Diagnosing respiratory disease by capturing aerosolized biomaterial particles using a packed bed system and method

Abstract

Disclosed are methods and devices for capturing and analyzing aerosolized particles in exhaled breath characteristic of respiratory diseases, enabling rapid, low-cost point-of-care assays for several diseases, including airway diseases such as COVID-19. The disclosed methods and systems selectively capture aerosolized particles using a packed bed column. The captured particles are eluted using a solvent and analyzed using an analytical device such as a MALDI-TOFMS.

Description

Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 169130, filed March 31, 2021, entitled "Diagnosis of Respiratory Disease by Capture of Aerosolized Biomaterial Particles Using a Packed Bed System and Method," U.S. Provisional Application No. 63 / 249357, filed September 28, 2021, entitled "Diagnosis of Respiratory Disease by Capture of Aerosolized Biomaterial Particles Using a Packed Bed System and Method," and U.S. Patent No. 63 / 325435, filed March 30, 2022, entitled "Diagnosis of Respiratory Disease by Capture of Aerosolized Biomaterial Particles Using a Packed Bed System and Method," the entire disclosures of which are incorporated herein by reference in their entireties. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] none. [Technical field]

[0003] This disclosure relates to methods and devices for capturing and analyzing aerosolized organic biomaterials, such as viral and bacterial particles, in exhaled breath and other aerosols, enabling rapid, low-cost point-of-care assays for several diseases, including airway diseases such as COVID-19. More specifically, but not by way of limitation, this disclosure relates to methods and devices for analyzing non-volatile organics in exhaled breath and other aerosols to detect respiratory diseases, such as COVID-19 and tuberculosis, using mass spectrometry, including MALDI-TOFMS. [Background technology]

[0004] Coronavirus disease 2019 (COVID-19) is a disease caused by the newly emerged coronavirus SARS-CoV-2. This novel coronavirus is a respiratory virus that spreads primarily through droplets produced when an infected person coughs or sneezes, or through droplets of saliva and nasal secretions. The novel 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 aid in mitigation.

[0005] Furthermore, tuberculosis (TB) has surpassed HIV / AIDS as a global cause of death (killer), with over 4000 deaths every day (Patterson, B et al., 2018). Incidence declines remain insufficient at a reported annual rate of 1.5%, making it unlikely that treatment alone will significantly reduce the burden of disease. In communities where HIV is highly prevalent, Mycobacterium tuberculosis (Mtb) genotyping studies show that recent infections, rather than relapses, account for the majority (54%) of TB cases. The physical process of TB transmission remains poorly understood, and the application of new techniques to elucidate key events in the generation, release, and inhalation of infectious aerosols has been slow. Empirical studies characterizing airborne infectious particles are scarce. Two major issues plaguing investigations are the low concentrations of naturally produced Mtb particles and the complication of environmental and patient-derived bacterial and fungal contamination of airborne samples. Nonetheless, many attempts at airborne detection have been made. A 2004 proof-of-concept study and subsequent feasibility study in Uganda sampled cough-generated aerosols from patients with pulmonary tuberculosis. Coughing directly into a sampling chamber equipped with two workable cascade impactors yielded positive cultures from more than a quarter of participants, despite having received 1–6 days of chemotherapy. A follow-up study using the same device found that participants with higher aerosol bacterial loads had higher rates of infection at home that may lead to disease detection, suggesting that quantitative airborne sampling may serve as a clinically relevant measure of infectivity. Thus, interruption of transmission could have a rapid and measurable impact on tuberculosis incidence.

[0006] The best way to control the transmission of TB is to rapidly identify and treat active TB cases (Wood, RC et al., 2015). The diagnosis of pulmonary TB is usually made by microbiological, microscopic, or molecular analysis of the patient's sputum. The "gold standard" test for TB infection in most developing countries is the smear culture based on a sputum sample. The sample is smeared on a culture plate, a stain specific for Mtb is added, and the stained cells are counted using a microscope. If the concentration of cells 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. The diagnosis may take several hours. The need for sputum as a diagnostic sample is a limiting factor due to the challenges of obtaining sputum from patients and its complex composition. The viscosity of the material limits the sensitivity of the test, increases the heterogeneity between samples, and increases the costs and labor associated with the test. Moreover, sputum (which requires coughing) is an occupational hazard for health care workers. Sputum has several disadvantages as a sample medium. First, only about 50% of patients are able to provide a good sputum sample. For example, children around the age of 8 are often unable to produce a sample on request, as they usually do not have the developed ability to "cough up" 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. Thus, diagnostic methods based on sputum analysis may not 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 is no longer representative of the disease deep in the lungs, and within a few days after the start of treatment, the number of live Mtb in sputum is greatly reduced. Urine and blood have been proposed as sample media for the diagnosis of tuberculosis infection. Blood is highly invasive and the costs of processing blood samples that are often HIV positive are high because in some parts of the world many TB patients are also co-infected with HIV. Moreover, patients with active TB infection may not have many TB cells circulating in their blood.Urine-based diagnostics have also been proposed, but these tests look for biomarkers of disease other than live TB bacteria and have not been validated for widespread clinical use.

[0007] A sample that is easier to collect and handle, safer, and more uniform would simplify TB diagnosis. Exhaled breath contains aerosols ("EBA") and vapors that can be collected noninvasively 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 derived primarily from water vapor, it contains dissolved nonvolatile compounds such as cytokines, lipids, surfactants, ions, oxidation products, adenosine, histamine, acetylcholine, and serotonin. In addition, 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 lining fluid and volatile compounds, providing a noninvasive indication of ongoing biochemical and inflammatory activity in the lungs. The rapid interest in EBCs has arisen from the realization that in pulmonary disease, EBCs have measurable characteristics that can be used to distinguish infected from 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 dilution, EBCs may not provide an accurate assessment of individual solute concentrations within the native airway lining fluid. However, they can provide useful information when concentrations differ substantially between health and disease or are based on the ratio of solutes present in the sample.

[0008] Patterson et al. (2018) used a custom-built Respiratory Aerosol Sampling Chamber (RASC), a novel device designed to optimize patient-derived exhaled aerosol sampling and to isolate and accumulate respirable aerosols from a single patient. Environmental sampling detects Mtb present after an aging period in the air within the chamber. Thirty-five newly diagnosed GeneXpert (Cepheid, Inc., Sunnyvale, CA) sputum-positive TB patients were sampled in approximately 1.4 m 3 Patients were monitored during one hour of confinement in a RASC chamber with a volume of 1000 x 1000 mm. GeneXpert genetic assays are based on polymerase chain reaction (PCR) and can be used to analyze samples for TB diagnosis and to indicate whether TB samples have drug resistance genes. The GeneXpert PCR assay for TB can accept sputum samples and provide positive or negative results in approximately one hour. The chamber includes aerodynamic particle size detection, viable and non-viable sampling devices, and real-time CO 2 Monitoring, and cough recordings were incorporated. Microbial culture 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, with 42% of samples positive by mycobacterial culture and 92% positive by ddPCR. A correlation was found between the incidence of cough and culturable bioaerosols. Mtb was detected in all viable cascade impactor stages, with a peak in aerosol sizes between 2.0 and 3.5 μm. This was consistent with a median aerosol culture-positive value of 0.09 CFU per liter of exhaled breath and an estimated median concentration of exhaled particulate bioaerosols of 4.5x10 7 CFU / ml. Mtb was detected in bioaerosols exhaled by the majority of untreated TB patients using the RASC chamber. Molecular detection was found to be more sensitive than Mtb culture on solid media.

[0009] Mtb can be identified in EBA by culture, ddPCR, electron microscopy, immunoassays, and cell staining (e.g., oramine and dmn-Tre). Of these, PCR and immunoassays have the potential to be rapid and specific to the species level. PCR and other genomics-based techniques are specific to the strain level. Mass spectrometry has also been shown to be specific to the strain level of cultures obtained from bacterial infections. For example, the Biotyper from Bruker Daltonics (Germany) has been shown to be able to identify up to 15,000 strains of bacteria that cause infections in humans. These techniques have been shown to be able to identify tuberculosis infections from EBA. Immunoassays for Mtb detection, such as those based on lipoarabinomannan, are also well known.

[0010] In the case of TB, people infected with TB are often diagnosed through passive case finding when individuals present at a clinic. Active case finding ("ACF") is generally considered to include other methods of contacting people suspected of having TB infection outside the primary health care system. According to the WHO, ACF is "the systematic identification of people suspected of having active TB using rapidly applicable tests, screening, or other procedures". The goal of ACF is to treat infected people early to shorten the average duration of infection and thereby reduce the spread of the disease. In the case of TB, by the time a person seeks help at a clinic, he or she may have transmitted TB to about 10 to about 115 other people. ACF helps reduce or prevent significant TB infections. Diagnostic systems and methods, such as sputum analysis and blood analysis, are not automated, do not operate autonomously, or are not rapid. Many involve expensive assays that are consumed per analysis, which means active case finding does not have general utility, especially in underdeveloped and developing countries. As mentioned above, EBA analysis appears to be a compelling diagnostic tool for tuberculosis detection offering rapid analysis, portability, and low cost since the need for expensive assays and consumables is eliminated. McDevitt et al. (2013) reported an EBA analysis device and method for influenza diagnosis. An impactor is used to remove large particles (>4 μm) from exhaled breath, followed by removal of small particles (<4 μm) with a wet film collector. Two size bins of collected particles were analyzed for influenza virus using reverse transcriptase polymerase chain reaction (rt-PCR), a genomics-based method. PCR technology uses biomolecular probes combined with other biomolecules, including enzymes, to amplify specific sequences of DNA if the specific sequence is present in the sample. The target sequence is believed to be specific for the disease being identified. McDevitt et al. showed that influenza can be diagnosed using EBA samples. The disclosed device and method have several drawbacks from a practical standpoint. First, exhaled aerosol samples are collected in individual samples of a few milliliters in volume, requiring significant effort to concentrate the samples.Furthermore, the diagnostic device is not coupled or integrated with the sample collector and is not suitable for use as an ACF tool. The ability to automate the RNA assay to create an autonomous diagnostic tool for tuberculosis analysis is not clear. A method to determine whether a sufficient amount of cough or exhaled aerosols was generated by a particular patient is not described. As a result, if a sample turns out to be negative for influenza, it may be due to a false negative resulting from improper sample collection. It is well known that there is a large variability between individuals regarding the amount of aerosolized lung fluid generated during various breathing maneuvers.

[0011] GeneXpert Ultra is a cutting-edge genomics-based point-of-care diagnostic device that uses PCR technology. Although 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 produced in EBA and can be collected in a 1-hour sampling period. A minimum of 1 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 mail for the United States Postal Service for the bacterial spores that cause anthrax as the mail passes through distribution centers. It combines a wet-wall cyclone with the GeneXpert PCR system to autonomously sample the air and report whether the pathogen is present. However, the GeneXpertUltra assay has a relatively high cost per test and takes approximately 1 hour to complete the assay and provide results. In general, PCR-based diagnostics are not suitable for TB screening in ACF applications due to the long time required for sampling and analysis and the relatively high cost per test.

[0012] The time associated with a diagnostic assay is a critical parameter for field or "point-of-care" testing. By definition, ACF is performed outside of the healthcare system, so ACF is an example of a field diagnostic assay. In the United States, point-of-care tests must provide an answer within 20 minutes; if not, the test is deemed too slow and unacceptable to achieve short patient wait times. In developing countries, especially those with a history of TB epidemics, a diagnosis can be made in about an hour using GeneXpert. As mentioned above, this assay is expensive to implement on a "cost per test" basis, and therefore has not yet been widely deployed. Because of the expense, it is not used to screen patients who appear healthy (asymptomatic) but may be infected with TB, but rather to confirm a diagnosis that is strongly suspected based on other tests and factors.

[0013] Fennelly et al. (2004) described a TB analysis using cough aerosols and a collection chamber containing two Anderson cascade impactors, using individuals known to be active patients. Individuals were asked to cough vigorously twice for 5 min each. Culturing the affected samples took 30–60 days, making this approach not amenable to automation. A challenging aspect of EBA as a clinical sample is the relatively small sample volume of exhaled particulates that can be collected from the breath. Furthermore, a significant portion of the collected mass is water. Molecules containing diagnostic information ("biomarkers") are only available in nanoliter or picogram quantities. Furthermore, the aerosol collection method must be effective at capturing the majority of the biomass in the exhaled breath. Exhaled breath includes air expelled from the lungs by various maneuvers, such as tidal breathing, deep breathing, coughing, and sneezing. Certain types of deep-breathing maneuvers, such as forced vital capacity (FVC), can be used to measure the maximum amount of lung capacity by inhaling as far as possible and exhaling as far (or deep) as possible to maximize vital capacity. Forced expiratory volume (FEV) measures the amount of air a person can forcefully exhale. The amount of air exhaled can be measured during the first second (FEV1), second second (FEV2), and / or third second (FEV3) of a forced breath. Forced vital capacity (FVC) is the total amount of air exhaled during an 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.

[0014] Although studies have shown that respiratory diseases can be detected from exhaled aerosols and exhaled condensate, current clinical testing for infectious diseases or illnesses such as tuberculosis, influenza, and pneumonia continues to utilize sputum, blood, or nasal swabs. Breath analysis tools have not been commercialized due to a lack of methods and devices to efficiently collect and concentrate the minute amounts of analytes present in exhaled breath. Furthermore, there are no standards or methodologies to evaluate exhaled volumes sufficient for a specific diagnosis. The disclosed exemplary devices and methods overcome these limitations by collecting exhaled aerosols and exhaled condensate into relatively concentrated samples at high flow rates and high efficiency. Additionally, size sorting of the aerosols can be incorporated to increase the signal-to-noise ratio of certain analytes prior to collection of the analytes. The concentrated samples can then be analyzed by several methods, preferably using methods that are sensitive, rapid, and highly specific for the analytes of interest. More preferably, the analysis is rapid and near real-time. Mass spectrometry, real-time PCR, and immunoassays are most likely to be sensitive, specific, and near real-time.

