Devices, methods and kits for capturing and processing biological samples - Patents.com

Abstract

The respiratory liquid particles and vapor are captured in a device that provides a surface and a chamber space for condensing or freezing the vapor and aerosol particles. One or more breaths are exhaled through the device. Capture can be on a freezing surface that immobilizes water upon contact. The chamber space in the device may freeze and collect the liquid respiratory particles and vapor. After collection, the liquid is collected and collected into a vial either by draining, pushing, or centrifugal force. The liquid may be collected and mixed with sample preparation reagents such as viral lysis reagents, internal standards, etc. After collection, the sample is analyzed. Analysis may be by PCR, qPCR, RT-PCR, RT-qPCR, LAMP or any nucleic acid detection method, mass spectrometry, absorbance spectrometry, or any analytical tool or method. The nucleic acid amplification reagent may include a lysis reagent such as acetonitrile.

Description

[Technical field]

[0001] The present invention relates to devices, methods and kits for capturing and processing biological samples from respiratory air, in particular for capturing biological samples that are liquid, particulate or vapor. [Background technology]

[0002] Collection of samples for detection of viruses and other markers can be difficult, especially in children. Respiratory samples are less intrusive and are the preferred method of sampling for viruses. In principle, if someone is shedding and exhaling a virus, they are infectious and can spread viral illness. However, liquid from respiratory breath is difficult to collect. Current respiratory liquid collection is a time-consuming and laborious process of placing an individual's respiratory breath into a device. In addition, respiratory breath generally contains a lower viral load than saliva or nasal swab samples.

[0003] Current techniques are limited in capturing exhaled breath quickly and efficiently. For the purpose of widespread rapid testing for infectivity, breath must be collected quickly from a large number of individuals. Many conventional systems recommend that 5-10 minutes of breath be collected prior to analysis, which typically results in a 1 mL liquid sample. Conventional systems also require the cooling sleeve to be cooled in a freezer several times to achieve cooling, which can make such systems impractical for large-scale use. Other devices for breath collection utilize similar or longer timelines, requiring at least 10 minutes each. Many such devices can only collect 1-2.4 μL / s of liquid from breath, which is too little sample for many purposes, and larger liquid volumes, e.g., 1 mL or more, must be collected to extract and recover a usable liquid biological sample for processing.

[0004] US Pat. No. 7,118,537 describes a device for condensing samples of fluid from respiratory air in which a sleeve surrounding a collection tube can be cooled, for example in a domestic refrigerator, to improve the efficiency of collection.

[0005] Thus, a need exists to rapidly capture liquid particles and vapors from breath samples, such as in one minute or less, for viral, bacterial, biological and chemical analysis. There is further a need to rapidly capture most (or all) of the liquid present in a breath sample and make it available for processing for detection. Summary of the Invention [Means for solving the problem]

[0006] Broadly speaking, the present invention provides methods, devices and kits for collecting biological samples, e.g., in the form of liquid particles, aerosol particles and / or vapor, by capturing the biological sample in a device that includes a surface and a chamber space that condenses or freezes the biological sample as the user exhales one or more breaths through the device. The capture can be on a freezing surface that immobilizes water on contact. The chamber space in the device may freeze and collect the liquid respiratory particles and vapor. The collected sample may be a frozen sample, a combination of liquid and frozen samples, or a liquid sample, depending, e.g., on the temperature of the capture surface and the time between collection and processing. After collection, the liquid biological sample is collected and collected in a vial, e.g., by draining, pushing, scraping, or centrifugal force. The liquid may be collected and mixed with sample preparation reagents, such as viral lysis reagents, internal standards, etc. After collection, the sample is analyzed. The analysis may be performed by PCR, qPCR, RT-PCR, RT-qPCR, digital PCR, LAMP or any nucleic acid detection method, mass spectrometry, spectrophotometry or any analytical tool or method. The nucleic acid amplification or related reagents may include a lysis reagent, such as acetonitrile.

[0007] In a first aspect, the present invention provides a method for detecting targets in a biological sample obtained from a user's breath, the method using a device for collecting a biological sample from breath comprising a tube adapted to allow a user to pass the breath through the device, a collection chamber in fluid communication with the tube and having a capture surface, the collection chamber being a closed-ended tube or terminating in a vial, a cooling element capable of cooling the capture surface to a temperature below the freezing point of water, and a turbulence inducer disposed in or around the tube to cause the flow of breath to be turbulent to enhance contact between the capture surface and the user's exhaled breath, the method comprising the steps of providing a biological sample by passing the breath through the tube as the user's breath condenses or freezes on the capture surface of the collection chamber, optionally allowing the frozen biological sample to melt to form a liquid biological sample for analysis, optionally processing the frozen or liquid biological sample, and analyzing a volume of the biological sample to detect the presence of a target.

[0008] In a further aspect, the present invention provides a method for detecting a target in a biological sample from a user's breath, the method comprising the steps of: (a) directing at least one breath of at least 10 seconds from the subject to a breath collection device, the breath collection device comprising a collection chamber capable of capturing at least a portion of the at least one breath as frost or ice to form a captured volume; (b) processing the captured volume to recover one or more components of the biological sample; and (c) analyzing the one or more components to detect the presence of a target, thereby detecting the target in the biological sample from the subject's breath.

[0009] In a further aspect, the present invention provides a device for collecting targets in a biological sample from a user's breath, the device comprising a tube adapted to allow a user to exhale their breath into the device, a collection chamber in fluid communication with the tube and having a capture surface, optionally the collection chamber being a closed-ended tube or terminating in a vial, a cooling element capable of cooling the capture surface to a temperature below the freezing point of water, and a turbulence inducer disposed in or around the tube to cause the flow of breath to be turbulent to enhance contact between the capture surface and the user's exhaled breath, and the biological sample from the user's breath condenses or freezes on the capture surface of the collection chamber.

[0010] In some cases, the tube has a first end for a user to exhale into the device, and the collection chamber is a vial with an internal capture surface, which is disposed over the second end of the tube, and the flow of breath air is reversed around the inner wall of the vial such that the biological sample condenses or freezes on the capture surface. The collection chamber may be one end of the tube, or the tube may incorporate a vial (e.g., a removable vial to facilitate processing of the collected sample).

