of 50 mM ammonium bicarbonate (pH 8.5) was added to each sample. Protein reduction was performed by adding dithiothreitol to a final concentration of 5 mM and incubating at 37°C for 30 minutes. Following reduction, iodoacetamide was added to a final concentration of 15 mM, followed by protein alkylation, and incubation at room temperature for 1 hour. The proteins were digested overnight using trypsin (Thermo Fisher Scientific). After digestion, the peptides were washed using C18 pack tips (Glygen, Columbia, MD). The final peptide samples were prepared with 20 μL of 0.1% formic acid for mass spectrometry. The samples were processed using an EASY-nLC1000 system (Thermo Fisher Scientific) coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). During tandem mass spectrometry, the peptides were loaded onto an Acclaim PepMap 100 C18 trap column (0.2 mm x 20 mm, Thermo Fisher Scientific) at a flow rate of 5 μl / min, and these peptides were separated using an EASY-Spray HPLC column (75 μm x 150 mm, Thermo Fisher Scientific). The HPLC gradient was performed for 60 minutes at a flow rate of 300 nL / min using a mobile phase of 5–55% (75% acetonitrile and 0.1% formic acid).Mass spectrometry data acquisition was performed in data-dependent acquisition mode. The precursor scan resolution was set to 60,000, and the product ion scan resolution to 15,000. Product ion fragmentation was achieved using high-energy collision-induced dissociation (HCD) at approximately 27% of the total energy. Bottom-up proteomics raw data files were processed against the UniProt human protein database / knowledge base using MaxQuant-Andromeda software.

[0057] Figure 6A shows silver-stained gel electrophoresis images of four subjects. "X" indicates a lane without a sample, used as an internal negative control (comparative example). No protein bands were observed in the condensate (EBC) samples collected with Trap 7003. The intensity of the protein band darkness suggests that the protein content in all samples exceeded 10 ng per protein band, which is suitable for sequential bottom-up proteomics. Furthermore, since the strongest protein bands appear between approximately 50 kDa and 75 kDa (band #1), 37 kDa (band #2), and between 10 kDa and 15 kDa (band #3), a similar protein band pattern should be observed in human subjects. Using tandem mass spectrometry, the proteins corresponding to band #1 were identified as serum albumin and keratin, the protein corresponding to band #2 as zinc-α-2-glycoprotein, and the proteins corresponding to band #3 as cystatin, dermcidin, and S100 protein. Furthermore, a protein cluster of approximately 37 kDa (band #2) suggests the possibility of post-translational modification of the protein, possibly glycosylation. Bottom-up proteomics and a search of protein databases confirmed that this protein is din-alpha-2-glycoprotein, a protein with four known N-linked glycosylation sites (UniProt Knowledge Base). The results demonstrate that silver-stained protein visualization combined with bottom-up proteomics can identify protein profiles in exhaled samples, even at very low protein concentrations. Additionally, the intensity of the protein bands likely correlates with the collected respiratory volume, as Subject 1 (producing approximately 144 liters of exhaled air) showed the darkest protein band, while Subject 2 (40 liters) showed the brightest. Differences in breathing techniques and protocols may also affect aerosol particle products from each subject, which could contribute to the heterogeneity of protein distribution among different subjects. In the example above, no specific instructions regarding breathing technique were used.A similar protein pattern was further confirmed in the TIC profile using tandem mass spectrometry (Figure 6B). By using various protein analysis techniques, the results strongly indicated that the column collection system could capture proteins from exhaled breath, and that the protein content of the exhaled breath sample facilitated serial protein identification using bottom-up proteomics. Furthermore, major proteins in saliva, such as alpha-amylase, were not identified, suggesting that the modification to the exemplary face mask 7007 prevented direct contact between the subject's mouth and the capture element 7001. The exemplary exhaled breath collection system disclosed herein may be used to effectively capture proteins from the lower respiratory tract.

[0058] The similarity in the protein patterns of exhaled breath collected from the four subjects was further confirmed by total ion chromatography (TIC) in LC-MS analysis, as shown in Figure 6B. The ion chromatography peak patterns were similar for all four subjects, with subject 1 showing a relatively stronger peak similar to the stronger band seen in the stained image in Figure 6A.