[0015] There is a need for sample collection methods that can deliver diagnostic assays that are faster and 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 fast, sensitive, specific, and preferably low cost per test. Such systems can be used for active case finding (ACF) of tuberculosis and other lung and airway diseases. To be effective, ACF systems must be fast and inexpensive "per diagnosis." A low cost per test is a prerequisite for screening large numbers of individuals to proactively prevent tuberculosis transmission and to search for the few who are actually infected with tuberculosis. Low-cost devices and methods will also be needed for point-of-care diagnostics of influenza and other pathogenic viruses, as patients with a supposedly "common cold" infection may be infected with rhinovirus. In some cases, respiratory infections are caused by bacterial or fungal microorganisms and may be treatable with antibiotics. In other cases, the microorganisms may be resistant to antibiotics, and diagnostic methods that can identify the resistance of the microorganism to antibiotics are desirable. A rapid EBA method is desired to distinguish between viral and bacterial infections in the respiratory tract while minimizing the occurrence of false negatives due to insufficient sample volume. Mass spectrometry, genomics methods including PCR, and immunoassays may have high sensitivity and specificity. Mass spectrometry, especially MALDI time-of-flight mass spectrometry (MALDI-TOFMS), has been demonstrated to be sensitive, specific, and near real-time, making it a suitable diagnostic tool for the analysis of EBA and EBC samples. Summary of the Invention

[0016] An exemplary breath collection system for monitoring a patient infected with a respiratory disease and connected to a ventilator to assist the infected patient's breathing is disclosed, the breath collection system having one or more sample capture elements, each of which has a packed bed column for selectively capturing aerosolized particles in exhaled breath produced by the patient, the sample capture elements being removably connected to an exhalation tube of the ventilator, and a subsystem including at least one of a pump, a power source, and a controller, the subsystem being disposed in fluid communication with the sample capture elements and configured to control operation of the sample capture elements, the pump being configured to draw exhaled aerosols into the sample capture elements, the subsystem being configured to control operation of the sample capture elements, the pump being configured to draw exhaled aerosols into the sample capture elements, the subsystem being configured to control operation of the sample capture elements, the sample capture elements ... 2 The system may further include at least one of a sensor and a particle counter. The subsystem may be disposed in a portable housing. The system may further include a trap disposed between the sample capture element and the pump and configured to capture exhaled breath condensate (EBC) passing through the packed bed, the exhaled breath condensate including at least one of water vapor, volatile organic components, and non-volatile organic components. The packed bed column may include a packed bed column having a packed bed of resin, cellulose, silica, agarose, and hydrated Fe. 3 O 4 The packed bed column may include solid particles having at least one of nanoparticles. The packed bed column may include at least one of resin beads having C18 functional groups on their surface, cellulose beads having sulfate functional groups on their surface, and mixtures thereof. The resin beads and cellulose beads may have a nominal diameter of at least about 20 μm. The resin beads and cellulose beads may have a nominal diameter of about 40 microns to about 150 microns. The beads may be packed between two porous polymer frit disks. The nominal flow rate drawn through the bed using the pump may be about 200 ml / min to about 3 L / min.

[0017] An exemplary system for monitoring patients infected with a respiratory disease is disclosed, the exemplary system including the breath collection system described above, a sample extraction system for extracting trapped aerosol particles specific to a respiratory disease from the packed bed column into one or more liquid samples, and an analyzer for analyzing the aerosol particles in the one or more liquid samples. The extraction system may include means for flushing the packed bed column with at least one solvent and collecting the solvent containing the aerosol particles from the packed bed. The at least one solvent may include at least one of acetonitrile, methanol, trifluoroacetic acid (TFA), isopropanol (IPA), and the balance is water. The one or more solvents may include about 50% to about 70% acetonitrile by volume in water, about 50% to about 70% isopropanol by volume in water, and about 0.05% TFA by volume in water. The analytical device may include at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS, and LC-MS / MS. The aerosol particles in the exhaled breath may include at least one of microbial, viral, metabolite biomarkers, lipid biomarkers, and proteomic biomarkers characteristic of respiratory diseases.

[0018] An exemplary method for monitoring a patient infected with a respiratory disease and connected to a ventilator to assist the infected patient's breathing is disclosed, the method comprising the steps of providing a sample collection system removably connected to an exhalation tube of a ventilator provided to assist the infected patient's breathing, the system having one or more sample capture elements, each having a packed bed column for selectively capturing aerosol particles in exhaled breath generated by the patient, the aerosol particles being characteristic of the respiratory disease, and a pump in fluid communication with the sample capture elements and configured to draw the exhaled aerosol into the sample capture elements; extracting the captured aerosol particles from the packed bed column into one or more liquid samples; and analyzing the aerosol particles in the one or more liquid samples to determine the presence or absence of the respiratory disease. The exemplary method may further include washing a column with at least one of 70% ACN, water, and 0.05% TFA prior to removably connecting the sample collection system to an exhalation tube of the ventilator. The extraction step may include flushing the packed bed column first with about 50% ACN by volume, followed by flushing with about 70% IPA by volume. The exemplary method may further include a sample processing step, which may include mixing the sample(s) with a MALDI matrix, and depositing the mixed sample(s) and MALDI matrix onto one or more sample plates. The exemplary method may further include a sample processing step, which may include subjecting the liquid sample(s) extracted from the sample extraction system to proteolytic digestion to generate peptide samples characteristic of the respiratory disease. The sample processing step may further include mixing the peptide sample with a MALDI matrix and depositing the mixed sample and MALDI matrix onto a sample plate.The analyzing step may analyze the sample plate using at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS, and LC-MS / MS.

[0019] An exemplary system for sampling ambient air in an enclosed space for the presence of respiratory disease and capturing aerosol particles in the ambient air is disclosed, the system comprising a sample collection module having one or more sample capture elements, each including a packed bed column for selectively capturing aerosolized particles from exhaled breath present in the ambient air, and a nebulizer for humidifying the ambient air before it enters the one or more sample capture elements, and a fluidics module having at least one of a water pump, a power source, a controller, an air pump for drawing ambient air through one or more sample capture elements, and a water container in fluid communication with the nebulizer and the water pump, the fluidics module configured to control operation of the sample collection module. The fluidics module includes a CO2 detector disposed between the sample capture elements and the pump. 2The sample collection module and the fluidic module may further include at least one of a sensor and a particle counter. The sample collection module and the fluidic module may each be disposed in a portable housing. The sample collection module and the fluidic module may be configured to be fluidly and electrically connected to each other using a quick connect / disconnect coupling that may be configured to detect proper mechanical and electrical contact and alert a user via at least one of a graphical user interface and an audible alarm disposed in one of the housings. The sample collection module and the fluidic module may be disposed in the same portable housing. Each packed bed column may include at least one of resin beads having C18 functional groups on their surface, cellulose beads having sulfate ester functional groups on their surface, and mixtures thereof. The resin beads and cellulose beads may have a nominal diameter of at least about 20 μm. The resin beads and cellulose beads may have a nominal diameter of about 40 microns to about 200 microns. A nominal flow rate drawn through each capture element using the pump may be at least about 20 L / min. The sample collection module may further include a water reservoir disposed in fluid communication with at least one of the water pump and the water container, and the nebulizer may be submerged in the water and disposed near an outlet of the water reservoir, and the nebulizer may be an ultrasonic mist nebulizer.

[0020] An exemplary system for detecting respiratory disease pathogens in ambient air is disclosed, the system including the system described above for sampling and trapping aerosol particles in ambient air, a sampling and extraction system for extracting trapped aerosol particles specific to respiratory disease from each packed bed column into one or more liquid samples, and an analyzer for analyzing the aerosol particles in the one or more liquid samples. The extraction system may include means for flushing each packed bed column with at least one solvent and collecting the solvent containing the aerosol particles from each packed bed. The at least one solvent may include at least one of acetonitrile, methanol, isopropanol (IPA), and the remainder may be water. The one or more solvents may include about 50% to about 70% acetonitrile in water by volume, and about 50% to about 70% isopropanol by volume in water. The analytical device may include at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS, and LC-MS / MS.

[0021] An exemplary method for detecting respiratory disease pathogens in ambient air in an enclosed space is disclosed, comprising selectively capturing aerosolized particles from exhaled breath in the ambient air using one or more sample capture elements each including a packed bed column, the ambient air being humidified before entering the one or more sample capture elements and drawn into the one or more sample capture elements using an air pump; extracting the captured aerosol particles specific to respiratory disease from each packed bed column into one or more liquid samples; and analyzing the aerosol particles in the one or more liquid samples. The packed bed column may include at least one of resin beads having C18 functional groups on their surface, cellulose beads having sulfate ester functional groups on their surface, and mixtures thereof. The extraction step may include flushing the packed bed column with at least one of acetonitrile, methanol, isopropanol (IPA), with the remainder being water. The exemplary method may further include a sample processing step, which may include mixing the one or more samples with a MALDI matrix and depositing the one or more mixed samples and MALDI matrix onto one or more sample plates. The exemplary method may further include a sample processing step, which may include subjecting the one or more liquid samples extracted from the sample extraction system to proteolytic digestion to generate peptide samples characteristic of the respiratory disease. The sample processing step may further include mixing the peptide samples with a MALDI matrix and depositing the mixed samples and MALDI matrix onto a sample plate. The analytical device may include at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS, and LC-MS / MS.

[0022] An exemplary sample capture element for capturing aerosolized bacteria and viruses is disclosed, which may include a packed bed column for selectively capturing aerosolized bacteria and virus particles in at least one of exhaled breath and ambient air drawn through the packed bed column using a pump, the packed bed column may include at least one of resin beads having C18 functional groups on their surface, cellulose beads having sulfate ester functional groups on their surface, and mixtures thereof, the resin beads and cellulose beads may have a nominal diameter of about 40 microns to about 200 microns, and the beads may be packed between two porous polymer frit disks in the packed bed column.

[0023] Other features and advantages of the disclosure will become apparent to those skilled in the art through examination of the following detailed description, which is set forth in part in the following description and the annexed drawings, in which preferred aspects of the disclosure are described and illustrated, and in part taken in conjunction with the accompanying drawings, in which: The advantages of the disclosure may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. [Brief description of the drawings]

[0024] The foregoing aspects and many of the attendant advantages of the present disclosure will be more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, in which: [Figure 1] FIG. 1 is a schematic diagram of an exemplary breath sample collection system having a packed bed column. [Diagram 2] FIG. 2 is a schematic diagram of an exemplary respiratory disease diagnostic system having a sample collection system. [Diagram 3] FIG. 3 is a schematic diagram of an exemplary diagnostic method using a system having a packed bed column. [Figure 4] FIG. 4 is a schematic diagram of an exemplary breath sample collection system including a packed bed column. [Diagram 5] FIG. 5 is a schematic diagram of an exemplary ambient aerosol sample collection system including a packed bed column. [Figure 6A] FIG. 6A shows the results of direct infusion mass spectrometry showing the ion intensity of MS2 capsid protein (A) in a control sample. [Figure 6B] FIG. 6B shows the results of direct infusion mass spectrometry showing the ion intensity of the MS2 capsid protein (B) in the trapped aerosol sample. [Figure 7A] FIG. 7A shows a selected ion chromatogram of MS2 capsid protein (A) in a control sample by Nanoflow-LC mass spectrometry. [Figure 7B] FIG. 7B shows the selected ion chromatogram of MS2 capsid protein (B) in the captured aerosol sample by Nanoflow-LC mass spectrometry. [Figure 8A] FIG. 8A shows the effect of C18 bead size (A) on the flow rate through an exemplary sample capture element. [Figure 8B] FIG. 8B shows the effect of bead volume (B) on the flow rate through an exemplary sample capture element. [Figure 8C] FIG. 8C shows the effect of inlet polymer frit pore size (C) on the flow rate through an exemplary sample capture element. [Figure 9A] FIG. 9A shows (A) a schematic diagram of an exemplary exhaled aerosol collection system for use with a ventilator connected to a patient diagnosed with COVID-19 in an intensive care unit. [Figure 9B] FIG. 9B shows a schematic diagram of an exemplary handheld accessory and control system configured to operate an aerosol collection system connected to a ventilator (B). [Figure 10] FIG. 10 is a schematic diagram of an exemplary aerosol collection system for ambient air sampling. [Figure 11A]FIG. 11A shows PCA and PLS-DA analysis showing separation of lipid and metabolite biomarkers between breath samples and blanks collected from 14 subjects (A). [Figure 11B] FIG. 11B shows the dynamic range distributions (B) with and without data filtering for both lipid and metabolite biomarkers in exhaled aerosol samples. [Figure 11C] FIG. 11C shows the ion intensities (C) of lipid and metabolite biomarkers with and without CO2 normalization. [Figure 12A] FIG. 12A is an isometric view (A) of an exemplary aerosol sample collection system for ambient air sampling showing the fluidic module and the sample collection module. [Figure 12B] FIG. 12B is an isometric view of the fluidic module (B). [Figure 12C] FIG. 12C is an isometric view (C) of the fluidic module. [Figure 12D] FIG. 12D shows a schematic diagram of mist generation using a fluidic module. [Figure 13A] FIG. 13A is the number of proteins identified using bottom-up proteomics of the four ambient air samples (A). [Figure 13B] FIG. 13B shows keratin 9 levels (B) in the four air samples. [Figure 14] FIG. 14 shows the results of protein blue staining of aerosolized BSA protein captured using an exemplary C18 packed bed column sample capture element and button sampler. [Figure 15A] FIG. 15A shows a MALDI TOF mass spectrum (A) of heat acid digested peptides in the medium of a SARS-CoV-2 virus sample that was not treated with an exemplary sample capture element. [Figure 15B] FIG. 15B shows a MALDI TOF mass spectrum (B) including peak assignments of heat acid digest peptides of SARS-CoV-2 viral particles extracted from an exemplary sample capture element. [Figure 15C] Figure 15C shows the distribution of peptide ion intensities (C) constructed using bottom-up proteomics of SARS-CoV-2 samples. [Figure 15D] FIG. 15D shows the distribution of peptide ion intensities (D) constructed using bottom-up proteomics of SARS-CoV-2 viral particles extracted from an exemplary sample capture element. [Figure 15E] Figure 15E shows peptide mass fingerprints (E) of SARS-CoV-2 viral particles extracted from exemplary sample capture elements using Mascot database searches with various numbers of allowed deletion site cleavage sites.

[0025] All reference numbers, identifiers, and callouts in the figures are incorporated herein by this reference as if fully set forth herein. Failure to number elements in the figures is not intended as a waiver of any rights. Unnumbered references may also be identified by the alphabetical letter of the figure or appendix.

[0026] The following detailed description includes references to the accompanying drawings, which form a part of the detailed description. The drawings show, by way of illustration, specific embodiments in which the disclosed systems and methods may be practiced. These embodiments, which should be understood as "examples" or "options," are described in sufficient detail to enable one skilled in the art to practice the invention. The embodiments may be combined, other embodiments may be utilized, or structural or logical changes may be made without departing from the scope of the invention. Therefore, the following detailed description is not to be taken in a limiting sense, and the scope of the invention is defined by the appended claims and their legal equivalents.