[0011] Additionally or alternatively, the device further comprises a turbulence inducer disposed within or around the tube to cause the flow of respiratory air to be turbulent to enhance contact between the capture surface and the user's exhaled respiratory air. This may be accomplished utilizing a turbulence inducer. The turbulence inducer may be a separate component from the tube or collection chamber, such as, for example, an insert, or may be provided by the tube or collection chamber having structure (e.g., uneven surfaces or protrusions) that influence the flow of respiratory air passing thereover to induce turbulence.

[0012] As described further herein, in some examples, the collection chamber is a syringe barrel, the tube fits into the syringe barrel, and the turbulence inducer fits around an exterior surface of the tube.

[0013] In some cases, the tube is open at a first end to allow a user to breathe into the device and has a wall towards the second end to deflect the user's breath onto the capture surface to increase contact between the capture surface and the user's breath.

[0014] Additionally or alternatively, the vials and / or tubes may be removable to facilitate processing of the biological sample or to provide a multi-use device through replacement of the vials and / or tubes.

[0015] In a further aspect, the present invention provides a device for collecting a biological sample from a user's breath, the device comprising a tube adapted to allow a user to exhale their breath into the device, a collection chamber in fluid communication with the tube and having a capture surface, optionally the collection chamber being a closed-ended tube or terminating in a vial, a turbulence-inducing insert disposed within the tube to enhance contact between the capture surface and the user's exhaled breath, and a cooling element capable of cooling the capture surface to a temperature below the freezing point of water, wherein a biological sample from the user's breath condenses or freezes on the capture surface of the collection chamber.

[0016] In a further aspect, the present invention provides a device for collecting biological samples from a user's exhaled breath, the device comprising a tube adapted to allow a user to exhale their breath into the device, a collection chamber in fluid communication with the tube and having a capture surface, and a cooling element capable of cooling the capture surface to a temperature below the freezing point of water, wherein the biological sample from the user's breath condenses or freezes on the capture surface of the collection chamber, the tube being open at a first end to allow a user to breathe into the device, and the tube comprising an end wall towards a second end to redirect the user's breath over the capture surface to enhance contact between the capture surface and the user's exhaled breath.

[0017] In a further aspect, the present invention provides a device for collecting frozen biological samples from a user's breath, the device comprising a tube adapted to enable a user to exhale breath into the device, the tube being in fluid communication with the tube and having a length of 50 cm. 2 and a cooling element capable of cooling the capture surface to a temperature of between about -10°C (optionally about -20°C) and about -40°C, wherein a biological sample from a user's breath condenses or freezes on the capture surface of the collection chamber for about 10 to 120 seconds to provide a biological sample having a volume of between about 20 μL and 250 μL, optionally 180 μL.

[0018] In a further aspect, the present invention provides a device for collecting frozen biological samples from a user's breath, the device comprising a tube adapted to allow the user to exhale breath into the device, a collection chamber in fluid communication with the tube, and a cooling element capable of cooling the capture surface to a temperature below the freezing point of water, the capture surface being cooled to a temperature between about -10°C (optionally about -20°C) and about -40°C, and / or the collection chamber being cooled to a temperature between about 50cm 2 and / or the biological sample from the user's breath condenses or freezes on the capture surface of the collection chamber for approximately 10 to 120 seconds to provide a biological sample having a volume of approximately 20 μL to 180 μL.

[0019] In a further aspect, the present invention provides the use of a device as defined herein for collecting a frozen or condensed biological sample from a user's breath and detecting a target in the biological sample.

[0020] In a further aspect, the invention provides a kit comprising a device as described herein, the kit comprising a plurality of disposable elements of the device and / or reagents for processing a biological sample. The disposable elements may comprise a collection tube, a turbulence inducer, a collection chamber, and optionally a plastic mouthpiece cover.

[0021] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually listed herein.

[0022] Unless the context dictates otherwise, the mechanism descriptions and definitions detailed above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0023] All references mentioned herein are expressly incorporated by reference in their entirety.

[0024] The present invention will now be described, by way of example and not limitation, with reference to the accompanying examples and drawings. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 is a schematic diagram of a device of the invention in which a Peltier element is used to freeze a biological sample in a vial and water aerosol particles and vapor are trapped by capturing breath air into the vial through a straw. [Diagram 2] FIG. 2 is a magnified portion of the device shown in FIG. 1 showing how respiratory air flows through the end of the straw and into the inside of the vial, with the biological sample captured inside the vial as liquid and / or ice. [Diagram 3] FIG. 1 is a diagram of one embodiment of the device of the present invention in which exhaled air is drawn through a vertical straw into a plastic, metal or glass tube vial adapted from a modified syringe. The airflow reverses near the end of the tube and passes through a turbulence inducer to increase contact with the wall surface. [Figure 4]FIG. 4 is a diagram of the device of FIG. 3 but with the respiratory air inlet horizontal rather than vertical. The horizontal tube may include a liquid trap. The respiratory air inlet of FIG. 3 and FIG. 4 may include a mouthpiece. [Figure 5a] FIG. 13 is a diagram of a plunger that redirects excess liquid, thereby equalizing the total volume of collected liquid. [Figure 5b] FIG. 13 is a diagram of a plunger that redirects excess liquid, thereby equalizing the total volume of collected liquid. [Figure 6] FIG. 1 is a diagram of a stacked Peltier collection cooler and reaction heater. [Figure 7] FIG. 1 illustrates collection of liquid from a tube vial of the present invention compared to a conventional tube. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Water vapor and water aerosol particles are present in the breath and may be deposited as liquid and / or ice / frost on the walls of the tube or vial when the temperature of the tube falls well below the freezing point of water. In the device of the present invention, the walls of the tube or vial include a collection surface that can be maintained at a temperature that is typically within the range of -10°C, -15°C, -20°C to -40°C or lower. This means that water vapor, particles, and / or aerosol particles in the breath are deposited on the collection chamber's capture surface where they may solidify and form ice crystals within the device of the present invention, or may otherwise condense on the cold capture surface, for example as liquid droplets.