[0059] Bottom-up proteomics was used to identify proteins in the collected breath samples. 197 proteins were identified from Subject 1, 47 from Subject 2, 25 from Subject 3, and 64 from Subject 4. Since Subject 1 had the most identified proteins, the protein identification numbers correspond to the protein content in the samples. In total, 303 proteins were identified from the four subjects. The most abundant proteins identified based on spectral matching are shown in Table 1, which include cystatin A, dermcidin, and several members of the S100 protein family. [Table 1. Top 20 proteins identified from breath samples of 4 subjects based on abundance] [Table 1]

[0060] [Table 2. Proteins corresponding to breath samples from 4 subjects] [Table 2] [Table 3]

[0061] Non-volatile organic molecules in exhaled breath can originate from both the upper and lower respiratory tracts. To clarify the tissue origin of the proteins identified in the previously described studies, the identified proteins were compared to five proteomic databases published from bronchoalveolar lavage fluid (BALF). It is well known that the BALF exhaled breath sampling method generates proteins from lower respiratory tract origins. The comparison showed that approximately 63 proteins identified in the aforementioned studies (Table 2) were reported in the BALF proteomics database, suggesting that the exemplary exhaled breath sample collection systems and methods disclosed herein were effective in capturing proteins originating from the lower respiratory tract, such as lung tissue. No proteins were identified that correlated with any bacteria or viruses suggesting that the volunteers were actually healthy. Therefore, the exemplary methods and systems disclosed herein may be used as diagnostic tools for detecting respiratory diseases based on the identification of proteins in exhaled breath.

[0062] [Example: Capture and analysis of aerosolized bacteria and viruses using exemplary packed bed column 1001 and MALDI TOF-MS]

[0063] One viral sample, bacteriophage MS2, and three bacterial samples—Escherichia coli (E. coli), Pseudomonas fluorescein, and Yersinia rhodei—were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia). 5 μL of each sample was prepared in 200 μL of HPLC-grade water and aerosolized into 50 mL tubes using a portable Aeroneb Go nebulizer (Philips). A collection column containing 25 g to 35 g of C18 beads was fluidically connected near the bottom of the 50 mL tube, and the nebulized sample was aspirated into and through the column using a pump. The pump flow rate was set to approximately 200 mL / min, and the collection time was approximately 10 minutes. After the completion of the collection step, the collection column was washed four times with 400 μL of water. Subsequently, the column was eluted with 400 μL of 70% isopropanol. Both the washing solution and eluate were collected and stored for MALDI-TOFMS analysis.

[0064] The CHCA (α-cyano-4-hydroxycinnamic acid) MALDI matrix was prepared in approximately 50% acetonitrile containing approximately 0.1% TFA (trifluoroacetic acid) at a concentration of approximately 10 mg / mL. Approximately 1.5 μL of sample was mixed with approximately 0.5 μL of CHCA matrix and deposited onto a MALDI plate. In all cases, after the sample was thoroughly dried, the plate was inserted into an Axima-CFR time-of-flight instrument (Kratos Analytical by Shimadzu Biotech, Manchester, UK). MALDI-TOF mass spectra were acquired in linear mode using a 337 nm N2 laser (laser power, 90 arbitrary units), and all spectra were collected as the average of 200 profiles from 1000 to 15,000 m / z.

[0065] Figure 8A shows the MALDI mass spectra of whole cell analysis of control, eluted, and washed samples of Escherichia coli (left), Pseudomonas fluorescein (center), and Yersinia roedei (right). Bacterial traces are clearly visible in the eluted samples but not in the washed samples. This indicates that the collection column was able to capture aerosolized bacteria. Figure 8B shows (A) the MALDI mass spectra of whole virus MS2 analysis and (B) the MALDI mass spectra of hot acid digestion analysis of virus MS2. The total mass of the MS2 capsid protein (P03612) and its bicharged ion were observed by MALDI-TOFMS. After hot acid digestion, intact capsid protein (red dots) and its digested peptides were observed and characterized by MALDI-TOFMS.