[0027] In this disclosure, aerosol generally means a suspension of particles dispersed in air or gas. "Autonomous" diagnostic systems and methods mean that they generate diagnostic test results "without or minimal intervention by a medical professional." The U.S. FDA classifies medical devices based on the risk associated with the device and by assessing the amount of regulation that would reasonably ensure the safety and effectiveness of the device. Devices are classified into one of three regulatory classes: Class I, Class II, or Class III. Class I includes devices with the lowest risk, while Class III includes devices with the highest risk. All classes of devices are subject to general regulation. General regulation is a basic requirement of the Food, Drug, and Cosmetic (FD&C) Act that applies to all medical devices. In vitro diagnostic products are reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including the determination of health status, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. Exemplary devices disclosed herein are capable of operating autonomously and producing reliable results, and therefore have the potential to be regulated as Class I devices. In some parts of the world with a high burden of TB infection, access to medically trained personnel is very limited. Autonomous diagnostic systems are preferred over non-autonomous diagnostic systems.

[0028] In this disclosure, the singular (equivalent to the English terms "a" or "an") is used to include one or more, and the term "or" is used to refer to a non-exclusive "or" unless otherwise specified. Furthermore, it is to be understood that expressions or terms used herein and not otherwise defined are for descriptive purposes only and not for limiting purposes. Unless otherwise specified in this disclosure, for purposes of interpreting the scope of the term "about," the margin of error associated with the disclosed values ​​(dimensions, operating conditions, etc.) is ±10% of the value stated in this disclosure. The margin of error associated with values ​​disclosed as percentages is ±1% of the stated percentage. The word "substantially" used before certain words includes the meanings "a significant portion of the specified range" and "most but not all of what is specified." Unless otherwise specified, concentrations of chemicals, solvents, etc. disclosed as percentages refer to volume %. Detailed Description

[0029] Exhaled aerosol particles include various non-volatile organic biomolecules such as metabolites, lipids, and proteins. Furthermore, non-volatile molecules have a wide particle size distribution, ranging from submicron size to about 10 microns in size. There is a need for a breath collection and disease diagnosis system and method that can efficiently capture various types of non-volatile molecules of various particle sizes from exhaled breath. Particular aspects of the invention are described below in considerable detail for the purpose of explaining the construction, principles, and operation of the disclosed method and system. However, various modifications can be made, and the scope of the invention is not limited to the exemplary embodiments described.

[0030] An exemplary exhaled breath analysis ("EBA") based diagnostic system 2000 (FIG. 2) can include an exhaled breath sample collection system 1000 disposed in fluid communication with a sampling system 2002 and an analysis system 2003.

[0031] An exemplary breath sample collection system 1000 (FIG. 1) may include a sample capture element 1001 with a packed bed column to selectively capture exhaled aerosols, including non-volatile organisms (including but not limited to bacteria and viruses) and molecules (including small molecules, lipids, and proteins), onto a highly efficient adsorbent material. The trap 1003 is in fluid communication with the column 1001 using tubing 1002. The trap 1003 may be made of glass or plastic material. The trap 1003 may be cooled to below ambient temperature using an ice bath or other suitable means. The trap 1003 may be used to collect water vapor and other volatile (chek) and non-volatile molecules that may pass through the collection column as exhaled breath condensate (EBC). During breath analysis of a patient's breath using a ventilator, the sample capture element 1001 is removably connected to a capnography port of the respirator tube of the ventilator and positioned close to or at the outlet from the patient's lungs. During breath analysis of a normally breathing person, the element 1001 may be removably connected to a mouthpiece (not shown) through which the patient is instructed to breathe or may perform a breathing maneuver as previously disclosed herein. For example, the capture element 1001 may be removably coupled downstream (at the outlet) of an exhaled breath collection element 1007 (FIG. 1), e.g., a first aid CPR rescue mask (e.g., model number EVR-CPR01, supplied by Dixie USA EMS Supply Co.), worn by the patient during breath analysis. A flow splitter 1008 may be disposed between the exhaled breath collection element 1007 and the capture element 1001 to split the flow of exhaled breath so that a first portion of the exhaled breath is directed to the capture element 1001 and a second portion is directed to a HEPA filter 1009. The flow splitter 1008 may be integrated into the collection element 1007. Additionally, a large particle trap 1012 may be positioned upstream of the capture element 1001 to remove large particles (larger than about 10 μm) of exhaled breath condensate from the exhaled breath stream before it enters the capture element 1001. A pump 1006 may be used to draw exhaled breath 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 the pump 1006 can be adjusted using a needle valve 1005 to achieve a desired flow rate. A check valve (one-way flow valve) 1011 can be placed between the pump 1006 and the capture element 1001 and is configured to be in an open position only when the pump 1006 is drawing breath through the packed bed column. When there is no flow, the valve 1011 is placed in a closed position. A nominal flow rate of about 200 ml / min to 600 ml / min can be used. Additionally, several capture elements 1001 can be used in parallel to increase the flow rate up to 12 L / min. Additionally, when one or more capture elements are in collection mode, one or more can be in elution mode and some can be in standby mode. CO2 can be introduced between the breath collection element 1007 and the sample capture element 1001 to determine if the breath sample volume was adequate. 2 Sensors and particle counters (not shown) may be provided. 2 Monitoring and particle counting allows an approximation of the percentage of exhaled air volume. A HEPA filter may be placed downstream of the trap 1003. The trapping element 1001 may be cooled using a cooling jacket or other means to reduce its temperature below ambient temperature to increase the efficiency of collection of non-volatile organic particles. The breath sample collection system may further comprise a humidifier 1010 placed upstream of the inlet to the trapping element to humidify the exhaled air and increase humidity within the packed bed column.

[0032] The breath collection element 1007 may comprise a tight-fitting mask configured to receive the individual's face and may be removably attached to the patient / 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 ambient air of the testing room or area. The element 1007 may be used to collect and direct the respiratory aerosol particles emitted from the patient's mouth and nose to the capture element 1001 using pump 1006 as described above, without depositing the aerosol particles on the walls of the element 1007. The element 1007 may be disposable to limit the risk of the patient becoming contaminated or infected with pathogens emitted by previous patients. Alternatively, the element 1007 may be reusable, in which case it may be sterilized.

[0033] An exemplary packed bed column in the capture element 1001 can contain Hamilton PRP-C18 resin beads supplied by Sigma Aldrich and other vendors. The bed can be held in place between two porous filter plates, such as fritted disks. For example, a polyethylene disk with an average pore size of more than 35 μm can be placed upstream of the bed, and a polyethylene disk with an average pore size of 10 μm (Boca Scientific, Dedham, MA) can be placed downstream of the bed. The 35 μm fritted disk allows for a faster air flow rate, while the smaller 10 μm fritted disk better traps all the C18 resin. In the exemplary element 1001, the packed bed can contain about 25 mg of C18 resin beads with a nominal diameter between about 12 μm and about 20 μm. Non-volatile organic components in the breath interact with the C18 functional groups on the beads and are removably captured. Water, volatiles, and other hydrophilic molecules can pass through the bed and be trapped in the glass trap 1003.

[0034] In addition to the C18 functional group, other functional groups that exhibit an affinity for non-volatile molecules may be used as adsorbents in columns immobilized on solid phase beads, such as resin beads. Solid phase beads can be resins, cellulose, silica, agarose, and hydrated Fe. 3 O4 The adsorbent material may be made of polymers and particles, such as nanoparticles. The adsorbent material may have other functional groups, including, but not limited to, octadecyl, octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethyl-aminopropyl, carboxypropyl, benzenesulfonic acid, and propylsulfonic acid, disposed on the solid phase beads. The functional groups may also include at least one of an ion exchange phase, a polymer phase, an antibody, a glycan, a lipid, DNA, and RNA.

[0035] An exemplary diagnostic system 2000 (FIG. 2) can include a breath sample collection system 2001 disposed in fluid communication with a sample extraction system 2002 and an analysis system 2003. The sample collection system 2001 can include the exemplary sample collection system 1000 (FIG. 2), previously described. The sample extraction system 2002 can be used to extract trapped non-volatile organics from the packed bed column of the system 1000 and can be deployed in-line or offline in the system 2000. If the system 2002 is deployed offline, at the end of breath sample collection, the capture element 1001 can be removed from the system 1000 and eluted with an organic solvent in the extraction system 2002 to remove non-volatile organics from the packed bed column. Exemplary organic solvents include, but are not limited to, about 50-70% acetonitrile in water for extracting trapped non-volatile organics (strong polar non-volatile organic molecules, proteins, etc.) from the packed bed column. The extraction may be repeated using the same or other solvents to extract less polar lipid molecules from the packed bed, including, 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 the system 2002 is placed in-line, CO 2At least one of the sensor and particle counter can be located upstream of the extraction system 2002. The system 2002 can include a solvent reservoir, a pump to transfer the solvent from the solvent reservoir to the packed bed column, and a container to collect the solvent containing the non-volatile biomarkers in another container or cup. Alternatively, the system 2002 can include an injector to inject the solvent into the packed bed column and collect the extract containing the non-volatile organics and biomarkers in a suitable cup or container or other laboratory tube having a small volume. The captured sample in the solvent can be further processed and analyzed in the analysis system 2003.

[0036] The analytical system 2003 can include a sample processing system 2004 and at least one diagnostic device 2005. The sample processing system 2004 can include the necessary components to perform one or more of the following steps:

[0037] (a) Placing a sample in at least one of a cup, a vial, and a sample plate. For example, the Series 110A Spot Sampler (aerosol device) uses a 32-well plate with a circular well geometry (75 μL well volume) or a teardrop well geometry (120 μL well volume), which is heated to evaporate the solvent and excess liquid / fluid to concentrate the sample. (b) placing the sample in a cup and exposing it to a vacuum source or a freeze-drying device to evaporate the solvent and concentrate the sample; and; (c) High temperature digestion step of proteins and virus particles.

[0038] The sample may be centrifuged to remove chemical contaminating particles. Many diagnostic devices may be adapted for use in the analytical system 2003, including but not limited to devices that perform genomics-based assays (e.g., PCR, rt-PCR, whole genome sequencing, etc.), biomarker recognition assays (e.g., ELISA, etc.), and spectral analysis such as mass spectrometry (MS). Among these diagnostic instruments, MS is preferred for its speed of analysis. 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 coupled with a high-resolution mass spectrometer. MALDI-TOFMS devices are compact and lightweight, consume less than 100 watts, 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 a laser pulse from an ultraviolet laser. This interaction between the sample and the laser produces large, informative, biological ion clusters that are characteristic of biological materials. If a concentrated sample having only trace levels of water or only trace levels of organic solvents, such as 50%-70% of either acetonitrile, methanol, and isopropanol in water, is provided by the sample processing system 2004, sample analysis using an MS will take less than 5 minutes (including sample pretreatment) because of the short time required to evaporate the water from the sample.

[0039] MALDI-TOFMS may be used to identify live / active agents including, but not limited to, Bacillus anthracis spores (strains), Y. aureus enterotoxin (SEA), Staphylococcus aureus enterotoxin B (SEB), ricin, abrin, Ebola Zaire strain, aflatoxin, saxitoxin, conotoxin, Enterobacteriaceae phage 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, fentanyl. Furthermore, the exemplary systems and methods disclosed herein can be used to achieve accurate detection and identification of SARS-CoV-2 from human breath samples.

[0040] In "matrix-assisted laser desorption / ionization" (MALDI), target particles (analytes) are coated with a matrix chemical that preferentially absorbs light from a laser (often ultraviolet wavelengths). In the absence of a matrix, biological molecules break down by pyrolysis when exposed to a laser beam in a mass spectrometer. The matrix chemical also transfers charge to the vaporized molecules, generating ions, which are accelerated down a flight tube by an electric field. Microbiology and proteomics have become major application areas of mass spectrometry. Examples include bacterial identification, discovery of chemical structure, and derivation of protein function. MALDI-MS has also been used for lipid profiling of algae. In MALDI-MS, a liquid, usually 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 suppress the effect of salt impurities on the mass spectrum of the sample. 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 the MALDI plate to obtain a uniform, homogenous 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 decomposes the cell membrane of the sample, making the proteins available for ionization and analysis in the 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. Pat. No. 8,409,870.

[0041] Additionally, the volatile organic compounds collected in trap 1003 (FIG. 1) can be warmed using a heater and swept to a diagnostic device that performs GC-MS, GC-IMS, volatile ion chromatography, or other type of analytical method suitable for analyzing volatile organic compounds.

[0042] Detection of viruses (e.g., SARS-CoV-2) centers on the detection of viral proteins, which is a challenging task. An exemplary method for virus detection may include a glycan-based capture matrix (beads) to extract the target virus from the background matrix (e.g., other non-viral biomolecules, contaminants). An aliquot of a sample that may contain the virus is collected, for example, using a sample collection system 1000, which may also contain other background contaminants, which may be deposited onto beads carrying capture probes. At least one of glycans, heparin, and carbohydrates may be used as capture materials or probes bound to resin beads or some other type of beads. An optional wash step may be used to remove non-target viral contaminants. The concentrated and purified virus may be eluted from the beads using a suitable solvent into an enclosed heating chamber containing an organic acid, which may include formic acid or acetic acid, and heated to about 120° C. for about 10 minutes to digest proteinaceous toxins into specific peptide fragments. This high temperature acidic protein digestion protocol cleaves proteins at aspartic acid residues, generating highly reproducible peptide patterns. The capture and digestion processes described here may be accomplished with antibodies and enzymes, respectively. Using this MALDI-TOFMS sample processing example, sensitivity for ricin biotoxin of greater than 100 ng / mL (with a S / N of approximately 50:1) was achieved in neat buffer. At a S / N (signal to noise ratio) of 3:1, a limit of detection (LOD) of less than 10 ng / mL may be achieved. For a 1 μL sample used in a MALDI-TOFMS analysis system, an LOD of approximately 10 ng / mL corresponds to approximately 10 pg (10 ―12g), which corresponds to a total mass of approximately 20,000 virus particles. An exemplary microfluidic sample processing system for carrying out the methods disclosed above can be configured to analyze samples collected from the air or from other sources such as nasal swabs. The glycan-based capture column and other microfluidic components can be reused. Large liquid reservoirs containing buffers, weak acids, and alcohols can be used to provide sufficient capacity to measure hundreds of samples in one channel of the system. Multiple systems can be run in parallel to process multiple samples simultaneously. The system is cost-effective because it does not require fragile and expensive biomolecular reagents.