[0027] Advantageously, in order to quickly and effectively collect the breath condensate, the frozen surface needs to be accessible to the exhaled air, for example by placing the collection chamber in fluid communication with a tube or straw through which the user of the device can exhale. Preferably, the device is adapted to maintain the collection surface at a cryogenic temperature to minimize the phenomenon in which the temperature of the frost increases at the collection surface of the collection chamber as it is collected thereon, potentially preventing further collection of moisture and possibly leading to inconsistent collection. It may also be advantageous to prevent the collection surface from coming into contact with ambient air before collection occurs, thereby preventing some of the collected frost-like sample from coming from the ambient air, rather than from the breath of the user. To this end, the collection surface, which is preferably frozen, may optionally be shielded from ambient air until the device sample is taken up by the collection surface, thereby protecting the collection surface from contact with air other than that in the breath exhaled by the user. For example, in the device of the invention, a tube or straw can be inserted into the device and collection vial beyond a barrier or shield, allowing delivery of a respiratory sample to a frozen capture surface. Prior to insertion of the tube or straw, the capture surface is effectively shielded from the surrounding air until the respiratory sample is drawn into the device. In one embodiment, the capture surface is a tubular vial with means for collecting liquid from the sample when removed from the cryogenic source, allowing collection and processing of samples having a volume of 200 μL or less, or alternatively having a volume of 250 μL or less.

[0028] Additionally, in the prior art, it is extremely difficult to collect very small sample volumes with conventional respiratory sampling, with the consequence that large sample volumes must be collected over many minutes to allow for downstream processing. The devices and methods of the present invention are capable of sample collection rates of up to 2 μL / s, more preferably up to 3-4 μL / s. After collection, the biological sample can be recovered as a liquid and subjected to further processing.

[0029] The collection structure of the device of the invention can take the form of a vial or a tube connected at an end to a vial. The terms "vial" or "tube vial" are used interchangeably herein. In some embodiments of the invention, the collection structure of the device is a tube. Alternatively, the collection structure of the device is a syringe with the plunger barrel removed and the bottom of the syringe capped or sealed. The tube and / or vial of the device is a cryogenic surface that may be flat or curved, smooth or uneven, and may include grooves and indentations to facilitate collection of liquid. The outside of the tube and / or vial is cooled while the breath is captured inside with the breath insert tube. In some embodiments of the invention, the breath insert is a (disposable) straw. The sample is guided into the tube and collected in the bottom of the vial. The frozen surface of the vial is protected from the ambient air until the breath is captured. The shielding material is removed and the frozen breath condensate is collected. Respiratory air enters the device through the mouth in a laminar flow. In some embodiments of the device, the laminar flow is interrupted to generate turbulent flow as the respiratory air flows across a cold surface. The introduction of turbulent flow may occur when the respiratory air flows in a reverse direction at the distal end of the collection tube vial. In some cases, the collection structure is a vial, e.g., a removable vial, but in the case of a tube, a collection vial is incorporated.

[0030] The invention described collects sufficient breath for analysis and virus detection in as little as 10 seconds up to 2 minutes of breath.

[0031] The material collected in the liquid and frozen condensate may potentially include viruses, bacteria, nucleic acids, organic compounds, volatile inorganic compounds, proteins or biological materials present in the respiratory air that will be collected by the device and method of the present invention.

[0032] After collection, the sample is analyzed. Analysis may be performed on the collected material to detect nucleic acids, utilizing devices that amplify and / or label, followed by detection and optionally quantification. Other detection devices and methods include mass spectrometry, LC / MS, spectrophotometry, UV and VIS spectrophotometry, IR spectrophotometry, gas chromatography, liquid chromatography, sequencing, next generation sequencing, culture, colony counting, isothermal and thermocycling nucleic acid amplification, labeling and induction, hybridization, CRISPR, respiratory panels, etc. Applications of the technique include detection of viral and bacterial infections spread by respiratory air, as well as detection of other diseases based on exhaled chemicals. Crucially, if an infectious agent is exhaled or comes out of a person's mouth, then by definition that person is infected. Viruses or bacteria can become airborne or droplets in the air and infect another person. The device may therefore be used as a tool for research and / or diagnosis.

[0033] The respiratory gas intake or sample inlet configuration of the device of the present invention may be vertical, horizontal, or between vertical and horizontal. A vertical respiratory gas intake may be placed straight down into the device, provided that the horizontal is 90 degrees to the instrument. The collection tube to which the intake is connected may be placed in any orientation. In some embodiments of the device, a horizontal or somewhat horizontal sample inlet may be employed. By somewhat horizontal, it is meant that the respiratory gas intake is within 45 degrees of horizontal. In some embodiments of the device, a vertical sample inlet may be employed. The device captures respiratory gas, water as gas, and liquid particulates. The horizontal configuration allows for the capture of respiratory gas without capturing saliva or drool. In addition to being horizontal, the respiratory gas intake may include a recess or trap to capture saliva or very large liquid particles.

[0034] In some embodiments of the device, a vertical sample inlet may be employed. A vertical inlet means that the breath air from the mouth is placed directly above the device and the breath air is guided down into the device. Somewhat vertical means that the tubing is within 45 degrees of vertical. A vertical or somewhat vertical breath air inlet orientation may be advantageous. In addition to capturing gaseous water and small airborne particles, larger liquid particles, saliva or droplets may also be captured. Some people spit or drool when breathing, speaking or singing. This may vary from person to person, with some people generating very large droplets when breathing, speaking or singing, whereas others generate very many droplets. In some embodiments of the device, the breath air inlet mouthpiece may be constructed to capture breath air when breathing out and when speaking or singing. In some embodiments of the invention, the mouthpiece covers a portion of the lips to facilitate sampling by a combination of breathing, speaking and / or singing. The vertical, or somewhat vertical, capture respiratory gas inlet directs respiratory gas / liquid, small respiratory particles and large respiratory particles, including airborne particles and saliva particles.

[0035] A vertical or near-vertical respiratory intake capture device can capture respiratory gases / liquids, small and large respiratory particles, saliva and drool, thus measuring the potential infectivity of different forms of disease exodus from an individual, whereas a horizontal or near-horizontal respiratory intake capture device will limit capture to respiratory gases and liquid particles large enough to remain in the respiratory gas.

[0036] Regardless of how the liquid particles are entrained in the air or what type of liquid particles are captured, the device of the present invention may be used as a tool for research and / or diagnosis. For example, the infectivity of a virus in a particular person depends not only on the virus's infectious ability, but also on the degree to which that person sheds the virus and carries it to another person. Public health safety is more affected by the presence of highly infectious individuals in a crowd than by the presence of infected individuals in a crowd. The capture and collection of water vapor and particles is efficient and effective. Collection is easy, which means that the procedure is performed quickly, with minimal effort, and without discomfort to the person providing the breath sample.