[0066] The abstract is provided in accordance with 37 C. FR § 1.72(b) to enable readers to quickly determine the nature and essence of the technical disclosure from a general understanding. It should not be used to interpret or limit the claims or their meaning.

[0067] Although this disclosure has been described in relation to preferred forms of implementation, those skilled in the art will understand that many modifications can be made to it without departing from the spirit of this disclosure. Therefore, the scope of this disclosure is not intended to be limited by the foregoing description.

[0068] It should be understood that various modifications can be made without deviating from the essence of this disclosure. Such modifications are implicitly included in the explanation. They remain within the scope of this disclosure. It should be understood that this disclosure is intended to result in patents that cover many aspects of the disclosure, both independently, as a system, and in both method and apparatus modes.

[0069] Furthermore, each of the various elements of this disclosure and claims may also be achieved in various ways. This disclosure should be understood to encompass each such variation, whether it be a variation of any device implementation, an implementation of a method or process, or merely a variation of any of these elements.

[0070] In particular, it should be understood that each element's word may be expressed by equivalent apparatus or method terminology, even if only the function or result is the same. Such equivalent, broader, or more general terms should be considered included in the description of each element or action. Such terms may be replaced where necessary to indicate the implicitly broad scope to which this disclosure is entitled. It should be understood that all actions may be expressed either as means to take that action or as elements that cause that action. Similarly, each physical element disclosed should be understood to encompass the disclosure of the action that the physical element facilitates.

[0071] Furthermore, with respect to each term used, a common dictionary definition, such as that found in at least one of the standard technical dictionaries recognized by the art and the latest edition of Random House-Webster's Unabridged Dictionary, should be understood to be incorporated herein for each term and all definitions, alternative terms, and synonyms, provided that its use in this application does not contradict such interpretation.