[0043] Hot acid digestion reproducibly cleaves proteins at aspartic acid residues, creating known peptide sequences with known masses. These peptide mass distributions are characteristic of precursor proteins. Thus, digestion provides excellent specificity if the protein of interest is highly divergent from background material. Furthermore, the mass distribution of peptides is directly determined by the genome, taking into account post-translational modifications. As soon as a new virus is isolated, its sequence is rapidly determined. The RNA sequence of the SARS-CoV-2 virus can be used to accurately predict protein sequences using modern bioinformatics tools (ExPASy bioinformatics portal). These proteins can be "digested" in silico (with a computer) using bioinformatics tools to create theoretical peptide maps. Thus, peptides resulting from SARS-CoV-2 digestion can be predicted and compared with experimental data to generate a specific MALDITOFMS signature of the organism. Reports suggest that the major proteins of SARS-CoV are characterized by a nucleocapsid protein of approximately 46 kDa and a spike protein of 139 kDa. Other proteins in reasonable abundance are the E, M, and N proteins.

[0044] The detection specificity of the target virus requires some degree of 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 mentioned earlier, the use of affinity capture probes for viral toxins based on glycan-modified agarose beads allows easy cleanup of the toxins, even when background proteins and other biomolecules are present in excess. When analyzing breath for viral targets such as SARS-CoV-2, other human proteins in the breath may interfere with the specificity of the detection. To ensure the highest specificity, affinity-based sample cleanup is necessary. For viral detection, bead materials that offer more selective affinity compared to the glycan-modified beads mentioned above may be required. For example, dextran-based adsorbents may be used for the purification of viruses, including coronaviruses, but the affinity of this resin for the target virus 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. Additionally, heparin and heparan sulfate may be used as binders to bind 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 in place of glycan capture beads. This resin may enable a bead-based capture affinity capture system to collect virus particles from exhaled breath. In an exemplary diagnostic system, a breath sample may be drawn through a capture bed in the 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 to the beads may then be eluted using a high concentration acid solution, such as at least one of about 12.5% ​​acetic acid, about 5% TFA, about 5% formic acid, and about 10% HCl, and sent to a high temperature acid digestion chamber to generate characteristic peptides.Peptide samples may be mixed with a MALDI matrix and deposited as a suitable substrate for MALDI TOFMS analysis. Samples may be deposited on a suitable substrate or disc pre-coated with the MALDI matrix.

[0045] FIG. 3 is a schematic diagram of an exemplary diagnostic method 3000 using the exemplary system 2000. The exemplary method 3000 may be used to perform autonomous point-of-care diagnostics based on exhaled breath. In step 3001, an individual (i.e., a patient) may be instructed to sit. The chair may be placed in a containment booth, if necessary. In step 3002, a sample breath collection element 1007 may be removably attached to the individual's head. The individual is then instructed to take a breath or perform one or more predefined maneuvers 3003, which may include a pre-set number of repetitions. Non-volatile organics in the breath are captured using the system 1000 and extracted in step 3004 using the system 2002 and eluted using a suitable solvent. During sample collection, the human breath passes through a column at a pre-defined flow rate, which is drawn by a suction pump. Non-volatile molecules in the breath interact with the functionalized beads (e.g., C18 functional groups immobilized on resin beads) of the capture element 1001, so that these molecules are captured in the column bed of the element 1001, while hydrophilic molecules, mostly consisting of water and aqueous electrolytes in the breath, pass through the column. Non-volatile organic molecules in human breath show a strong affinity for alkyl chains through 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, including but not limited to acetonitrile, methanol, and isopropanol as mentioned above. In step 3005, the sample may be further processed using component 2004. The type of sample processing depends on the type of diagnostic device and the non-volatile analyte particles of interest. As mentioned above, the virus sample may be treated in a hot 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 be deposited on a suitable substrate or disk pre-coated with a MALDI matrix. The sample is then analyzed by the diagnostic device in step 3006.If the diagnostic device is a MALDI-TOFMS, sample processing may also include plating the sample onto a MALDI-TOFMS sample disk, heating the disk to concentrate the sample, and drying the disk. The sample disk is analyzed using the MALDI-TOFMS. The TOFMS detector may be modified to incorporate an ion gate and reflectron to allow analysis and sequencing of fragmented COVID-19 viral peptides during MALDI-TOF / MS. The resulting spectrum is compared to spectra of samples known to be positive for a particular respiratory infection, spectra of 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 patient.

[0046] Once the breath collection element 1007 is attached to the patient and sampling begins, the exemplary system and method is preferably autonomous (except for asking the patient to leave the chair after performing necessary operations) and produces a diagnostic test result on its own. For viral particles such as SARS-CoV-2, the particle diameter is about 0.1 microns, and the sensitivity is about 10 3 From 10 4 It may be between two virus particles.

[0047] The report said that analysis of nasal and throat swabs from influenza and COVID-19 patients revealed that approximately 10 3 From 10 10 The results suggest that influenza patients produce 10 ... 4 If the SARS-CoV-2 output is similar to that of influenza, then it is possible that the exhaled particles are greater than 10 3 From 10 4A particle output and particle collection efficiency of greater than 99.9% should be sufficient to identify target virus particles in exhaled breath using the exemplary techniques and systems disclosed herein. Detection times using the exemplary systems and methods will be between about 10 and 20 minutes, including the steps of sample extraction (breathing), sample collection, sample processing (digestion), and analysis using MALDI TOF-MS. This detection time is very fast compared to existing detection systems.

[0048] An exemplary sample processing component can include a thermal acid digestion module or cartridge to autonomously extract sample from the packed bed column 1001, perform sample cleanup, perform thermal acid digestion, and provide the sample ready for plating onto a MALDI-TOFS sample substrate or disk. The cartridge can be designed to be reusable with the addition of the ability to flush the cartridge after each use.

[0049] Another exemplary sample collection system 7000 (FIG. 4) is disclosed. The exemplary sample capture element 7001 can include a packed bed column containing C18-bonded resin beads. These resin beads have C18 functional groups immobilized on the surface. The capture element 7001 can be connected or removably attached 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, can be modified to removably connect to a HEPA filter 7009. The HEPA filter prevents contamination of exhaled air with contaminants from ambient air. An oxygen inlet 7010 to the mask, typically located below the stem and configured to be adjacent to the chin of a human subject when the subject wears the mask, can be modified to removably connect to the capture element 7001. The element 7001 can be removably inserted into the mask 7007 through the inlet 7010 or can be removably connected or inserted into the mask 7007 to form a substantially leak-free fit 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 the mask fits snugly against the patient's face. The mask 7007 configured as described above prevents direct contact between the mouth of the element 7001 and the inlet of the column, minimizing or eliminating contamination of the column inlet with saliva, and also maximizing collection of non-volatile organic particles from the exhaled air. A trap 7003 submerged in ice water may be placed after (downstream of) the capture element 7001. The flow rate (air aspiration rate) using the pump 7006 may be controlled using a needle valve 7005 and may aspirate 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 fluidic components, such as a check valve (see FIG. 1 ), may be installed in the system 7000 to prevent backflow of the element 7001 into the column bed. CO in the exhaled air 2 CO passes through the column bed of element 7001. CO is then pumped between the outlet of breath capture element 7001 and trap 7003 to determine whether the volume of the breath sample and / or the breath maneuver is adequate. 2Sensors can be placed. 2 Monitoring allows approximation of the percentage of exhaled air volume. A particle counter can be placed between the outlet of element 7001 and trap 7003 to detect the size and number of particles exiting the column bed, and can also be used to detect saturation of the column bed and breakthrough of non-volatile organic molecules from the column bed. The exemplary system 7000 can also include a bypass line (not shown) of the trapping element 7001 to allow normalization of the breath volume before routing to the column bed of element 7001. 2 Sensors and particle counters may also be fluidly connected to the bypass line. The capacity of the functionalized solid beads in the column bed of the capture element 7001 to capture non-volatile organic molecules may be between about 0.05 mg (non-volatile organic) / mg beads and about 0.5 mg / mg. The capacity of C18-bonded resin beads in the column bed of the exemplary capture element may be about 0.1 mg / mg. That is, a column bed with 25 mg of C18 beads will have a capacity to capture or adsorb about 2.5 mg of non-volatile organic molecules.

[0050] In addition to the C18 functional group, other functional groups that exhibit an affinity for non-volatile molecules may be used as adsorbents in columns immobilized on solid phase beads, such as resin beads. Solid phase beads can be resins, cellulose, silica, agarose, and hydrated Fe. 3 O 4 The adsorbent material may be made of polymers and particles, such as nanoparticles. The adsorbent material may include other functional groups disposed on the solid phase beads, including, but not limited to, octadecyl, octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl, 2,3-dihydroxypropoxypropyl, trimethyl-aminopropyl, carboxypropyl, benzenesulfonic acid, and propylsulfonic acid. The functional groups may also include at least one of an ion exchange phase, a polymer phase, an antibody, a glycan, a lipid, DNA, and RNA.

[0051] The exemplary systems and methods described herein are not necessarily limited in their diagnostic capabilities for respiratory infections. For example, lung cancer may also release biomarkers into the peripheral lung fluid, and these biomarkers may be easily detected by the disclosed systems and methods. Furthermore, because blood is in close contact with the alveolar lining of the lungs, biomarkers for infections or cancers in other parts of the body (other than the lungs) may migrate beyond the alveolar lining into the peripheral lung fluid, which may be detectable by analysis of EBA. As a result, the scope of the invention is not limited to the detection and diagnosis of respiratory diseases. The exemplary systems and methods may be used to capture aerosolized chemical particles, such as ricin, and analyze the particles to prevent the threat of chemical attack.

[0052] In the exemplary systems and methods described herein, the length (L) of the packed bed column in the sample capture element 1001 is about 3 mm. The nominal inner diameter of the tube is about 7 mm (D). An exemplary packed bed containing about 25 mg of C18 resin beads having a nominal particle size (Dp) of about 12 μm to 20 μm results in an L / Dp ratio of about 150 to 250 with a D / Dp ratio of about 350 to about 580. These column parameters have been found to prevent undesirable local flow distributions within the bed and ensure that substantially all of the resin beads are exposed to the aerosol flow through the bed.

[0053] Exemplary systems and methods are disclosed for capturing (or collecting) ambient aerosol particles for diagnosis of at least one respiratory disease. An exemplary aerosol collection system 900 (FIG. 5) can include an ambient aerosol discharge chamber 901 and a sample capture element 902, preferably fluidly and removably connected directly to the discharge chamber. The capture element 902 has a packed bed for selectively capturing non-volatile organic components, such as bacteria and virus particles, in the aerosol. A pump 903 is disposed in fluid communication with the sample capture element and configured to draw an aerosol flow through the sample capture element. The particle capture efficiency of the collection system 900 can be at least about 99% for particle sizes ranging from about 0.3 μm to about 10 μm, which covers the range of most organic particles in ambient air. Although naked virus particles are very small in size, often on the order of 100 nm, exhaled respiratory particles (exhaled aerosol, EBA) that may constitute virus particles collected from ambient air (e.g., SARS-CoV-2 virus causing COVID-19) vary in size. ) are often measured to range in size from about 100 nm to about 5 μm. Furthermore, a significant portion of the aerosol mass is composed of particles larger than about 2 μm in diameter. Virus particles are typically suspended in aqueous lung fluid that contains water, surfactants, proteins, salts, and other chemicals. Particle generation is greatest during conversation and other activities that cause deep breathing. After these particles are exhaled, they typically shrink due to loss of water. If then captured using the exemplary ambient aerosol collection system 900, the majority of the EBA mass is expected to contain particles between about 1 μm and about 5 μm in size. Prior to use, the packed bed column may be washed (or flushed) with about 70% acetonitrile (ACN), followed by about 0.05% trifluoroacetic acid (TFA). About 400 μL of 70% acetonitrile and about 400 μL of 0.05% trifluoroacetic acid (TFA) may be used to wash an exemplary column containing about 25 mg of C18 beads. Additionally, the exemplary column may be washed once with 70% ACN and three times with 0.05% TFA. The exemplary column is kept moist or wet prior to use.For a packed bed column containing about 25 mg of C18 beads, about 100 μL of 0.05% TFA may be used to load the beads after the wash step. To avoid freezing, the column may be preferably capped on both ends and stored at about 4° C.

[0054] In other exemplary aspects, multiple packed bed columns may be configured as a bundle of columns, e.g., a sample collection cartridge. The cartridge may have a suitable inlet distribution element disposed in fluid communication with the aerosol sample flow to distribute the aerosol sample substantially uniformly to the multiple packed bed columns as the flow is drawn through the cartridge using a pump. The cartridge may have a suitable outlet distribution element disposed in fluid communication with the pump to draw the aerosol sample through the multiple packed bed columns. The exemplary sample collection cartridge may be used to capture the aerosol sample using methods and apparatus for high air flow rates (e.g., about 200 L / min to about 500 L / min) collection of exhaled aerosol present in room air. These methods and devices may be combined with MALDI-TOFMS and sensitive and specific on-site genomic analysis suitable for active case finding (ACF) of diseases such as COVID-19 from air samples, as disclosed in commonly owned U.S. Patent Application No. 17 / 586,679, entitled "Respiratory Disease Surveillance System and Method Using High Flow Aerosol Capture for Rapid Analysis on-site," the disclosure of which is incorporated herein by reference in its entirety. Alternatively, exemplary sample collection systems including at least one packed bed column or capture element may be arranged in parallel with each other to selectively capture non-volatile particles in the exhaled aerosol in each column or capture element. Each packed bed column may have about 200 mg of C18 resin beads. One or more appropriate pumps downstream of the capture element may be selected to provide the required flow rate for each packed bed column. An exemplary flow rate of about 20 L / min can be passed through each column of the aerosol sample to achieve a capture capacity of about 10% by weight (20 mg non-volatile particles / 200 mg C18 beads) in each bed. A sample capture element having four beds arranged parallel to one another can provide a total flow rate of about 80 L / min through the four beds. The number of beds can be scaled up or down to achieve a desired aerosol sample flow rate through the sample capture element.For example, ten of the exemplary packed bed columns can be arranged parallel to one another in a bundle or cartridge to process approximately 200 L / min of aerosol sample through the cartridge. Reducing the amount of C18 beads in each bed can reduce the bed length and potentially increase the flow rate through each bed as a result of reducing the pressure drop through each bed.