[0037] In addition to collecting liquid from breath, sample liquid may be collected from the ambient space: air may be pumped through the device to collect and detect substances that may be present in the ambient air of a room or building, or even outside the building.

[0038] definition Efficient capture or collection means that most or all of the water vapour and liquid present in the breath sample is captured.

[0039] Efficient capture or collection means that the collection procedure can be performed quickly, with sample collection and preparation for processing in less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than 1 minute.

[0040] Ease of collection means that the procedure can be carried out quickly, with minimal effort, and without discomfort to the person providing the breath sample.

[0041] The collection vial in the device of the present invention is any type of closed tube or structure that allows liquid to be collected directly from the collected sample. The tubular vial of the present invention has a means for collecting liquid from the sample. Any method may be used to combine and collect liquid from the breath in the vial, including gravity, scraping, force or centrifugal force.

[0042] The collection tube of the apparatus / device of the present invention may be any type of tube or structure that allows for the direct collection of liquid from the collected sample. In some cases, the collection tube may be a closed-ended tube or may terminate in a vial.

[0043] Frosted or frozen breath is defined as any water vapor or water aerosol collected from exhaled breath in the devices and methods of the present invention. The collected water may be mostly or partially solid, but some portion may be in liquid form or may dissolve quickly when the device is removed from the cold source or as the sample collection progresses and the device warms up.

[0044] Cryogenic temperatures may be defined as -10°C or below, or as being cold enough to trap at least a portion of the respiratory vapor or liquid particles as ice or frost, i.e., to provide a frozen or partially frozen sample. Cryogenic temperatures may range from about -10°C to -40°C.

[0045] A collection vial is defined as a chamber or vial into which liquid from a breath sample can be directed for collection, further processing or storage.

[0046] Although invisible to the human eye, water vapor and water particles are always present in the respiratory air. The dew point is the temperature at which liquid forms condensate from the respiratory air. When the temperature falls below the dew point and below the freezing point, frost will be collected. Respiratory air frost is water vapor and particles that have solidified and formed ice crystals within the device of the present invention or condensed on a cold capture surface. In the device and method of the present invention, frost is formed and collected from water and air that is at ambient or body temperature when captured within the device.

[0047] The devices and methods of the present invention easily, efficiently and effectively capture liquid water from aerosol particles and vapors from a respiratory sample for viral, bacterial, biological and chemical analysis. Efficient capture means that most (or all) of the water liquid present in the respiratory sample is captured and available to be processed for detection. It is important to collect all of the respiratory sample liquid in which viruses, bacteria or chemicals may be present. If only the most easily collectable parts of the sample are collected, such as the larger liquid particles, a non-representative sample may be collected.

[0048] Since the present invention uses a device with a cryogenic surface temperature (e.g., at the sample capture surface or area), collection is generally more efficient at the beginning of the collection process, and collection efficiency decreases as the volume of collected breath increases and samples are collected. Surface ice, frost, or liquid formed will reduce collection efficiency because the surface temperature is warmed and cannot be cooled as much or as quickly. In addition, since the devices of the present invention are generally small, this allows the water captured by the device to be more easily coalesced and collected for processing. This works against liquid capture, since the mass of the collection device is small because the device is small. As the size of the device decreases, the amount of liquid that can be collected may also decrease. Capture of all or most of the liquid water in the breath may only be efficient for 10, 15, 20, 25, or 30 seconds, or for 1, 2, or 3 minutes, and then will decrease. However, by this time, sufficient breath liquid and vapor will be collected for detection of the desired substance. All of the captured liquid may be processed for detection. In some cases, the method of the present invention includes a further step of processing all or part of the captured liquid biological sample, for example, to enable detection of targets present in the sample. In some cases, at least 25% of the captured liquid sample is used for downstream processing. In some embodiments, at least 50%, 75%, 80%, 85%, 90%, 95% or more of the sample is processed, for example, to increase the sensitivity of detection of targets present in the sample.

[0049] The capture or collection of water vapor and particles is efficient in the device and method of the present invention, which means that most or all of the water vapor and liquid present in the breath sample is captured. The capture or collection of water vapor and water aerosol particles is efficient, which means that the collection process can be performed quickly. The collection of the sample can be performed in less than 5 minutes, less than 2 minutes, less than 1 minute, less than 45 seconds, less than 30 seconds, less than 20 seconds, less than 15 seconds, or less than 10 seconds. The collection and preparation of the sample for processing can be performed in less than 10 minutes, less than 5 minutes, or less than 2 minutes. Effective means for rapid collection and processing and the detection process can be started immediately after the start of sample collection, often within a few minutes. The detection process can be started in less than 10 minutes or less than 5 minutes. This includes dissolving the sample in an organic solvent. PCR detection or LAMP detection can be performed as fast as 20 minutes, but this technology is rapidly improving and the detection time may be further reduced.

[0050] The cold surface area of ​​the devices of the invention is small due to the desire to capture and process small volumes of liquid. In some embodiments of the invention, the volume of the vial into which liquid is collected is 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 0.5 mL, 0.2 mL, 0.1 mL, 0.05 mL or less. In some embodiments of the invention, the cold surface area on which ice forms is less than 100 cm. 2 , 75cm 2 , 50cm 2 , 40cm 2 , 30cm 2 , 20cm 2 , 10cm 2or less. The temperature of the collection surface may increase as the sample is collected, but in some embodiments of the invention, the initial temperature of the cold surface is below 0°C. In some embodiments, the initial temperature of the cold surface may be -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C or lower. The capture surface of the collection chamber may be maintained between 0°C and -80°C, between -15°C and -70°C, or between -20°C and -40°C.

[0051] In order to quickly and effectively collect respiratory frost, the frozen surface needs to be easily accessible. However, if the frozen surface is exposed, frost and liquid can be inadvertently collected from the surrounding air. To prevent this, the surface may be shielded, for example with a shielding material, until a sample is taken to the surface. However, once ice collection begins, collected ice on the surface will increase the collection temperature, which will reduce the efficiency of further collection. Further collection is possible, but collection may occur at a slower rate if the cool interior surface temperature cannot be maintained and increases.