[0072] Furthermore, the use of the transitional phrase “comprising” or “comprise” is used to maintain the “open-ended” claims of this specification, in accordance with conventional claim interpretation. Therefore, unless otherwise required by context, “comprising” is intended to mean including the described element or step or group of elements or steps, but not to exclude other elements or steps or groups of elements or steps. Such terminology should be interpreted in the broadest manner to provide the applicant with the broadest legally permissible scope. The following lists the technical features described here. [Technical Feature 1] In an exhaled breath sample collection system for diagnosing at least one respiratory disease using exhaled breath, A breath collection element configured to accommodate an individual's face in order to collect exhaled breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds, A sample capture element is provided, which has a packed bed column for selectively capturing non-volatile organic components in exhaled breath, and is detachably and fluidly connected to the exhaled breath collection element. A breath sample collection system characterized by having a pump configured to communicate with the sample capture element and draw exhaled breath into the sample capture element. [Technical Feature 2] The system according to technical feature 1, wherein the non-volatile component in exhaled breath comprises exhaled aerosol particles having at least one of the following: microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteomic biomarkers characteristic of respiratory diseases. [Technical Feature 3] The system according to technical feature 1, further comprising a flow splitter positioned between the exhaled air collection element and the sample capture element to split the exhaled airflow such that a first portion of the exhaled airflow is directed toward the sample capture element and a second portion of the exhaled airflow is directed toward the HEPA filter. [Technical Feature 4] The system according to technical feature 1, further comprising a one-way valve located downstream of the sample capture element and positioned to be in an open position under the flow exiting the sample capture element toward the pump, and otherwise in a closed position. [Technical Feature 5] The system according to technical feature 1, further comprising a large particle trap for capturing large particles of exhaled breath condensates so that they do not reach the above-mentioned filled sample capture element. [Technical Feature 6] The system described in technical feature 5, wherein the size of the above-mentioned large particles is at least about 10 microns. [Technical Feature 7] The system according to technical feature 1, wherein the packed bed column has solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. [Technical Feature 8] The system according to technical feature 1, wherein the packed bed column has resin beads having a C18 functional group on its surface. [Technical Feature 9] The system according to technical feature 8, wherein the resin beads have a nominal diameter between approximately 12 μm and approximately 20 μm. [Technical Feature 10] The system according to technical feature 8, wherein the above-mentioned resin beads are filled between two porous polymer frit discs. [Technical Feature 11] The system according to technical feature 10, wherein the polymer frit disk positioned at the inlet end of the packed column is characterized by an average pore size of at least 35 μm. [Technical Feature 12] The system according to technical feature 10, wherein the polymer frit disk positioned at the outlet end of the packed column is characterized by an average pore size of approximately 10 μm. [Technical Feature 13] The system described in Technical Feature 1, wherein the weight of the backed bed is approximately 25 mg. [Technical Feature 14] The system described in Technical Feature 1, wherein the above-mentioned pump is a diaphragm pump. [Technical Feature 15] The system described in technical feature 14, wherein the nominal flow rate of the above pump is between approximately 200 ml / min and approximately 600 ml / min. [Technical Feature 16] The system according to technical feature 1, wherein the exhalation extraction element comprises at least one of a CPR rescue mask, a CPAP mask, a ventilator mask, and a medical universal mouthpiece. [Technical Feature 17] The system according to technical feature 1, further comprising a trap positioned between the sample capture element and the pump, configured to capture exhaled condensate (EBC) containing at least one of water vapor, volatile organic components, and non-volatile organic components passing through the packed bed. [Technical Feature 18] The system described in technical feature 17, wherein the trap is cooled below ambient temperature. [Technical Feature 19] The system according to technical feature 7, wherein the solid particles include a functional group immobilized on the surface of the particles, and the functional group is at least one of C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA. [Technical Feature 20] The system according to technical feature 19, wherein the ion exchange phase comprises at least one of diethylaminoethylcellulose, QAE Sephadex, Q Sepharose, and carboxymethylcellulose. [Technical Feature 21] The system according to technical feature 19, wherein the polymer phase comprises at least one of polystyrene-co-1,4-divinylbenzene, methacrylate, polyvinyl alcohol, starch, and agarose. [Technical Feature 22] The system according to technical feature 19, wherein the antibody has at least one of anti-human albumin and anti-influenza A virus NP. [Technical Feature 23] The system according to technical feature 1, wherein the