[0055] As described above, a source of an aerosol sample containing non-volatile organic aerosol particles in exhaled breath may include an exhaled breath sample collected using an exhaled breath collection element, such as a CPAP mask configured to receive an individual's face. The exhaled aerosol particles may then be drawn through a packed bed column containing C18 beads to capture the non-volatile exhaled breath particles. An exemplary sample capture element with a packed bed column may be used to capture non-volatile organic particles from exhaled breath present in ambient air of a target area, such as ambient air in a hospital or clinic waiting room, ambulance, operating room, or intensive care unit. An exemplary aerosol collection system 1400 (FIG. 10) is disclosed for collecting non-volatile organic particles in ambient air present in a room, such as an operating room in a hospital. The non-volatile particles may include exhaled breath aerosol particles dispersed in ambient air and may include at least one of microorganisms, viruses, metabolite biomarkers, lipid biomarkers, and proteomic biomarkers characteristic of respiratory disease. The system 1400 can include at least one sample capture element 1407 having a packed bed column of C18 resin beads to selectively capture non-volatile particles as ambient air is drawn (or pulled) through the at least one element 1407 using a suitable pump 1406. The capture element 1407 can be removably disposed within the housing 1401 using quick connect / disconnect couplings or fittings at the inlet end 1408 and outlet end 1409. Exemplary quick connect / disconnect couplings can include plug and socket couplings (latch lock sockets, twist lock sockets, etc.) supplied by McMaster-Carr (Cleveland, Ohio). The pump 1406 can be disposed within the housing 1401 and driven using power from the power supply 1402. The couplings and mating fittings within the housing 1401 can have electrical contacts to detect appropriate mechanical and electrical connections / contacts and alert a user, for example, via a graphical user interface, which can use an interface disposed on the enclosure 1401, such as via an audible alarm.Alternatively, the pump 1406 may be located outside the housing 1401 and may include a room suction machine or pump powered by an external power source. The aerosol collection system 1400 system may further include a nebulizer element 1404 that humidifies the ambient air before it enters the collection element 1407 to increase the humidity in the packed bed column to about 95% RH, which may increase the particle capture efficiency of the element 1407. Alternatively, the nebulizer 1404 may be in the form of a sprayer or humidifier that converts water into a fine spray or mist. Water stored in a container 1405 may be supplied to the element 1404 using a pump 1403. The power source 1402 may provide power to operate the pump 1403 and the nebulizer 1404. An exemplary nebulizer is the Aeroneb lab micropump nebulizer and control module (Aerogen, Ireland). Exemplary pumps include diaphragm pumps and peristaltic pumps. The diameter of the trapping element 1407 can be between about 10 mm and about 20 mm. The inner diameter of the trapping element 1407 can be about 16 mm. The packed bed can include C18 resin beads with a nominal particle size of about 75 μm to about 150 μm. The bed weight of each trapping element can be about 200 mg, and the bed length can be between about 3 mm and about 5 mm. The C18 particles can be packed between an upstream porous polymer frit disk and a downstream porous polymer frit disk. The pore sizes of the upstream and downstream disks can be about 90 μm and 35 μm, respectively. The dimensions of the exemplary housing 1401 can be about 11 inches by 11 inches by 5 inches (length by height by depth). As described in Example 8, greater than 99.8% of the ambient air particles were captured using the trapping element 1407 of the system 1400, suggesting that the particle capture efficiency was at least 99% even at high gas hourly space velocities of about 2×106 per hour. Alternatively, the water pump 1403, the pump 1406, the power supply 1402, the nebulizer 1404, and the water container 1405 may be located outside the housing 1401, for example in a separate housing or subsystem to reduce the size of the housing 1401, such that essentially no noise is generated from the housing 1401.In this exemplary aspect, the housing 1401 can include one or more sample capture elements 1407 and can be placed near a patient in a hospital operating room.

[0056] In another exemplary aerosol collection system 1400A (FIGS. 12A-D) for collecting non-volatile organic particles in ambient air present in a room, the water pump 1403, the water container 1405, the power module 1402, and the air pump 1406 (not shown) can be disposed in a fluidic module 1410. The water container 1405 can have a water capacity of about 1.3 liters. The module 1410 can be a Pelican case or other suitable enclosure that is portable or easily portable. Input power can be provided via a cable 1411 that can be removably connected to a wall electrical outlet (120 VAC). The module 1410 can also have an on / off power switch 1412. The module 1410 is configured to be in fluid communication with the sample collection module 1413 using suitable flexible tubing. The modules 1410 and 1413 can be disposed near each other or at a distance from each other. For example, the module 1413 may be located in an operating room (OR) of a hospital, but the fluidics module may be located outside the room or in a remote location to minimize noise generated by the pump in the module 1410. Water in the container 1405 is transferred to the reservoir 1414 using the pump 1403. The reservoir 1414 may be configured to hold a predetermined volume of water, with excess water spilling back into the container 1405. The nebulizer 1404 may be located near the outlet of the reservoir 1414. The nebulizer 1404 may be an ultrasonic mist atomizer (e.g., manufactured by DHTS), which is powered by a 3.7-12 VDC power supply or a battery. The atomizer may be powered by a power supply located in the collection module 1413. The nebulizer 1404 may be 20 mm in diameter and may operate at approximately 113 kHz. The water mist generated by the nebulizer 1404 is delivered through a conduit 1415 to a manifold 1416. The manifold 1416 can have multiple outlet ports 1417 located proximate to an air sample inlet port 1418 in a second manifold 1419. The manifold 1419 can support multiple sample captures. Details regarding the exemplary sample element 1407 are described above.During sample collection, water mist is continually generated to humidify the ambient air that exits through port 1417 and is drawn into the capture element 1407 through port 1418 using air pump 1406 (FIG. 10). Any water that condenses from the mist within the collection module 1413 (condensation exiting the sample capture element) is collected in a reservoir 1420 appropriately positioned below the collection module 1413.

[0057] The packed bed in each capture element 1407 is kept moist during sample collection as ambient air is drawn into each capture element using pump 1406 at a flow rate of about 20 L / min. Pump 1403 can deliver water to reservoir 1414 at about 1.3 ml / min. The water flow rate can be between about 1 ml / min and about 2 ml / min. At this flow rate, and using a starting water volume of about 1.3 liters, fluidics module 1410 can deliver water mist to capture element 1407 for about 16 hours. After sample collection, the capture element is removed from collection module 1413 and the aerosol particles are extracted using a suitable organic solvent. The organic solvent can include at least one of acetonitrile (ACN), methanol, and isopropanol (IPA), with the remainder being water. The solvent can include about 50% to about 70% acetonitrile by volume in water to elute polar particles such as small metabolites and proteins from the bed. Other solvents may include about 50% to about 70% by volume isopropanol in water to elute non-polar particles such as lipids from the bed. The diameter of the capture element 1407 may be between about 10 mm and about 20 mm. The inner diameter of the capture element 1407 may be about 16 mm. The packed bed may include C18 resin beads with a nominal particle size of about 75 μm to about 150 μm. The bed weight of each capture element may be about 200 mg, and the bed length may be between about 3 mm and about 5 mm. The C18 particles may be packed between the upstream and downstream porous polymer frit disks. The pore sizes of the upstream and downstream disks may be about 90 μm and 35 μm, respectively. To capture aerosolized virus particles, the exemplary sample capture elements 1001, 7001, 1301, 902, and 1407 may include sulfate ester immobilized cellulose beads. Alternatively, in the sample collection module 1413, one or more capture elements can include a packed bed of C18 beads and one or more capture elements 1407 can include a packed bed of sulfate immobilized cellulose beads. Alternatively, in the sample collection module 1413, one or more capture elements can include a packed bed of a mixture of C18 beads and sulfate immobilized cellulose beads.An exemplary sulfate bead may be Cellufine sulfate beads (JKC Corp., Japan). The particle diameter may be between about 40 μm and about 130 μm. An exemplary sample capture element may have about 100 mg of sulfate ester-immobilized cellulose beads arranged as a packed bed column. An exemplary sample capture element may have an inner diameter of about 6.8 mm and a length of about 30 mm.

[0058] In an exemplary aerosol sample collection method using system 1400 or 1400A, sample capture elements 1407 are inserted into port 1421 of collection module 1413. Water pump 1403 and nebulizer 1404 are energized to generate water mist to humidify ambient air drawn through port 1418. Ambient air samples are then drawn into each sample capture element 1407 using pump 1406 for a predetermined sampling time. The sampling time can be varied to cover the duration of an operation in a hospital operating room. In some cases, the sampling time could be about 8 hours. Each capture element 1407 is then removed from housing 1401 and washed with an organic solvent to elute the captured non-volatile organics from the packed bed within each element 1407. As described above, the organic solvent can include at least one of acetonitrile (ACN), methanol, and isopropanol (IPA), with the remainder being water. The solvent may comprise about 50% to about 70% by volume of acetonitrile in water to elute polar particles such as small metabolites and proteins from the bed. Another solvent may comprise about 50% to about 70% by volume of isopropanol in water to elute non-polar particles such as lipids from the bed. The bed may be washed first with ACN in water, followed by IPA in water. The particles may then be concentrated using lyophilization and analyzed using diagnostic instruments having at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS. The extracted particles may also be subjected to a high temperature digestion step to generate characteristic peptides, for example, from virus particles.

[0059] An exemplary system 1300 (FIG. 9A) and method for capturing exhaled aerosols is disclosed by placing an exemplary sample capture element 1301 with a packed bed column in fluid communication with a ventilator 1305. The ventilator 1305 is a life support device used in intensive care units for patients who are unable to breathe on their own. For example, patients with severe symptoms of COVID-19 may require ventilator assistance to breathe. A tube 1306 is inserted directly into the trachea through the patient's mouth or nose. The ventilator pushes air through this tube into the lungs, forcing the person to inhale. The ventilator typically forces air in for one second, pauses for about three seconds to allow the patient to exhale through the same tube, and then repeats the cycle. The inlet end 1302 of the capture element 1301 is removably connected, preferably directly to the exhalation tube of the ventilator, to minimize particle loss. The outlet end 1303 is removably connected to a pump 1304 using tubing to draw exhaled air through the packed bed column in element 1301 at a flow rate of about 200 ml / min to about 2.5 L / min. Similar to system 7000 (FIG. 7), system 1300 can include a trap disposed between end 1303 and pump 1304 to collect condensate. The trap can be cooled to a temperature below ambient temperature. An optional HEPA filter and needle valve or flow meter can be installed between the trap and the pump. CO in the exhaled air 2 The CO is passed through the packed bed column. 2 The sensor may be placed in 2The monitoring allows an estimate of the exhaled volume. A particle counter can be placed between the outlet end 1303 and the trap to detect the size and number of particles exiting the packed bed column, and can be used to detect bed saturation and breakthrough of non-volatile organic molecules (bacterial and viral particles) from the column bed. The non-volatile organic molecule capture capacity of the C18 beads in the element 1301 can be between about 0.05 mg (non-volatile organic) / mg beads to about 0.5 mg / mg. The capacity of the C18-bound resin beads in the column bed in the exemplary capture element can be about 0.1 mg / mg. That is, a column bed with 25 mg of C18 beads would be characterized by a capacity to capture or adsorb about 2.5 mg of non-volatile organic molecules. The pump 1304 can be a diaphragm pump. CO 2 Data from the sensor can be stored on a non-volatile memory card, such as an SD card commonly used in portable devices. A flow sensor can be installed to monitor the flow rate through the C18 packed bed column. Alternatively, a flow controller can be used to achieve a constant flow rate, for example, 500 mL / min through the packed bed column. To enable exhaled aerosol sampling from a ventilator located in a hospital intensive care unit using the exemplary capture element 1301, a pump 1304 can be configured to pump CO 2 The sensor 1311, associated power supply 1307, system control components, and necessary fluidic components (tubing, quick couplings, etc.) are connected to a portable system 1313 (FIG. 9B). The capture element 1301 may be placed in fluid communication with the system 1313 (FIG. 9B) via a port 1314, which may have a quick connect / disconnect coupling. A portion of the exhaled air leaving the capture element 1301 using a pump 1304 is pumped to a CO 2 The CO may be delivered to a reservoir 1312 that is in fluid communication with the sensor 1311. The reservoir 1312 may be a substantially sealed container. 2It is used to prevent air leakage from the sensor. The system 1313 may have a user interface and an on-off switch to start and stop sampling of the breath using the element 1301. Additionally, components such as a flow controller and a flow restrictor 1309 may also be packaged in the portable system 1313. The pump 1304 may be a diaphragm pump. The portable system 1313 may be 11 inches by 7.5 inches by 5.5 inches (length by depth by height) and may have noise canceling materials such as foam padding to reduce the noise level caused by the pump to less than 45 dB. After a predetermined sample collection period, the sample capture element 1301 may be removed from the system 1300. The element 1301 may then be autoclaved at 110° C. for about 10 minutes to disinfect the element 1301 before extracting the captured aerosol particles. The trapped non-volatile aerosol particles may be extracted by flushing the column with about 200 μL to about 400 μL of solvent having 70% acetonitrile (ACN), about 50% to about 70% methanol, and about 50% to about 70% isopropyl alcohol (IPA). For example, a 50% ACN flush may be used to elute metabolites and proteins in a first stage flush, followed by a 70% IPA flush to elute lipids from the packed bed column. To preserve the trapped bioaerosol particles, the organic solvent may be removed from the packed bed column by lyophilization overnight, if necessary. The organic solvent may also be removed by incubation on a heating block at about 70° C. for about 30 minutes. Finally, the bed may be washed with about 0.05% TFA. Methods for analyzing metabolites, proteins, and lipids include silver staining for protein profiling, protein assays for protein content, bottom-up proteomics and LC-MS / MS for metabolomics and lipid omics, and MALDI-TOF mass spectrometry for molecular profiling. In an exemplary study, exhaled aerosols from a patient infected with pneumonia were collected using a capture element 1301 connected to a ventilator. During subsequent analysis, the total exhaled air volume collected, the CO in the exhaled air, and the amount of CO in the exhaled air were analyzed.2 As revealed by the Pearson correlation heatmap consisting of the variables of content, protein content, MALDI-TOF total ion intensity, and MALDI-TOF MS single peak (4820 m / z) intensity, protein content measured using protein assay and molecular profiling measured using MALDI-TOF MS were found to be good indicators of pneumonia infection in patients.