[0052] In addition, sampling of very small volumes of available liquid from breath is difficult with normal respiratory device sampling. The formation of frost on the surface prevents further collection of frost. With the device of the present invention, very small volumes of liquid are collected and processed in an efficient manner. The volume of collected liquid may be less than 200 μL, less than 100 μL, less than 50 μL, less than 25 μL, less than 20 μL, less than 15 μL, less than 10 μL, or less than 5 μL. With the device and method of the present invention, sufficient liquid can be collected from less than 10 exhaled breaths, i.e., less than 9, 8, 7, 6, 5, 4, 3, or less than 2 exhaled breaths. With the device and method of the present invention, usable liquid can be collected even from a single exhaled breath. Usable liquid from a single exhaled breath of an adult can exceed 100 μL, and a significant portion can be captured, typically in the range of 20-80 μL.

[0053] To achieve capture of these sample volumes, the collection chamber volumes of the devices of the invention are generally smaller than those used in prior art devices for collecting breath samples. In some cases, the collection chamber volumes are between 0.5 mL and 50 mL, or between 1 mL and 30 mL, and between 5 mL and 20 mL, or as set forth in the table below.

[0054] [Table 1]

[0055] In one example, the collection chamber has an inner diameter of 8.3 mm, a 0.54 cm 2 Cross-sectional area of ​​19.56 cm 2 The design is made from a 3 mL syringe with a surface area of ​​0.2 mm and a volume of approximately 4.06 mL. The inlet tube has an inner diameter of 0.4 cm and an outer diameter of 0.54 cm. The turbulence inducer consists of a flange that extends outside the inlet tube to a width of 0.72 cm.

[0056] In another example, the collection chamber has an inner diameter of 1.18 cm, 2 The internal surface area of ​​the collection chamber is 22.24 cm. 2 The inlet tube and the turbulence inducer surrounding the inlet tube inserted into the collection chamber occupy approximately half of this volume. The internal volume of the central airway is 0.32 cm 2 The inlet tube has an outer diameter of 7.9 mm and the turbulence inducer flange protrudes to 10.75 mm. The cross-sectional area of ​​the collection tube outside the turbulence inducer is therefore slightly larger than the cross-sectional area inside the central air passage of the turbulence inducer. This difference in cross-sectional area compensates for the increased turbulence in the airflow once the breathed air leaves the central air passage, and allows the breathed air to flow easily without back pressure and contact the cold surface. Due to ease of use and speed of capture, this is used in many of the embodiments of the present invention.

[0057] In another example, a 10 mL syringe has an inner diameter of 1.45 cm and an inner diameter of 1.65 cm. 2 Cross-sectional area of ​​30.52 cm 2 The inlet tube for this example has an inner diameter of 1.0 cm and a working volume of 0.79 cm. 2 The flange on the turbulence inducer extends to 1.4 cm to induce turbulent respiratory airflow.

[0058] In another example, a 35 mL syringe serves as the collection chamber. The syringe has an inner diameter of 2.29 cm, a length of 4.12 cm 2 cross-sectional area of ​​43.12 mL and volume of 75.32 cm 2 In this example, the inlet tube has an inner diameter of 1.5 cm and a surface area of ​​1.77 cm 2 cross-sectional area of ​​18.5 mL and a volume of 49.34 cm 2 This larger option has a lower initial efficiency than the smaller version, but the efficiency does not decrease significantly over the several minutes it takes to collect breath air.

[0059] In the device of the present invention, a freezing capture surface is provided to the respiratory air by a vial or tubular fixture or straw. The tube or straw inlet is effectively shielded from ambient air until the respiratory air can be captured and provided into the device. This can be accomplished by having a barrier at the end of the straw, or simply by having a straw that is long enough so that ambient air cannot easily enter the device.

[0060] In one embodiment of the device, the capture surface is a tubular vial equipped with means for collecting liquid from the sample when it is removed from the freezing source.

[0061] The freezing vial, tube or surface of the device of the invention may be smooth or uneven, such as flat or curved, and may include grooves, baffles or indentations to facilitate collection of liquid. In some cases, the tube may be metal, glass or plastic, and the vial or tube wall thickness may be 5, 3, 2, 1, 0.05 mm or less. The outside of the vial is cooled and the breath air is drawn into the interior of the tube or vial. A disposable straw or tube inlet may be used to draw the exhaled breath air into the vial. The sample is collected in the vial. The frozen surface of the vial is protected from the ambient air until the breath air is drawn. The shielding material is removed, and the frozen and liquid condensate of the breath air is collected.

[0062] Materials collected within the frozen and / or liquid condensate of respiratory air may include viruses, bacteria, spores, organic compounds, volatile inorganic compounds, proteins, and any other biological compounds or materials.

[0063] The collected respiratory liquids may be analyzed to detect nucleic acids. This may be done by amplification and labeling. They may be quantified by various methods including LAMP, PCR, qPCR, RT-qPCR and any other detection device including next generation sequencing. Other detection devices and methods include mass spectrometry, LC / MS, UV, IR, etc. Applications of the technique include detection of viral or bacterial infections spread by exhaled pathogens, which, if present in the respiratory air, are by definition infectious to the subject, or detection of organic molecules. The device may be used as a tool for diagnosis or research. The nucleic acid sample may be RNA or DNA. Preferably, the target nucleic acid is a virus, for example the virus is selected from the group consisting of COVID-19 (caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)), Acquired Immune Deficiency Syndrome (AIDS, caused by Human Immunodeficiency Virus (HIV)), Herpes, Chickenpox, Measles, Influenza, any type of cancer, and others. Other examples include herpes simplex, varicella virus (VZV), respiratory syncytial virus (RSV), Epstein-Barr virus, cytomegalovirus (CMV), coronavirus, rotavirus, hepatitis, monkeypox, Marburg disease, genital warts (human papillomavirus (HPV)) and BK virus. Examples of bacteria which may be detected using the present invention include Mycobacterium tuberculosis (TB) or Staphylococcus aureus.

[0064] In addition to collecting liquid from breath, liquid may be collected from the surrounding space: air may be pumped through the device to collect and detect substances that may be present in the ambient air in a room or building, or even outside the building.

[0065] Cooling of the frost collecting tube or vial may be accomplished using a number of different methods, including but not limited to Peltier coolers, circulating coolers containing liquids at temperatures below the freezing point of water, circulating evaporative coolers, devices that cool by releasing a gas such as compressed carbon dioxide, cryogenically cooled cold surfaces including the use of devices containing or treated with liquid nitrogen or dry ice, and other methods.

[0066] The device of the present invention may be a stand-alone surface where a person approaches the device to deposit a sample. The device may be placed in front of a mouth or in a room. The device may have one sampling inlet or multiple sampling inlets operated in parallel.