capture element is cooled to the ambient temperature or below. [Technical Feature 24] The system according to technical feature 1, further comprising a humidifier positioned upstream of the inlet to the capture element in order to humidify the exhaled air and increase the humidity within the packed bed column. [Technical Feature 25] In a sample capture element for the diagnosis of respiratory diseases using exhaled breath, It has a packed bed column for selectively capturing non-volatile organic components in breath, The above packed bed column, Solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles, A sample capture element characterized by having a functional group immobilized on the surface of the above particles, wherein the functional group has at least one of the following: C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA. [Technical Feature 26] The element according to technical feature 25, wherein the solid particles have a nominal diameter between approximately 12 μm and approximately 20 μm. [Technical Feature 27] The element described in technical feature 25, wherein the above-mentioned solid particles are packed between two porous polymer frit disks. [Technical Feature 28] In a respiratory disease diagnostic system for diagnosing respiratory diseases using exhaled breath, The respiratory disease diagnostic system comprises a breath sample collection system, a sample extraction system, and a sample analysis system. The above breath sample collection system is: A breath collection element configured to accommodate an individual's face in order to collect exhaled breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds, A sample capture element having a packed bed column for selectively capturing non-volatile organic components, and which is detachably and fluidly connected to the exhaled breath collection element, The pump has fluid communication with the sample capture element and is configured to draw exhaled air into the sample capture element. The above sample extraction system extracts non-volatile organic substances from the packed bed column. The above sample analysis system is A sample processing system for processing and concentrating samples collected on a sample plate, A respiratory disease diagnostic system characterized by having a diagnostic device for analyzing samples. [Technical Feature 29] The system according to technical feature 28, wherein the diagnostic device has at least one of PCR, ELISA, rt-PCR, mass spectrometer (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS. [Technical Feature 30] The system according to technical feature 28, wherein the diagnostic device has MALDI-TOFMS. [Technical Feature 31] The system according to technical feature 28, wherein the extraction system has means for flushing the packed bed column with a solvent and removing the solvent containing non-volatile organic matter from the packed bed. [Technical Feature 32] The system according to technical feature 31, wherein the solvent comprises at least one of acetonitrile, methanol, acid, and isopropanol, with the remainder being water. [Technical Feature 33] The system according to technical feature 31, wherein the solvent contains acetonitrile in water at a concentration between approximately 50% and approximately 70% by volume. [Technical Feature 34] The system according to technical feature 31, wherein the solvent contains isopropanol in water at a concentration between approximately 50% and approximately 70% by volume. [Technical Feature 35] The system according to technical feature 31, wherein the solvent contains methanol in water at a concentration between approximately 50% and approximately 70% by volume. [Technical Feature 36] The system according to technical feature 31, wherein the extraction system has means for flushing the packed bed column with at least one of about 12.5% ​​acetic acid, about 5% TFA, about 5% formic acid, and about 10% HCl. [Technical Feature 37] In a respiratory disease diagnostic system for diagnosing respiratory diseases caused by viruses having at least one of SARS-CoV, MERS-CoV, and SARS-CoV-2 in exhaled breath, The respiratory disease diagnostic system comprises a breath sample collection system, a sample extraction system, a sample processing system, and a diagnostic device. The above breath sample collection system is: A breath collection element configured to accommodate an individual's face in order to collect exhaled breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds, A sample capture element having a packed bed column for selectively capturing non-volatile organic components, and which is detachably and fluidly connected to the exhaled breath collection element, The pump has fluid communication with the sample capture element and is configured to draw exhaled air into the sample capture element. The above sample extraction system extracts non-volatile organic substances from the packed bed column. The above sample processing system has means for thermal acid digestion of nonvolatile organic matter containing virus particles extracted from the above sample extraction system in order to generate a peptide sample characteristic of the virus. The above-mentioned diagnostic device is a respiratory disease diagnostic system characterized by analyzing the above-mentioned peptide sample. [Technical Feature 38] The system according to technical feature 37, wherein the extraction system has means for flushing the packed bed column with at least one of about 12.5% ​​acetic acid, about 5% TFA, about 5% formic acid, and about 10% HCl. [Technical Feature 39] The system according to technical feature 37, wherein the packed bed column has solid particles having a functional group immobilized on the surface of the particles, and the functional group has at least one of carbohydrates