[0060] As previously described herein, the packed bed column of the sample capture element 902 (FIG. 5) may comprise Hamilton PRP-C18 resin beads supplied by Sigma Aldrich and other vendors. The resin beads may be characterized by a nominal diameter between about 12 μm and about 20 μm. Preferably, the nominal diameter of the C18 beads is about 20 μm. The packed bed may be held in place between two porous filter plates, such as fritted disks. For example, a polyethylene disk with an average pore size of more than 35 μm may be placed upstream of the bed, and a polyethylene disk with an average pore size of 10 μm (Boca Scientific, Dedham, Massachusetts) may be placed downstream of the bed. The 35 μm fritted disk increases the air flow rate through the bed, minimizing undesirable pressure drop through the bed. On the other hand, the smaller 10 μm fritted disk prevents the C18 resin beads from leaking out of the column. The packed bed may comprise about 25 mg of C18 resin beads. Non-volatile organic components in the exhaled breath, including bacteria and virus particles, removably interact with the C18 functional groups on the beads and are captured. Water, volatile organic molecules and other hydrophilic molecules pass through the bed and can be captured in a suitable trap 904 located between the sample capture element 902 and the pump 903. The trap 904 can be cooled to below ambient temperature. The sample capture element 902 can be cooled to a temperature below ambient temperature. A portable laser particle counter (MetOne Instruments, Grants Pass, Oregon) 905 can be installed downstream of the capture element 902 to measure the particle number and particle size of any particles not captured by the capture element 902. The particle number and particle size data can be used to estimate the capture efficiency of the element 902 as a function of particle size. The C18 resin beads provide a surface affinity between the carbon chains on the beads and the organic molecules present on the surface of viruses and microorganisms. For example, the bacterial cell surface is composed of glycans of various structures. In addition, waxy mycolic acids cover the surface of the tuberculosis bacillus. These organic molecules are generally hydrophobic and show a significant affinity for the alkyl chains of the C18 beads in the packed bed.In addition to the chemical properties, the particle size and bed length of the packed bed column are carefully selected to achieve particle capture by physical adsorption. To capture aerosolized virus particles and virus particles in solution, the exemplary sample capture elements 1001, 7001, 1301, 902, and 1407 can include sulfate ester immobilized cellulose beads. Exemplary sulfate beads can include Cellufine sulfate beads (JKC Corp., Japan). The particle diameter can be between about 40 μm and about 130 μm. The exemplary sample capture element can include about 100 mg of sulfate ester immobilized cellulose beads arranged as a packed bed column. The exemplary sample capture element can have an inner diameter of about 6.8 mm and a length of about 30 mm. As shown in Example 8, heat digestion of SARS-CoV-2 viral particles extracted using a packed bed column of sulfate ester immobilized beads, analysis using MALDI TOFMS, and processing of the spectra using a peptide fingerprinting protocol will allow matching of peptide fingerprints for the nucleoprotein (N) and for rapid identification of SARS-CoV-2.

[0061] The trapped aerosol particles from the packed bed column of the sample capture element 902 can then be extracted using a solvent suitable for analysis. A suitable extraction system can include means for flushing the packed bed column with a solvent to remove the solvent, including non-volatile organics, from the packed bed. The solvent can include at least one of acetonitrile, methanol, acid, isopropanol, and the remainder water. The solvent can include about 50% to about 70% acetonitrile by volume in water. The solvent can include about 50% to about 70% isopropanol by volume in water. The solvent can include about 50% to about 70% methanol by volume in water. The solvent can include at least one of about 12.5% ​​acetic acid by volume, about 5% TFA by volume, about 5% formic acid by volume, and about 10% HCl by volume. The packed bed can be washed at least once with water prior to solvent extraction to remove water-soluble inorganic contaminant particles. The collected samples may then be analyzed using diagnostic instruments having at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS. Alternatively, after the collection step, the C18 beads may be transferred to a capped tube with a suitable extraction solvent, such as 70% isopropanol, and shaken by hand or centrifuged to extract the trapped aerosol into the solvent. In the exemplary systems and methods disclosed herein, the C18 beads and / or columns may be reused after first washing the beads with an organic solvent, followed by washing with an inorganic solvent, such as water or PBS buffer. Diagnostic instruments that enable top-down proteomics, such as MALDI-TOFMS, may be used. In top-down proteomics, intact protein ions or large protein fragments from bacteria or viruses are subjected to gas-phase fragmentation for MS analysis.

[0062] The disclosed exemplary systems and methods can be used to establish a baseline profile of proteins, metabolites, and lipids in exhaled breath, which can then be used to differentiate breath from patients with various respiratory diseases, providing a powerful diagnostic tool for disease detection based on analysis of non-volatile aerosols in exhaled breath. [example]

[0063] Example 1. Capture and Analysis of Aerosolized Bacterial and Viral Particles Using an Exemplary Packed Bed Column 900 and MALDI TOF-MS

[0064] The capture and analysis of aerosolized Escherichia coli K12 strain, bacteriophage MS2, Pseudomonas fluorescens 1013, and Yersinia rhodei CDC3022-85 were investigated. Aerosol particles from approximately 20 μL of virus- or bacteria-containing water samples were generated using a Sono-Tek ultrasonic nozzle (Milton, NY). The nozzle was adjusted to generate aerosol particles ranging in size from approximately 0.3 μm to approximately 10 μm, with a median diameter of approximately 2 μm. The particles were directed into a release chamber 901 (50 ml conical tube) with a nominal volume of approximately 50 ml and drawn through a packed bed column at a flow rate of approximately 500 ml / min for approximately 10 min. The packed bed column in the capture element 902 contained approximately 25 mg of C18 resin beads with a size of approximately 20 μm. The beads were washed once with 70% acetonitrile and three times with 0.05% trifluoroacetic acid. After washing, the beads were stored moist prior to use. To this end, the column 902 may be capped at both ends and stored in a refrigerator to prevent the beads from drying out prior to use. The column bed was approximately 3 mm long and 7 mm in diameter. The bed volume was approximately 0.115 cc. Thus, at a flow rate of 500 ml / min, the gas hourly space velocity (GHSV, the ratio of flow rate to bed volume) was calculated to be approximately 260,000 per hour. The trapping element 902 was removably mounted near the bottom of a 50 ml conical tube. A portable laser particle counter (MetOne Instruments, Grants Pass, OR) 905 was used to measure particle sizes ranging from approximately 0.3 μm to approximately 10 μm upstream and downstream of the trapping element 902. The trap 904 consisted of a laboratory glass reservoir cooled with ice water and was also placed downstream of the element 902 to collect aerosol condensate (e.g., water vapor) passing through the column. The particle counts upstream and downstream of the capture element 902 for bacteriophage MS2 are shown in Table 1.

[0065] [Table 1]

[0066] Based on the particle counts shown in Table 1, about 99.8% of the MS2 particles are captured in the packed bed column of capture element 902, suggesting that the particle capture efficiency is at least 99%, even when the gas hourly space velocity is about 260,000 per hour. The dimensions (length, diameter) of the packed bed column and the flow rate through the bed can be modified to achieve a gas hourly space velocity of at least 250,000 / h. Without being bound to any particular theory, particle capture efficiencies of at least 99% can be measured at gas hourly space velocities between about 250,000 / h and about 3,000,000 / h. A capture efficiency of at least 99% has also been measured for aerosolized E. coli particles. Thus, an exemplary packed bed column of about 3 mm in length, containing particles as small as about 0.3 μm, provides significantly higher particle capture efficiencies, exceeding capture efficiencies using filter substrates such as electret and Teflon filters with thicknesses between about 0.05 mm and about 0.15 m. As previously mentioned, the L / Dp of the packed bed is approximately 150-250, preventing localized flow through the bed and exposing virtually all of the beads and surface area to capture (chemisorb or physisorb) bioaerosol particles. After capturing particles for approximately 10 min, the packed bed column was washed three times with approximately 400 µL of water, and then the captured biomaterial was eluted with 200 µL of 70% isopropanol. Samples collected during the washing and elution steps were analyzed using mass spectrometry.

[0067] MALDI-TOF mass spectra of samples were acquired using a Shimadzu Axima CFR-plus mass spectrometer operated in linear mode from 1000 to 15000 m / z. For direct injection and nanoflow LC-mass analysis, an LTQ Orbitrap system coupled with an EASY-nLC1000 system (Thermo Fisher Scientific) was used. The flow rate for direct injection was approximately 3 μL / min. For LC-MS analysis, samples were injected onto a microflow C18 column (Acclaim™ PepMap™ 100, 75 μm x 2 μm x 25 cm, Thermo Fisher Scientific) and proteins were separated using a gradient of solvent B (99% acetonitrile, 0.1% formic acid) from 5% to 65% in 90 min. Ion fragmentation was performed using the collision-induced dissociation (CID) method. To improve ion fragmentation coverage, a stepwise CID approach was used and top-down mass spectra were acquired using collision energies of 0%, 10%, 15%, 20%, 25%, 30%, and 35%, respectively. During top-down mass spectrometry data analysis, monoisotopic masses were deconvoluted using Xcalibur software (Thermo Fisher Scientific) and fragmented ions were inspected and identified using ProSight Lite (Northwestern University).

[0068] The wash samples and elution samples (after extraction of the packed column bed) were analyzed using MALDI-TOFMS. The results showed that bacterial features were well represented in the elution samples, suggesting that the aerosol capture and elution methods and systems disclosed herein provide analysis of intact or original (whole cell) biomaterial. Furthermore, the signal-to-noise ratios between the control and elution samples were indistinguishable, suggesting that capture and extraction using the packed column bed was highly effective. Indeed, after a quick centrifugation of the elution samples, bacterial material was visualized as a pale pellet at the bottom of the tube, suggesting that the capture capacity of the collection system was overly powerful.

[0069] Most viruses have a protein shell. MS2 was used as a representative virus model to evaluate the ability of the disclosed exemplary systems and methods to capture viruses. MALDI-TOF mass spectrometry showed that the capsid protein, a historical biomarker for MS2, was observed in the eluted sample. The identity of the MS2 capsid protein (13729 Da, MH+) was confirmed using a top-down mass spectrometry approach, where a reliable statistical score is constructed when matching experimental fragmentation ions with in silico protein fragmentation patterns. Thus, using MALDI-TOFMS whole cell characterization and top-down protein identification, we confirmed that the disclosed aerosol capture system and method can be used to capture aerosolized virus particles. To further confirm the results observed using top-down mass spectrometry, direct infusion mass spectrometry and nanoflow LC mass spectrometry were used for quantitative analysis. Direct infusion mass spectrometry showed that the signal intensity of referee insulin was approximately 3.6E+6 (Figure 6A-B). The signal intensity of the MS2 capsid protein was approximately 1.1E+5 for both the control and capture samples (samples obtained by extracting aerosols from a packed bed column), suggesting that no obvious sample loss was observed using the exemplary aerosol capture system and method, as well as MALDI-TOF MS analysis. This observation was further confirmed using nanoflow LC mass spectrometry, the gold standard for molecular quantitative analysis. The LC mass spectrometry results (Figure 7A-B) showed that the peak area and ion intensity of the MS2 capsid protein were virtually identical for both the control and capture samples, suggesting that the exemplary aerosol capture system and method maintained high biochemical capture efficiency.Without being bound by any particular theory, several factors contribute to the superior capture efficiency of the disclosed aerosol capture method, including, but not limited to, the high retention capacity of the C18 resin beads for aerosolized organic biomaterials such as viruses and bacterial particles, preparation of the packed bed prior to use, placement of the capture element directly in the aerosol particle source such as the aerosol release chamber 901, and the unique packed bed column parameters used to capture the aerosolized particles. As discussed above, the dimensions of the packed bed column provide sufficient retention time at a flow rate of approximately 500 ml / min to ensure good contact between the carbon chains on the resin beads and the organic molecules on the virus surface. Additionally, because the aerosol particles, including viruses and microorganisms, are directly exposed to the C18 beads, "wall loss" was minimized. Wall loss is typically present in other aerosol collection methods where particles are deposited on the sides or walls of the device. Quantitative analysis of MS2 capsid protein demonstrated the superior capture efficiency of the disclosed aerosol collection system and method. Furthermore, because no vacuum pump is used, the disclosed system and method may be treated as a "soft harvest technique" since it preserves the viability of whole cell viruses, bacteria, and other microorganisms necessary for other microbial culture studies.

[0070] Example 2. Exemplary packed bed column characteristics

[0071] Commercial use of membrane-based aerosol collection devices suffers from problems of clogging and pressure drop caused by the accumulation of water droplets and other environmental particles during aerosol collection. This requires a large membrane surface area, which increases costs, has high parasitic power, and requires noisy air pumps. Aerosol capture tests were performed using C18 beads with nominal diameters of about 10 μm and about 20 μm. The beads were packed between polymer frit disks. The average pore size of the upstream and downstream disks was about 35 μm and 10 μm, respectively. HPLC grade water and E. coli vegetative cells were aerosolized into chamber 901 and drawn through an exemplary packed bed column 902 using a pump 903, preferably a diaphragm pump. E. coli is a rod-shaped bacterium with nominal dimensions of about 1 μm to about 2 μm in length and about 1 μm in diameter. The pump flow rate was set at 2.5 L / min, and the actual flow rate through the bed was measured using a flow meter (see Figure 5). Tests were performed using a packed bed column of C18 beads. As shown in Figure 8A, the measured flow rate through a bed containing approximately 25 mg, 20 μm C18 beads was approximately 750 ml / min when the bed (approximately 3 mm long) was exposed to HPLC grade water aerosol and approximately 600 ml / min when exposed to HPLC grade water aerosol. The bed was exposed to E. coli aerosol for a 30 minute capture time. This result suggests that the 25 mg bed of approximately 20 μm C18 beads did not saturate during these tests, indicating that the amount of beads in the bed could be reduced. Alternatively, a 25 mg bed could be used to extend the capture time. In contrast, when the bed contained 10 μm C18 beads, the flow rate decreased from approximately 200 ml / min to approximately 50 ml / min in approximately 30 minutes when the bed was exposed to HPLC grade water aerosol. Furthermore, when the bed was exposed to E. coli aerosol, the flow rate decreased from approximately 100 ml / min to approximately 3 ml / min, indicating a significant pressure drop through the bed containing the 10 μm beads. These results suggest that C18 beads of at least about 20 μm particle size in the packed bed column of the capture element 902 are preferred.