[0067] The duration of harvesting may be a few minutes, but can be fairly rapid. In some methods of the invention, harvesting times are 5 minutes or less, 3 minutes or less, 2 minutes or less, 60 seconds or less, 45 seconds or less, 30 seconds or less, 15 seconds or less, 10 seconds or less, or even 5 seconds or less to harvest usable ice and liquid.

[0068] The sampling and detection process utilizes freezing, scraping, or melting ice with a solvent, and optionally adding a lysis reagent followed by collection by analysis. Samples collected in vials or tubes may be centrifuged to combine the liquid and may be picked up for transfer with a pipette. Samples may be collected remotely and mailed, or collected at the point of care. One sample may be collected, or multiple samples may be collected in parallel and processed in a 96-well or 384-well sampling device.

[0069] It is possible to capture and detect viruses directly without lysis or sample preparation. Breath collection by freezing can keep viruses stable, capture all chemicals, and can be done quickly. Capture and processing can be done reproducibly since more than 70%, more than 80%, or more than 90% of the viruses, bacteria, or chemicals can be collected. All of the sample can be processed and detected. A portion of the virus may release nucleic acid that can be detected. However, the virus can be killed or inactivated by adding an organic solvent such as acetonitrile, for example, making the collected liquid safe to handle. Methods, devices, and kits useful for processing nucleic acid samples for storage and analysis, particularly by amplification techniques, are described in the inventor's co-pending International Patent Publication No. 2021 / 209564 (PCT / EP2021 / 059815, filed April 15, 2021), the entire contents of which are incorporated by reference in their entirety.

[0070] Column sample preparation for nucleic acids can be used. Enzymatic degradation of viral proteins can be used to release nucleic acids prior to detection. In other approaches, the detection method involves essentially no sample preparation and nucleic acids may be detected directly from viruses or other materials that contain nucleic acids. Other organics can be detected directly using mass spectrometry and other methods.

[0071] Processing small liquid volumes from breath air is difficult and novel. Efficient capture and manipulation makes detection sensitive and rapid. There is little or no sample preparation for spectroscopic and chromatographic analysis. The collection chambers or vials of the present invention may be placed in a 96-well or 384-well configuration after collection. Analysis of the samples may be automated. Samples may be directly introduced into a mass spectrometer or LC-MS, a microvolume UV spectrometer or other light absorption spectrometer. Nucleic acid detection requires only dissolution in an organic liquid such as acetonitrile for viral and bacterial detection. Detection may involve LAMP, RT-PCR, LC, LC-MS, GC, GC-MS, MS, IR, UV, FTIR, NMR or any analytical technique. Detection may be performed with loop-mediated isothermal amplification, whole genome amplification and multiple displacement amplification, strand displacement amplification and cleavage enzyme amplification reactions, helicase-dependent amplification, recombinase polymerase amplification and SIBA nucleic acid sequence-based amplification and transcription-mediated amplification.

[0072] To be most beneficial and provide a safety margin of infectivity, the method and device of the present invention may detect a virus shedding rate that is 10 times lower than the probability of causing infection for a given situation. For example, a teacher or student at school will be infectious if they are shedding approximately 600 virus particles per minute. Using this method, 30 seconds of breath can be collected from each student to assess the viral load. If the viral load exceeds 300, the subject will be considered infectious, and therefore the present invention will report more than 30 viral particles from this sample. In the most susceptible situation, such as traveling by plane or train, the method of the present invention may test a full minute of breath and detect only 5 viral particles.

[0073] Many LAMP studies have demonstrated limits of detection (LOD) in the range of 100 virus particles, but various techniques are available to increase this sensitivity to the level of detecting 2-3 virus particles. The present invention can increase sensitivity and specificity by incorporating conventional techniques, including fluorescent detection. Novel viral lysis reagents, such as acetonitrile, will increase recovery of viral nucleic acid, further increasing sensitivity.

[0074] In the case of very small volumes, the entire sample may be collected and processed. Detection may be quantitative or may be a simple "yes" or "no" that a substance is present above or below a defined detection limit. Sampling may be rapid, i.e., sampling time may be 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds or less.

[0075] In some instances, the number of exhaled breaths is limited to one exhaled breath capture, two exhaled breath captures, three exhaled breath captures, or between one and ten exhaled breath captures.

[0076] In the present invention, small liquid volumes may be collected and processed from the respiratory air, i.e., in the range of 500 μL, 400 μL, 300 μL, 200 μL, 100 μL, 80 μL, 50 μL, 40 μL, 30 μL, 20 μL or 10 μL or in the range of 5-100, 10-100, 15-300 or 20-100 μL.

[0077] Respiratory gas liquid particles and vapor are captured as frost or ice and liquid in tube vials utilizing surface temperatures of -10° C., -15° C., -20° C., -25° C., -30° C., -35° C., -40° C., -45° C., -50° C., -55° C., -60° C., -65° C., -70° C., -80° C. or lower. Capture may be performed with an active cooling device that removes heat from the cooled surface at the same time the sample is collected.

[0078] The ends of the capture device may be sealed so that air does not pass through the vial, tube or syringe. Because the ends are sealed, the breath capture device reverses or changes the flow through the device. The reverse breath flow reduces the diffusion distance to the cold surface. The air flow may be laminar or turbulent. Breath liquid from particles and vapors is deposited directly in a vial drain or drained directly into the vial. The vial containing the captured liquid may be used directly for processing and detection, including nucleic acid detection. The vial may contain a dissolution solvent. The vial may contain an amplification reagent.

[0079] Cryogenic cooling and freezing of a Peltier device or vial is shown in FIG. 1. Ambient air is shielded from the device by shielding 10 and freezer cover 14. Collection vial 30 is cooled directly by Peltier device 18. Collection vial 30 can be metal or plastic or another material or combination of materials. Breath air is exhaled and drawn into the device by straw 12, and liquid aerosol particles and vapor are captured in collection vial 30. The device provides cryogenic cooling of the walls of collection vial 30, allowing rapid collection of the sample. However, the device has a limited capacity. A heat sink 20 removes heat from the hot side of the Peltier device, and a fan 22 blows air to cool the heat sink to ambient temperature.