such as glycan, heparin, heparan sulfate, and dextran. [Technical Feature 40] In a method for diagnosing respiratory illness caused by a virus having at least one of SARS-CoV, MERS-CoV, and SARS-CoV-2 in exhaled breath, A breath sample collection step in which a breath sample is collected from an individual, A step of providing a breath collection element configured to accept an individual's face in order to collect breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds, The exhaled sample collection step comprises the step of drawing exhaled breath into a sample capture element having a packed bed column in order to selectively capture non-volatile organic components using a pump, The sample extraction system includes the step of extracting non-volatile organic matter containing virus particles from the packed bed column using at least one of approximately 12.5% ​​acetic acid, approximately 5% TFA, approximately 5% formic acid, and approximately 10% HCl, The steps include digesting the extracted non-volatile organic matter to generate a peptide sample characteristic of the virus, A method for diagnosing respiratory diseases, characterized by comprising the step of analyzing the peptide sample using a diagnostic device. [Technical Feature 41] The method according to technical feature 40, wherein the above analysis step comprises the step of plating the peptide sample onto a MALDI matrix-coated sample plate and analyzing the plated sample using MALDI-TOFMS. [Technical Feature 42] The method according to technical feature 40, wherein the packed bed column has solid particles having a functional group immobilized on the surface of the particles, and the functional group has at least one of glycan, heparin, heparan sulfate, and carbohydrate. [Technical Feature 43] In an exhaled breath sample collection system for diagnosing at least one respiratory disease using exhaled breath, A breath collection element configured to accommodate an individual's face in order to collect exhaled breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds, If the system includes two or more capture elements, at least one sample capture element is arranged parallel to each other, each element comprising a packed bed column for selectively capturing non-volatile organic components, and is removably and fluidly connected to an exhaled breath collection element, A breath sample collection system comprising at least one pump configured to communicate fluidly with at least one sample capture element and to draw exhaled breath into the at least one sample capture element. [Technical Feature 44] The system described in technical feature 43, wherein the nominal flow rate of at least one of the above-mentioned pumps is approximately 2.5 liters / minute. [Technical Feature 45] The system described in technical feature 44, wherein the above antibody is immobilized on protein A / G agarose beads. [Technical Feature 46] In an exhaled breath sample collection system for diagnosing at least one respiratory disease using exhaled breath, A mask configured to accommodate an individual's face for collecting exhaled breath containing water, volatile organic compounds (VOCs), and non-volatile organic compounds, the mask comprising a stem and a port located beneath the stem, A HEPA filter is detachably and fluidly connected to the stem of the mask described above, A packed bed column for selectively capturing non-volatile organic components in exhaled breath, and a sample capture element that is detachably and fluidly connected to the port, A breath sample collection system characterized by having a pump configured to communicate with the sample capture element via fluid and to draw exhaled breath into the sample capture element. [Technical Feature 47] The system according to technical feature 46, wherein the non-volatile component in the exhaled breath comprises exhaled aerosol particles having at least one of the microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteomics biomarkers characteristic of the above-mentioned respiratory diseases. [Technical Feature 48] The system according to technical feature 46, wherein the packed bed column has solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. [Technical Feature 49] The system according to technical feature 46, wherein the packed bed column has resin beads having a C18 functional group on its surface. [Technical Features 50] The system according to technical feature 49, wherein the resin beads have a nominal diameter between approximately 12 μm and approximately 20 μm. [Technical Feature 51] The system according to technical feature 49, wherein the above-mentioned resin beads are filled between two porous polymer frit discs. [Technical Feature 52] The system according to technical feature 46, further comprising a trap positioned between the sample capture element and the pump, configured to capture exhaled condensate (EBC) containing at least one of water vapor, volatile organic components, and non-volatile organic components passing through the packed bed. [Technical Feature 53] The system described in technical feature 52, wherein the above trap is cooled below ambient temperature. [Technical Feature 54] The system according to technical feature 48, wherein the solid particles include a functional group immobilized on the surface of the particles, and the functional group is at least one of C18 (octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethylaminopropyl, carboxypropyl, benzenesulfonic acid, propylsulfonic acid, ion exchange phase, polymer phase, antibody, glycan, lipid, DNA, and RNA. [Technical Feature 55] In a sample collection system for collecting aerosol particles for the diagnosis of at least one respiratory disease, A sample capture element having a packed bed column for selectively