[0072] Additionally, the effect of increasing the pressure drop across the bed on the flow rate reduction was investigated by varying the amount of C18 beads in the packed bed column, and the pore size of the polymer frit disk placed at the inlet end of the bed. The pump flow rate was set at 2.5 L / min, and the actual flow rate through the bed was measured using a flow meter. The amount of C18 beads, nominally 20 μm in diameter, was increased from 25 mg to 40 mg in the packed bed column. The average pore sizes of the upstream and downstream disks were 35 μm and 10 μm, respectively. Increasing the amount of bed from 25 mg to 40 mg increased the bed length from 3 mm to 5 mm. In each case, the flow rate through the bed was monitored after exposing the bed to aerosolized HPLC-grade water aerosol. The results (Figure 8B) show that increasing the amount of C18 beads in the bed from 25 mg to 40 mg reduced the flow rate from 700 ml / min to 150 ml / min after a 30-minute collection time. Furthermore, in another series of tests, the pore size of the inlet polymer frit disk was changed from 35 μm to about 10 μm. Upon exposure to an aerosol of E. coli (vegetative cells), the flow rate decreased from about 600 ml / min to about 250 ml / min over a 30 min collection time (Figure 8C).

[0073] Example 3. Capture and analysis of exhaled aerosols from patients diagnosed with COVID-19 using an exemplary packed bed column connected to a ventilator

[0074] The exemplary system 1300 (FIG. 9A) was evaluated in a hospital intensive care unit (ICU) dedicated to the treatment of patients diagnosed with COVID-19 disease. The flow rate through a packed bed column containing approximately 25 mg of C18 beads (nominal diameter 20 μm) in the sample capture element 1301 was set to 500 ml / min. Before installation in the system 1300, the capture element was washed once with 70% acetonitrile and then three times with 0.05% TFA. The capture element was stored at 4°C before use to prevent drying of the C18 beads in the packed bed. Exhaled aerosols were then collected from each patient for approximately 4 hours at a flow rate of 500 ml / min. After the collection period, the packed bed column was removed from the collection system. The column was washed with approximately 200 μL to approximately 400 μL of 70% ACN or 70% IPA. The organic solvent was removed from the packed bed column by lyophilization overnight. The organic solvent can also be removed by placing the device 1301 on a heating block at about 70° C. for about 30 minutes. The trapped aerosol particles were extracted or separated using about 40 μL to 100 μL of 0.05% TFA. The samples were then analyzed using SDS-PAGE electrophoresis and silver staining, MALDI-TOFMS (whole cell top-down proteomics), and bottom-up proteomics.

[0075] Approximately 5 μl of all collected samples were used for SDS-PAGE electrophoresis, which was performed using the Criterion Tris-HCl gel system (Bio-Rad Laboratories, Hercules, CA). After SDS-PAGE electrophoresis, SDS-PAGE gels were prepared using a silver staining kit (Thermo Fisher Scientific) to visualize protein bands. Bovine serum albumin was used as an internal positive control. Protein bands were observed in all three patient samples. Based on the BSA control sample, the protein content of the three samples was estimated to be at least 100 ng.

[0076] For whole cell MALDI-TOF MS analysis, 0.2 μL of analyte was mixed with 0.2 μL of α-cyano-4-hydroxycinnamic acid MALDI matrix (CHCA) prepared in 70% ACN. The mixture was deposited on a MALDI sample cap and mass spectra were collected using an exemplary MALDI-TOF mass spectrometry system disclosed in co-pending patent application PCT / US20 / 48042, entitled "System and Method for Rapid and Autonomous Detection of Aerosol Particles," which is incorporated herein by reference in its entirety. MALDI-TOF spectra were collected from samples of patients #3 and #4. Mass peaks were observed in both samples. Peak patterns generated from MALDI-TOF MS were examined using pattern recognition algorithms for detection and classification.

[0077] For bottom-up proteomics, 5 μl of each sample was used. 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 min. After reduction, protein alkylation was performed by adding iodoacetamide to a final concentration of 15 mM and incubating at room temperature for 1 h. Trypsin (Thermo Fisher Scientific) was used for overnight protein digestion. After digestion, peptides were cleaned up using a C18 pack tip (Glygen, Columbia, MD). Peptide samples were then prepared in 20 μl of 0.1% formic acid for mass spectrometry analysis, including MALDI-TOF mass spectrometry. Samples were processed using an EASY-nLC 1000 system (Thermo Fisher Scientific) connected to an LTQ Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). For tandem mass spectrometry, peptides were loaded onto an Acclaim PepMap 100 C18 trap column (0.2 mm × 20 mm, Thermo Fisher Scientific) at a flow rate of 5 μl / min and separated on an EASY-Spray HPLC column (75 μm × 150 mm, Thermo Fisher Scientific). The HPLC gradient was run at a flow rate of 300 nl / min for 60 min using a 5%-55% mobile phase (75% acetonitrile and 0.1% formic acid). Mass spectrometry data collection was performed in data-dependent acquisition mode. The resolution of the precursor scan was set to 30,000 and the scan resolution of the product ions was set to 15,000. Fragmentation of the product ions was achieved using higher-energy collision-induced dissociation at 30% of the total energy. Bottom-up proteomics raw data files were processed with MaxQuant Andromeda software (maxquant.org) against the “human” and “SARS-COV-2” protein databases (uniprot.org) following standard recommendations and instructions.The human protein database contained 20,395 proteins reviewed, and the SARS-CoV-2 protein database contained 13 proteins reviewed. Peptide fingerprints generated from liquid chromatography profiles and digested peptides were identified using LC-MS and MALDI-TOF MS for all three patient samples. A total of 222 proteins were identified in all three patient samples. Most of the proteins were found to be derived from human blood, indicating active interactions between the lungs and blood. Typical lung proteins and SARS-CoV-2 proteins were identified, as shown in Table 2.

[0078] [Table 2]

[0079] Example 4. Capturing Ambient Air Aerosol Particles Using Exemplary Collection System 1400

[0080] Capture of particles in ambient air was tested using an exemplary system 1400 with four capture elements 1407. Each element 1407 contained about 200 mg of C18 beads with a nominal diameter between about 75 μm and 150 μm packed between two porous fritted disks in a polymer tube with an inner diameter of about 16 mm. The packed bed length was about 3 mm and the bed volume was about 0.6 cc. Air was passed through each column at a flow rate of about 20 L / min using a pump 1406. The packed bed was wetted with water prior to sample capture. Particle size and particle count were measured using a Met One Instruments particle counter. Particle counts upstream and downstream of the capture element 1407 are shown in Table 3.

[0081] [Table 3]

[0082] Based on the particle counts shown in Table 3, greater than 99.8% of the ambient air particles were captured using capture element 1407 of system 1400, which represents a particle capture efficiency of approximately 2×10 per hour gas hourly space velocity. 6 When used to capture non-volatile organic aerosols such as protein, lipid, and metabolite biomarkers or whole pathogens, the capture capacity of the beds could be around 10 wt.-%.

[0083] Example 5. Analysis of breath collected from healthy human subjects to establish baseline breath metabolites and lipids

[0084] In the exemplary system 7000, the column of element 7001 contained about 35 mg of C18 beads with a nominal diameter between about 55 μm and 105 μm. A packed bed of C18 beads was placed between an upstream porous frit with a pore size of about 90 μm and a downstream frit with a pore size of about 35 μm. The diameter of element 7001 was about 6 mm and the bed length was about 3 mm. With a bed volume of about 0.09 cc and a flow rate through the bed of about 3 L / min, the gas hourly space velocity (GHSV) through the bed was about 2×10 per hour. 6 , allowing flexibility in increasing the flow rate; either by passing more through the bed or reducing the bed volume. As described below, even at this high space velocity, efficient capture of non-volatile organic particles such as metabolites and lipids was demonstrated. Three capture elements (columns) 7001 were used to capture exhaled aerosols from each subject sequentially in time: 10 min for the first column, then 10 min for the second column, then 10 min for the third column. The first column was washed using ACN to elute metabolites. The second column was washed with 300 μL of 70% IPA to elute lipids. The third column was used as a control, where only HEPA filtered air was collected. During sample collection, CO 2 Using sensors to measure CO in each individual's breath 2 The levels were sensed and recorded. 2 The sensor was located between the outlet of the column and the pump.

[0085] Eluted metabolite and lipid samples were lyophilized overnight. Dried samples were prepared as solutions for LC-MS / MS analysis. Total ion chromatography (TIC) in LC-MS analysis was used for peak alignment and intensity normalization. Identification of metabolites and lipids was based on accurate mass measurement and MS / MS spectrum matching using the open source software MS-DIAL (PRIMe platform from Riken Metabolomics). Data were filtered to remove minor peaks as follows: Peaks with low ion intensity were ignored, and if peaks were identified in only one subject, these peaks were treated as artifacts and ignored. Ion peaks from the third column (HEPA-filtered air) were used as a baseline filter to exclude these peaks from all exhaled breath samples. Individual CO measured during sample collection from each subject was analyzed using the ion peaks from the third column (HEPA-filtered air) and the ion peaks from the third column (HEPA-filtered air) were used as a baseline filter to exclude these peaks from all exhaled breath samples. 2 The CO concentrations were used to normalize the ion intensities of the individual breath samples. 2 The individual CO concentrations during sample collection using the first column (sample capture element) 2 The concentrations (ppm) were between a minimum of about 6850 ppm and a maximum of about 12799 ppm. During sample collection using the second column, CO 2 Concentrations fell between 4925 ppm and 16453 ppm. The final human breath sample database constructed from this study included molecular identification, retention time, total mass, and ion fragmentation information (MS / MS).

[0086] The data science software RStudio was used to visualize the exhaled breath and blank samples using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). As shown in Figure 11A, a clear separation was observed between the exhaled breath samples and the HEPA-filtered air (blank) samples for both metabolites and lipids in human exhaled breath. Similar separation is expected during the analysis of exhaled breath samples from healthy subjects and subjects infected with respiratory diseases. Also, as shown in Figure 11B, about 1804 metabolites and 1150 lipids were identified in the exhaled breath samples but not in the HEPA-filtered air (blank) samples. The relative standard deviation (RSD, 0-300%) indicates that lipids are characterized by a large number of identifications below 100% RSD, suggesting that the standard deviation from the mean of lipids associated with the exhaled breath samples from the 14 subjects is very small. This gives great confidence that lipid biomarkers from exhaled breath aerosols collected and eluted using the exemplary system and method can be used to discriminate between healthy and respiratory disease-infected patients. Finally, the ion intensities from the individual captured samples (of the 14 subjects) were calculated based on the CO 2 As shown in Figure 11C, this normalization protocol highlights the specific variations in ion intensities of both lipid and metabolite biomarkers for a given subject (top 5 ion intensities in each case) and can be used to better understand the characteristics of metabolite and lipid biomarkers in healthy individuals, which is used during the interpretation of data collected from infected patients.

[0087] Example 6. Ambient Air Aerosol Particle Capture and Bottom-Up Proteomics Using Exemplary Collection System 1400A

[0088] Capture of particles in ambient air was tested using an exemplary system 1400A with four capture elements 1407. Each element 1407 had approximately 200 mg of C18 beads with a nominal diameter between approximately 75 μm and 150 μm packed between two porous fritted disks in a polymer tube with an inner diameter of approximately 16 mm. The packed bed length was approximately 3 mm and the bed volume was approximately 0.6 cc. Air was drawn through each column at a flow rate of approximately 20 L / min using pump 1406 and was humidified before entering the sample capture element. Four samples were collected: (1) from a single-occupancy (1 person) office with 8 hours of air sampling, (2) from a cafeteria with 8 people, with 2 hours of air sampling, and (3) and two samples from a low-traffic, unattended operating room in a hospital with 7 hours of air sampling. After sample collection, the aerosol particles in each packed bed column were eluted using 2 mL of 10-80% organic solvent, followed by overnight freeze-drying to remove the organic solvent. For each sample, residual proteins were assessed by protein silver staining and bottom-up proteomics.

[0089] Sixty proteins were identified from the office air sampling sample, 72 proteins were identified from the cafeteria air sample, 84 proteins were identified from low traffic operating room (OR) air sample #1, and 38 proteins were identified from low traffic OR air sample #2 (Figure 13A). Typical proteins were identified and contained keratin in all four samples (Table 4), which was evidence of proteins from human activity. Keratin-9 levels were assessed and showed that the office sample collected from a room occupied by one person contained the most keratin-9 (Figure 13B).

[0090] [Table 4]

[0091] Example 7. Capture of aerosolized proteins using an exemplary sample capture element with a C18 packed bed column and a commercially available button sampler

[0092] An exemplary sample capture element consisted of a packed bed column of C18 beads with a nominal diameter of about 45 μm to 120 μm. The amount of C18 beads in the column was about 200 mg, and the bed length was about 3 mm. The length and diameter (ID) of the capture element were about 60 mm and about 15.9 mm, respectively. The button sampler (SKC Ltd, UK) consisted of a filter sampler (25 mm filter with pore size >1 μm) with a porous curved inlet designed to improve the collection characteristics of inhalable dust (aerodynamic diameter 100 μm). The dust contains bioaerosols for total microbial counts by bioaerosol.

[0093] Protein solutions containing BSA protein in artificial saliva (NCZ-APS-0012) background solution with a BSA concentration of approximately 2 mg / ml were aerosolized using a dynamic concentration aerosol generator at the Johns Hopkins University Applied Physics Laboratory. For the exemplary sample capture element, a mechanical pump was used to draw the aerosol through the packed bed column at approximately 10 L / min. After sample collection, the C18 bead packed bed column was eluted with 2 mL of 70% CAN, and the organic solvent was removed by lyophilization overnight. Samples were suspended in 50 μL of water and used for SDS-PAGE electrophoresis, blue staining, and bottom-up proteomics.

[0094] FIG. 14 shows protein blue staining of samples collected using the exemplary C18 packed bed column and the button sampler. As can be seen, the protein capture efficiency of the C18 exemplary packed bed column was significantly better than that obtained using the button sampler. Furthermore, in the case of silver staining, the BSA protein band (approximately 66 kDa) was not seen in the samples collected with the button sampler. In contrast, the BSA protein band was seen in all samples collected with the exemplary C18 packed bed column. In addition to the intact BSA protein, its dimer form (approximately 140 kDa) and trimer form (approximately 200 kDa) were seen in all samples collected with the C18 packed bed column.

[0095] Example 8. Capture and analysis of SARS-CoV-2 virus using sulfate functional groups

[0096] SARS-CoV-2 South African strain was propagated using the cell line Vero E6 TMPR332. 0.3 mL of virus was mixed with PBS buffer to a final volume of 4.7 mL. Growth medium was decanted from the tissue culture vessels and gently washed three times with 20 mL of PBS. 5 mL of diluted virus was pipetted into the vessels and incubated at 37°C for 15 min. 20 mL of unsupplemented medium was added to each vessel and incubated for 2 days until approximately 80% CPE was achieved. CPE at harvest was 90%. TCID50 was concentrated to 2.32e+7 (calculated by Reed & Muench method). For the purification step, the virus was ultracentrifuged at 100,000g for 60 min, the supernatant was decanted, and the pellet was resuspended in 1 mL of water. The virus was then heat inactivated at 70°C for 30 min.