[0080] FIG. 2 shows that respiratory air flowing in direction 24 can be captured in collection vial 30. The air flow reverses at the bottom of the vial and then travels along the walls of collection vial 30. The flow can be laminar. A turbulent air flow increases the collection speed and volume. A turbulence inducer 26 can be placed on the outside of the inlet tube 13 as shown here, or on the inside surface of collection vial 30. The direction of the turbulence is indicated by arrow 27 and can be linear and / or irregular, or can incorporate a swirling motion in different directions. Ice and liquid 28 are collected inside collection vial 30 at the walls.

[0081] FIG. 3 shows a device formed of a syringe 34 and a vial with a closed end 38, essentially making the entire syringe into a vial with liquid collection at the end. A copper or metal tube 40 is placed on top of the Peltier device 18 to cool the syringe 34 and the closed end vial 38. Breath air is captured in a vertical tube 32. The inlet of the vertical tube 32 may be circular to place lips around the tube or may be a mouthpiece into which or on which a mouth may be placed. The air breath flow into the tube 32 reverses near the end of the closed end vial 38 and passes through a turbulence inducer 26 to increase contact with the inner wall surface of the syringe 34 and its spaces as well as the closed end vial 38. Frost and liquid may be collected by gravity drainage, solvent washing, centrifugal force, or a plunger may be inserted into the syringe 34. For example, a rapid plunger scraping motion may be used to force the liquid into the closed end vial 38. If a quick plunger motion is used, the closed end vial 38 can be loosened to allow air to vent without allowing liquid to escape.The heat sink 20 and fan 22 are as shown in FIG.

[0082] The device of FIG. 4 has all the same mechanisms as shown in FIG. 3. A horizontal respiratory inlet 33 is used instead of a vertical respiratory inlet. The horizontal tube may include a liquid trap (not shown). The entrance of the horizontal respiratory inlet 33 may be circular for placing lips around the tube, or it may be a mouthpiece into which or on which the mouth may be placed. FIG. 5a and FIG. 5B show a device used to standardize the volume of liquid collected in a syringe. FIG. 5a shows a modified plunger 41 collecting liquid 47 from a syringe 34 into a closed-end vial 38. A relief tube 45 with a top check valve 43 allows air to pass into the modified plunger 41 when the modified plunger 41 is inserted. The collected liquid 47 is scraped by a scraper and discharged into the closed-end vial 38. FIG. 5b shows further downward movement of the modified plunger 41 into the syringe 34 and the closed-end vial 38. A relief tube 45 with a top check valve 43 now allows liquid to pass through the plunger 41 when the modified plunger 41 is inserted, rather than allowing it to go in the opposite direction. Excess liquid 44 passes through the plunger, leaving only a predetermined amount of the collected liquid 49 in the closed-ended vial 38. The closed-ended vial 38 can be removed to access the liquid for further processing.

[0083] A stacked Peltier 58 with collection cooler 54 and reaction heater 52 supported by legs 60 is shown in FIG. 6. A single Peltier wafer would achieve the deep freezing temperature required. However, stacking thermoelectric module wafers can increase cooling capacity if heat is efficiently removed from the other side of the module. The hot side of the reaction heater module 52 can be used to heat the reaction block. Heat is removed by the heat sink 20 and fan 22. For example, the heated side of the device may be used to achieve LAMP detection by maintaining the detection LAMP mixture at 65° C.

[0084] FIG. 7 compares the collection of liquid from the syringe and vial of the present invention in curve 62 compared to a conventional EBC collection tube shown as a straight line 64. The figure shows the difference in collection of liquid aerosol particles and liquid vapor in the device of the present invention and in the conventional device in the figure. The conventional device has a large capacity to capture the breath as a liquid, which is also necessary to be able to collect and process the liquid. The device and method of the present invention collects quickly, but the small size of the device allows the rate of collection to drop quickly. The figure shows the exponential collection of the present invention 62 and the linear collection of the conventional device 64. The total amount of liquid that can be collected with the device of the present invention is low, but sufficient liquid can be collected quickly. The exponential collection is rapid at first and then levels off. Rapid collection is possible, but collection slows down quickly by insulating the ice and liquid from the collected breath or cold surface area. In addition, the surface must be shielded to prevent ambient liquid vapor from collecting as ice and to prevent or slow further capture of respiratory liquid and vapor. Because the volume of the device of the present invention is small relative to prior art devices, R-tubes and other devices, capture of liquid from the ambient air will reduce the ability to capture additional respiratory liquid particles and vapor.

[0085] The workflow steps to the complete process for detecting viruses and bacteria include frost freezing and / or liquid capture, optional lysis with organic solvents and direct detection without additional sample cleanup. Organic solvents kill and deactivate viruses, rendering them incapable of infecting. The workflow of the present invention can detect RNA, DNA, chemicals, proteins, carbohydrates, viruses, bacteria, spores and all biomolecules.

[0086] Visible or fluorescent detection may be used. For high sensitivity, digital PCR may be used. Detection may be one sample at a time, or multiple samples may be in parallel. In some embodiments, groups are examined.

[0087] Sample processing and reporting may be performed using the instrument or using a cell phone or smartphone.

[0088] Detection and reporting may occur on the smart device associated with the presented sample and on the subject's phone along with identification. The subject's smart device may present the sample with a scan barcode, QR code to associate the sample with a specific person using a reporting mechanism. A "yes" or "no" report can be given along with a report giving guidance at a safe distance. If a highly infectious individual was present, an initial report can be provided in the LAMP report, but the LAMP analysis can continue to provide "yes" and "no" answers even at low infectiousness. This technique can quantify the amount of virus, DNA, RNA, bacteria or organic chemicals in a room, airplane or any interior space.

[0089] Active cooling is defined herein as the process of adding cooling while a sample is being collected. Examples of active cooling include salt ice, melting dry ice, or a Peltier cooler. A cryogenic cooler is a cold object that cools another while its own temperature increases. Examples include keeping a cold (not frozen) drink, or a block of metal in a refrigerator or freezer. In some examples, the device may utilize a cryogenic cooler at -10°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C.

[0090] In some embodiments, ethanol or another liquid with a very low freezing temperature may facilitate heat transfer between the vial and the cooling device, hi other embodiments, the cooling surface may fit snugly against the vial.

[0091] Any smartphone or smart device equipped with a camera, internet connection, and capable of running the application can be used for data analysis and reporting. The camera continuously monitors the reaction tubes for changes in color or fluorescence that indicate a positive result. The application (app) processes the data from the camera and reports it to interested parties.