capturing non-volatile organic components in the above aerosol, A sample collection system characterized by having a pump configured to communicate with the above-mentioned sample capture element and to draw the above-mentioned aerosol into the sample capture element. [Technical Feature 56] The system according to technical feature 55, wherein the non-volatile component in the aerosol contains at least one of the following: microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteomics biomarkers characteristic of respiratory diseases. [Technical Feature 57] The system according to technical feature 55, wherein the packed bed column has solid particles having at least one of resin, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles. [Technical Feature 58] The system according to technical feature 55, wherein the packed bed column has resin beads having a C18 functional group on its surface. [Technical Feature 59] The system according to technical feature 58, wherein the resin beads have a nominal diameter between approximately 12 μm and approximately 20 μm. [Technical Feature 60] In a system for diagnosing respiratory illnesses caused by aerosolized viral and bacterial particles, The sample collection system described in technical feature 55, A sample extraction system for extracting non-volatile organic substances from the above packed bed column, A respiratory disease diagnostic system characterized by comprising a diagnostic device for analyzing the extracted non-volatile organic substance sample. [Technical Feature 61] The system according to technical feature 60, wherein the extraction system has means for flushing the packed bed column with at least one of about 12.5% ​​acetic acid, about 5% TFA, about 70% isopropanol, about 5% formic acid, and about 10% hydrochloric acid. [Technical Feature 62] The system according to technical feature 60, wherein the diagnostic device has at least one of PCR, ELISA, rt-PCR, mass spectrometer (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS. [Technical Feature 63] The system according to technical feature 60, further comprising a sample processing system for processing and concentrating a sample collected on a sample plate. [Technical Feature 64] The above sample processing system, The above sample is mixed with the MALDI matrix, The system according to technical feature 63, comprising the step of adhering the mixed sample and the MALDI matrix to a sample plate. [Technical Feature 65] The system according to technical feature 64, further comprising the step of drying the sample plate after the above mixed sample and the above MALDI matrix have been applied to the sample plate. [Technical Feature 66] The above sample processing system, A means for generating a peptide sample characteristic of the virus by thermal acid digestion of a non-volatile organic substance containing virus particles extracted from the above sample extraction system, The above peptide sample is mixed with the MALDI matrix, The system according to technical feature 63, comprising the step of adhering the mixed sample and the MALDI matrix to a sample plate. [Technical Feature 67] The system according to technical feature 66, further comprising the step of drying the sample plate after the above-mentioned mixed sample and MALDI matrix have been applied to the sample plate. [Technical Feature 68] The system according to technical feature 66, wherein the above MALDI matrix comprises α-cyano-4-hydroxycinnamic acid, acetonitrile, TFA, and water. [Technical Feature 69] The system according to technical feature 60, wherein the aerosolized virus particles have at least one of SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0073] [References] 1. B. Bake, P. Larsson, G. Ljungkvist, E. Ljungstrom, and AC Olin, "Exhaled particles and small airways," Respiratory Research (2019)20:8. 2. Fennelly KP, Martyny JW, Fulton KE, Orme IM, Cave DM, et al.(2004) Cough-generated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness. Am J Respir Crit Care Med 169: 604-609. 3. Dina Hashoul and Hossam Haick, "Sensors for detecting pulmonary diseases from exhaled breath," Eur. Respir. Rev. 2019; 28: 190011. 4. Hunt, J., "Exhaled breath condensate: An evolving tool for noninvasiveevaluation of lung disease," J. Allergy Clin. Immunol. 2002; 110:28-34. 5. Maria D. King, Andrew R. McFarland, "Bioaerosol Sampling with a WettedWall Cyclone: Cell Culturability and DNA Integrity of Escherichia coliBacteria," Aerosol Sci. Technol., 46:82-93, 2012. 6. James J. McDevitt, Petros Koutrakis, Stephen T. Ferguson, Jack M. Wolfson,M. Patricia Fabian, Marco Martins, Jovan Pantelic, and Donald K. Milton,"Development and Performance Evaluation of an Exhaled-Breath BioaerosolCollector for Influenza Virus," Aerosol Sci. Technol. 2013 January 1;47(4): 444-451. 7. Benjamin Patterson, Carl Morrow, Vinayak Singh, Atica Moosa, Melitta Gqada,Jeremy Woodward, Valerie Mizrahi, Wayne Bryden, Charles Call, Shwetak Patel,Digby Warner, Robin Wood, "Detection of Mycobacterium tuberculosis bacilliin bio-aerosols from untreated TB patients," Gates Open Research 2018,1:11. 8. Wood R., Morrow C., Barry C.E., III, Bryden W.A., Call C.J., Hickey A.J., etal.: Real-Time Investigation of Tuberculosis Transmission: Developing theRespiratory Aerosol Sampling Chamber (RASC). PLoS One. 2016; 11(1): e0146658. 9. Rachel C. Wood, Angelique K. Luabeya, Kris M. Weigel, Alicia K. Wilbur, LisaJones-Engel, Mark Hatherill, and Gerard A. Cangelosi, "Detection ofMycobacterium tuberculosis DNA on the oral mucosa of tuberculosispatients," Sci. Rep. 5, 8668 (2015). 10. Fatima B. Wurie, Stephen D. Lawn, Helen Booth, Pam Sonnenberg, Andrew C.Hayward, "Bioaerosol production by patients with tuberculosis duringnormal tidal breathing: implications for transmission risk," Thorax 2016;71: 549-554.