[0097] The sample capture element contained sulfate ester immobilized cellulose beads (Cellufine® Sulfate). The packed bed column contained beads with diameters of about 40 mm to about 130 mm. The packed bed column (30 mm length × 6.8 mm internal diameter) contained about 100 mg of beads. The column was washed once with 1 mL of wash solution (0.2 M NaCl in 1x PBS). 900 μL of SAR-CoV-2 virus sample was prepared in 100 μL of 10x PBS to create a final solution of 1x PBS solution. The entire sample solution containing the virus was loaded onto the column using a syringe and pushed into the column. The sample exiting the column was collected and pushed into the column an ​​additional five times to maximize virus capture within the bed. To completely remove unbound proteins, 2 mL of 0.2 M NaCl in 1x PBS wash solution was pushed into the column three times. Air was then pumped into the column to ensure that the wash solution remained within the column. The captured viral particles were then eluted using 900 μL of 1.5 M NaCl in 1×PBS elution solution.

[0098] The eluted sample was split into two samples and each sample was subjected to hot acid digestion. Prior to this step, the eluted sample was filtered in a centrifuge. 200 μL of eluted virus was mixed with 250 μL of HPLC water and loaded onto a 3k (3 kDa MWCO) filter column. The sample was centrifuged at 14,000 g for 25 minutes in a microcentrifuge. The 3k column was filled with 450 μL of HPLC water and centrifuged again. This process was repeated three times. The column was turned upside down in a new hold tube and centrifuged at 10,000 g for 10 minutes. This sample was used for hot acid digestion. Approximately 50.5 μL of water was added to the sample produced by 3k filtration to bring the final sample volume to 87.5 μL. 12.5 μL of acetic acid was added to create a 12.5% ​​acid solution (total of 100 μL). The sample was subjected to hot acid digestion at approximately 140 °C for approximately 15 minutes.

[0099] The samples were then analyzed using MALDI-TOF mass spectrometry. CHCA matrix was prepared at a concentration of approximately 9 mg / mL in 70% acetonitrile. Approximately 1 μl of the hot acid sample was placed on the MALDI plate. Once the sample was partially dry, approximately 1 μl of CHCA solution was placed on top and mixed by pipetting up and down. The samples were analyzed using a commercial Bruker Daltonics microflex LRF MALDI-TOF mass spectrometer. MALDI-TOF mass spectra were acquired in positive linear mode, and an average of 600 profiles were collected in the mass range of 700–21,000 m / z for every spectrum. The online program MASCOT Peptide Mass Fingerprint (Matrix Science, Boston, MA) was used to identify peptide mass fingerprints. Mass peaks with S / N>10 were extracted to “mass values” for analysis. The “SwissProt” protein database was used, and “all entries” was selected for classification. The enzyme was defined as "Formic_acid" and various deletion cleavage numbers (0–9) were allowed. Peptide tolerance was set to 0.5 Da and the monoisotopic type was selected.

[0100] Figure 15A-B shows that the MALDI TOF mass spectrum of heat acid digested peptides from virus samples extracted from the sulfate packed bed column showed a clearer profile compared to the spectrum of the sample not treated with the sulfate packed bed column. Bottom-up proteomics was used for peptide identification and showed that peptides of VeroE6 cell proteins were most abundant in the sample not treated with the sulfate packed bed column (Figure 15C). In contrast, the sample extracted from the sulfate packed bed column showed that peptides of SARS-CoV-2 proteins were most abundant (Figure 15D-E). Viral peptides were also characterized by significantly higher ion intensity in the sample extracted from the sulfate packed bed column (Figure 15D). These results indicate that sulfate immobilized cellulose beads can be used to capture aerosolized virus particles. Furthermore, bottom-up proteomics showed that the identified peptides correspond to the N protein characteristic of SARS-CoV-2. Using peptide identification information from bottom-up proteomics, six mass peaks were identified in the MALDI-TOF MS spectrum (Figure 15B) that are characteristic of SARS-CoV-2. A typical mass shift characteristic of thermal acid hydrolysis, +115.1, was observed in these peak assignments.

[0101] Since most of the peptides identified after enrichment correspond to the N protein, the MALDI-TOF MS mass peaks from the sample extracted from the exemplary capture element can be used to identify the N protein following SARS-CoV-2 detection using peptide mass fingerprinting. Thirty-seven mass peaks with a signal-to-noise ratio greater than 10 were selected and processed with the MASCOT peptide mass fingerprinting program. Because hydrolysis of proteins by thermal acid causes missed cleavages, the effect of allowed missed cleavages on the identification score was evaluated. The results showed that the N protein can be reliably identified using the MALDI-TOF mass spectral profile when the missed cleavages are greater than 0 (Figure 15E). Among the identification results, the three allowed missed cleavage sites yielded the best score, and the score did not change significantly even when a larger number of missed cleavages was used. The results indicated that the combination of high-speed MALDI-TOF MS and peptide mass fingerprinting can be used for the detection of SARS-CoV-2.

[0102] The exemplary sulfate packed bed column and sample extraction, processing and analysis methods may also be used to capture and identify other viral particles. Peptides identified using MALDI-TOF MS and Mascot database searching may also be used to distinguish different strains of SARS-CoV-2 virus. For example, Eta / B.1.525 strain has SD>Y transitions at amino acid positions 2 and 3 from the natural variant in the reference database UniProtKB-P0DTC9(NCAP_SARS2). Alpha / B.1.1.7 has a D>L transition at amino acid position 3 of the natural variant. Eta / B.1.525 has an A>G transition at amino acid position 12 from the natural variant. Omicron / B.1.1.529 has a P>L transition at amino acid position 13 from the natural variant. Omicron / B.1.1.529 is missing amino acid positions 31-33 from the natural variant. Delta / B.1.617.2 has a D>G transition at amino acid position 63 from the natural variant. Gamma / P.1 has a P>R transition at amino acid position 80 from the natural variant. Delta / B.1.617.2 and kappa / B.1.617.1 have a D>Y transition at amino acid position 377 from the natural variant.

[0103] The Abstract is provided to comply with 37 C.FR § 1.72(b) to enable the reader to quickly determine the nature and gist of the technical disclosure from a general overview perspective, and is not intended to be used to interpret or limit the scope or meaning of the claims.

[0104] Although this disclosure has been described in connection with preferred modes of practicing it, those skilled in the art will appreciate that numerous modifications can be made thereto without departing from the spirit of this disclosure, and therefore, it is not intended that the scope of this disclosure be limited by the foregoing description.

[0105] It is also understood that various modifications can be made without departing from the essence of this disclosure. Such modifications are implicitly included in the description. They still fall within the scope of this disclosure. It is understood that this disclosure is intended to result in patents covering many aspects of the disclosure, both individually and as a system as a whole, and in method and apparatus modes.

[0106] Moreover, each of the various elements of this disclosure and claims may also be achieved in a variety of ways, and this disclosure should be understood to encompass each such variation, whether that variation be in any apparatus implementation, method or process implementation, or simply variations of any of these elements.

[0107] In particular, it should be understood that each element word may be expressed by equivalent apparatus terms or method terms 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 operation. Such terms may be substituted where necessary to make clear the implicitly broader scope to which this disclosure is entitled. It should be understood that every operation may be expressed as a means for taking that operation or as an element that causes that operation. Similarly, each physical element disclosed should be understood to encompass a disclosure of the operation that the physical element facilitates.

[0108] Furthermore, for each term used, unless its usage in this application is inconsistent with such interpretation, a common dictionary definition, as contained in, for example, at least one of the standard technical dictionaries recognized by artisans and the most recent 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.

[0109] Additionally, the use of the transitional phrase "comprising" is used to maintain the "open-ended" claims herein in accordance with conventional claim interpretation. Thus, unless the context requires otherwise, "comprising" is intended to mean the inclusion of a recited element or step or group of elements or steps, but not the exclusion of other elements or steps or group of elements or steps. Such terms should be interpreted in the broadest manner to afford the applicant the broadest scope legally permissible.

[0110] [References] 1. 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. 2. Hunt, J., "Exhaled breath condensate: An evolving tool for noninvasive evaluation of lung disease," J. Allergy Clin. Immunol. 2002; 110:28-34. 3. 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 bacilli in bio-aerosols from untreated TB patients," Gates Open Research 2018, 1:11. 4. Wood R., Morrow C., Barry C.E., III, Bryden W.A., Call C.J., Hickey A.J., et al.: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC). PLoS One. 2016; 11(1): e0146658. 5. Rachel C. Wood, Angelique K. Luabeya, Kris M. Weigel, Alicia K. Wilbur, Lisa Jones-Engel, Mark Hatherill, and Gerard A. Cangelosi, "Detection of Mycobacterium tuberculosis DNA on the oral mucosa of tuberculosis patients," Sci. Rep. 5, 8668

Claims

1. 1. A breath collection system for monitoring a patient infected with a respiratory disease and breathing using a ventilator, comprising: one or more sample capture elements, each having an inlet end, an outlet end, and a packed bed column for selectively capturing aerosol particles in exhaled air produced by a patient breathing using the ventilator, the inlet end of the one or more sample capture elements being removably connected directly to one end of an exhaled air tube of the ventilator, the packed bed column being kept wet by washing with one or more organic solvents and water prior to use, and a second end of the exhaled air tube being inserted directly into the patient's organ through the mouth or nose; A subsystem comprising at least one of a pump, a power source, and a controller configured to draw exhaled air containing aerosol particles into the one or more sample capture elements, the subsystem being arranged in fluid communication with the outlet end of the one or more sample capture elements and configured to control the operation of the one or more sample capture elements.

2. The subsystem includes a CO ２ 2. The system of claim 1, further comprising at least one of a sensor and a particle counter, the particle counter configured to detect saturation of the bed and breakthrough of aerosolized particles from the packed bed column in each of the one or more sample capture elements.

3. 3. The system of claim 2, further comprising a trap disposed between the sample capture element and the pump and configured to capture exhaled breath condensate (EBC) including at least one of water vapor, volatile organic components, and non-volatile organic components passing through the packed bed column in each of the one or more sample capture elements.

4. The system described in claim 1, wherein in each of the one or more sample capture elements, the packed bed column has one or more of resin beads having C18 functional groups on their surface, cellulose beads having sulfate ester functional groups on their surface, or a mixture thereof.

5. 5. The system of claim 4, wherein the resin beads and cellulose beads have a nominal diameter of at least about 20 μm.

6. 2. The system of claim 1, wherein the nominal flow rate drawn through the packed bed column in each of the one or more sample capture elements using the pump is between about 200 ml / min and about 3 L / min.

7. 1. A system for monitoring a patient infected with a respiratory disease and breathing using a ventilator, comprising: The breath collection system of claim 1 ; a sample extraction system for extracting the aerosol particles from the packed bed column in each of the one or more sample capture elements into one or more liquid samples; and an analyzer for analyzing aerosol particles in the one or more liquid samples.

8. 8. The system of claim 7, wherein the extraction system comprises means for flushing the packed bed column in each of the one or more sample capture elements with at least one solvent and collecting the solvent containing the aerosol particles from the packed bed.

9. 9. The system of claim 8, wherein the one or more solvents comprise about 50% to about 70% by volume acetonitrile in water, about 50% to about 70% by volume isopropanol in water, about 0.05% by volume trifluoroacetic acid (TFA) in water.

10. 8. The system of claim 7, wherein the analytical device comprises at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS, and LC-MS / MS.

11. A patient condition monitoring system for monitoring the condition of a patient infected with a respiratory disease and breathing using a ventilator, selectively capturing aerosolized particles in exhaled breath produced by the patient using a sample collection system; The sample collection system comprises: one or more sample capture elements, each having an inlet end, an outlet end, and a packed bed column for selectively capturing aerosol particles in exhaled air produced by a patient breathing using the ventilator, the inlet end of the one or more sample capture elements being removably connected directly to one end of an exhaled air tube of the ventilator, the packed bed column being kept wet by washing with one or more organic solvents and water prior to use, and a second end of the exhaled air tube being inserted directly into the patient's organ through the mouth or nose; a pump in fluid communication with the outlet end of the one or more sample capture elements and configured to draw the exhaled air containing aerosol particles into the one or more sample capture elements; a sample extraction system is used to extract aerosol particles from the packed bed column in the one or more sample capture elements into one or more liquid samples; A patient condition monitoring system, characterized in that an analysis system is used to analyze aerosol particles in the one or more liquid samples to determine the presence or absence of a respiratory disease.

12. The patient status monitoring system of claim 11, wherein the packed bed column in the one or more sample capture elements is washed using at least one of 70% ACN, water, and 0.05% TFA prior to removably connecting the sample collection system to the expiratory tube of the ventilator.

13. A patient status monitoring system as described in claim 11, wherein the packed bed column in the one or more sample capture elements is first flushed with about 50 volume percent acetonitrile (ACN) and then flushed with about 70 volume percent isopropyl alcohol (IPA).

14. The one or more liquid samples are mixed with a MALDI matrix to obtain one or more mixed samples; 12. The patient condition monitoring system of claim 11, wherein the one or more mixed samples are deposited on one or more sample plates.

15. A patient status monitoring system as described in claim 11, wherein the one or more liquid samples extracted using the sample extraction system are subjected to proteolytic digestion to produce one or more peptide samples characteristic of the respiratory disease.

16. The one or more peptide samples are mixed with a MALDI matrix to obtain one or more mixed samples; 16. The patient condition monitoring system of claim 15, wherein the one or more mixed samples are deposited on one or more sample plates.

17. The patient status monitoring system of claim 14, wherein the one or more mixed samples are analyzed using at least one of PCR, ELISA, rt-PCR, mass spectrometry (MS), MALDI-MS, ESI-MS, and MALDI-TOFMS, and LC-MS / MS.

18. A sample capture element for capturing aerosolized bacteria and viruses in exhaled air produced by a patient breathing using a ventilator, comprising: a packed bed column configured to selectively capture aerosolized bacterial and viral particles in the exhaust air as the exhaust air is drawn through the packed bed column using a pump; The packed bed column comprises: A sample capture element comprising at least one of resin beads having C18 functional groups on the surface, cellulose beads having sulfate ester functional groups on the surface, and mixtures thereof, the beads being kept wet prior to being used for sample capture, the resin beads and cellulose beads having a nominal diameter of about 40 μm to about 200 μm, and a particle capture efficiency of at least 99%.

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