Claims

1. In an exhaled breath sample collection system for diagnosing at least one respiratory disease using exhaled breath, The system is A mouthpiece into which a person is instructed to breathe or perform some kind of breathing action, A sample capture element having a packed bed column configured to selectively capture bacteria and viruses, as well as aerosol particles including small molecules, lipids, and proteins, wherein the sample capture element is configured to be detachably attached to the mouthpiece, A humidifier is positioned upstream of the inlet to the sample capture element and humidifies the exhaled air to increase the humidity inside the packed bed column. A breath sample collection system characterized by having a pump configured to communicate with the sample capture element and draw exhaled breath into the sample capture element.

2. The breath sample collection system according to claim 1, wherein the sample capture element is cooled to a temperature below the ambient temperature.

3. The breath sample collection system according to claim 1, wherein the mouthpiece is a general-purpose medical mouthpiece.

4. The breath sample collection system according to claim 1, wherein the protein comprises a virus-related protein including one or more of SARS-CoV, MERS-CoV, or SARS-CoV-2.

5. The exhaled sample collection system according to claim 1, wherein the protein comprises one or more proteins derived from the lower respiratory tract, including serum albumin, keratin, glycoprotein, cystatin, dermicidin, or S100 protein.

6. The breath sample collection system according to claim 1, wherein the packed bed column comprises resin beads having a C18 functional group on its surface.

7. The breath sample collection system according to claim 1, wherein the packed bed column comprises heparin covalently bound to Sepharose beads.

8. The breath sample collection system according to claim 1, wherein the packed bed column comprises solid particles on which a functional group is immobilized on the particle surface, and the functional group comprises one or more carbohydrates including glycan, heparin, heparan sulfate, or dextran.

9. The breath sample collection system according to claim 6, wherein the nominal diameter of the resin beads is approximately 12 μm to approximately 20 μm.

10. The breath sample collection system according to claim 1, wherein the nominal flow rate through the packed bed column by the pump is approximately 200 ml / min to approximately 600 ml / min.

11. The breath sample collection system according to claim 1, further comprising a trap positioned between the sample capture element and the pump, configured to capture breath condensate (EBC) containing at least one of water vapor, volatile organic components, and non-volatile organic components passing through the packed bed column, wherein the trap is cooled to a temperature below ambient temperature.

12. The breath sample collection system according to claim 1, wherein the particle capture efficiency of the breath sample collection system is 99% or more.

13. In a respiratory disease diagnostic system using exhaled breath, A breath sample collection system according to claim 1, A sample extraction system that uses a solvent to extract captured bacteria and viruses, as well as molecules including low molecular weights, lipids, and proteins, from the packed bed column and produce a collected liquid sample. It has a sample analysis system, The above sample analysis system is A sample processing system for processing and concentrating the collected samples on a sample plate, A diagnostic system for respiratory diseases, characterized by having a diagnostic device for analyzing the above-mentioned sample.

14. The respiratory disease diagnostic system according to claim 13, wherein the solvent comprises at least one of acetonitrile, methanol, an acid, and isopropanol, with the remainder being water.

15. The respiratory disease diagnostic system according to claim 13, wherein the solvent comprises at least one of about 12.5% ​​acetic acid, about 5% trifluoroacetic acid, about 5% formic acid, and about 10% hydrochloric acid.

16. The respiratory disease diagnostic system according to claim 13, wherein the sample extraction system comprises means for washing the packed bed column with a solvent, and means for removing from the packed bed column aerosolized bacteria and viruses, as well as the solvent containing molecules including small molecules, lipids, and proteins.

17. 18. The respiratory disease diagnostic system according to claim 13, wherein the diagnostic device comprises at least one of PCR, ELISA, rt-PCR, mass spectrometer (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS.

18. The aforementioned sample processing system is A means for generating a peptide sample characteristic of particles by thermal acid digestion of the bacteria and viruses, as well as molecules including low molecular weights, lipids, and proteins, extracted from the above sample extraction system, A means for mixing the peptide sample with the MALDI matrix, A respiratory disease diagnostic system according to claim 13, further comprising means for applying the mixed sample and the MALDI matrix to a sample plate.

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