[0092] A cell phone camera may continuously monitor, for example, 96 vial positions and identify them as either fluorescent tubes, non-fluorescent or empty wells. The app records the time when a new tube is added to the rack and when the fluorescence becomes bright enough to be detected. Depending on the processing power, the app may quantify the brightness over time from individual wells and calculate the time of maximum increase in fluorescence. Any time point can estimate the viral load in the sample. By monitoring both the beginning and the end, different samples may be run independently in parallel, with monitoring starting as soon as each tube is added. If there is no fluorescence at a given end time point, for example an hour, the sample is considered negative. If the sample fluoresces, it is considered positive.

[0093] Once a positive or negative result is determined, various predefined groups may be automatically notified. The test subject typically receives a message either through the app or via a text message generated by the app. To receive the message, the test subject must enter their contact information and indicate informed consent for the test. Others who are tested at the same location for a given period of time may also be alerted that they have been exposed. If the viral load is quantified, the level of exposure can be estimated. Depending on the technical capabilities of the location tracking, the exposure may be more accurately estimated. For example, the person who was tested may receive a message saying, "You were likely within one meter of a person shedding 10,000 viral particles per minute for 30 minutes. Your risk of infection is 50%." The test subject may be advised to wear a mask.

[0094] The data may also be sent to the organization conducting the testing as well as local health officials. If the testing was done at a cinema or airport, the cinema or airport may be notified so that they can take action to protect patrons and follow hygiene measures. The airport, among others, may further contact the airline, including those managing the specific flight to take action depending on whether the subject is already on the plane. The destination airport may also be contacted in advance to prepare for potential exposure when the plane unloads passengers. Companies may choose to make the testing status of employees publicly available on the app. For example, "Cashier Steve tested negative at 10:30 a.m."

[0095] The data may also be reported to local health authorities or researchers if desired. If the tests are widely used and recorded, they may add to a growing population of statistical samples for asymptomatic monitoring.

[0096] Test subjects may provide personal contact information and consent to report at the time of testing. In one example, a phone number may be used as it is a unique identifier and a convenient means of making contact. Test subjects may also download an app for more detailed information. If a user chooses not to use the app, they may receive text notifications regarding their test results as well as notifications of possible exposure. If a user chooses to use the app, they may access the current status of their test as well as publicly available test data. Ongoing testing may be expressed in terms of a potential drop in viral load. For example, a high viral load of 40,000 may indicate a positive result after 10 minutes, so if there is no positive result in 10 minutes, the app may report that the viral load is below 40,000. As time progresses, this maximum possible viral load will drop. These numbers can also be expressed in terms of the amount of time and distance one can safely spend with another person, for example, "It is safe for you to talk 6 feet away from someone for 1 hour, and it is safe for you to stand 3 feet away from someone for 1 hour."

[0097] Because time is a function of both the likelihood of transmission and viral load, a "safe distance" can be calculated. As time progresses from the start of testing, the possible viral load exponentially declines if no positive results are detected. The app may calculate the maximum possible viral load in real time and derive the shortest distance the subject can safely maintain for that time period. For example, the app may display in real time, "You can safely stand 5 feet or 1.5 meters away from other people!"

[0098] Experts believe that as few as 300 viral particles are sufficient to cause an infection with SARS-CoV-2. COVID patients are reported to exhale between 60 and 25,000 viral particles per minute, leading to the possibility of widespread transmission. The possibility of transmission from one person to another depends on the amount of virus being shed, the distance between the infectious person and the subject, the duration of contact, and the volume of the room in which they may have been together. These factors can be expressed by the following formula:

number

[0099] This formula can be used to determine the viral shedding rate required to infect another person under given conditions of time, distance, and room volume. For example, a person who maintains a distance of 2 meters for an hour in a large supermarket will not infect another person unless that person is shedding at least 40,000 viral particles per minute, which is considered an extremely high level. In contrast, a person sitting 50 cm from another person for an hour in a medium-sized church can infect someone even if that person is only shedding 300 viral particles per minute. Passengers on long train or plane trips can spread their infection over a 12-hour journey with a shedding rate of only 52 viral particles per minute.

[0100] Quantitative viral load studies have noted that viral loads tend to peak in the first few days of infection, whether sampling is from breath, saliva, or nasopharyngeal swabs, then drop to lower levels before tapering off over several days. Currently, the general public has no way to estimate their own level of infection and is encouraged to remain in isolation for 10 to 14 days as a precaution. A rapid, simple, and affordable quantitative test could allow recovering patients to estimate their own level of infection or to screen for asymptomatic spread in large groups.

[0101] In some aspects, the invention may be to detect illnesses where the infectious agent is exhaled, whether that infectious agent is a virus, bacteria, fungus, etc. Alternatively, in other aspects, the invention may be used as a research tool to develop diagnostics.

[0102] Samples may be collected in less than 5 minutes to capture respiratory liquid particles as frost or ice. Capture is performed in a vial or tube to collect the respiratory gas and then combine the liquid. The use of a vial is convenient for centrifuging the liquid, combining, and then pipetting to a detection device. This process may be automated. The use of a syringe tube to collect the frost is convenient for combining the liquid and depositing it into a processing vial or detection device.

[0103] The breath gas may be directed into a device such as a disposable straw and collected in a disposable vial or tube. Several different turbulence inducers may be used to direct the breath gas so that it can optimally contact the cold surface. In some examples, the cold surface is the inside surface of a 3 or 5 mL syringe barrel. In these examples, the syringe is sealed at the tip and the turbulence inducer can be inserted into the syringe. In one example, the inner barrel of a 3 mL syringe is 6.5 cm long with an inside diameter of 0.9 cm. The turbulence inducer is 8 cm long and its tip is fully inserted into the bottom of the syringe barrel. The base protrudes 1.5 cm out from the syringe and is flared to fit a paper straw into its opening. The interior of the breath gas induction device is a hollow tube with four vents at the tip. The outside of the tube may have various surface structures to optimize the contact of the breath gas with the freezing exterior surface. Examples of similar devices are shown in Figures 1 to 4.

[0104] In one embodiment, the exterior surface of the turbulence inducer is a screw-like helix that directs the respiratory air along the longest possible route back from the syringe barrel. The helix does not fit snugly against the inside of the syringe barrel, allowing some of the respiratory air to leak out, which brings this air into direct contact with the freezing surface. This embodiment may be twisted when removed to minimize accidental removal of respiratory air condensate. In another embodiment, a series of baffles with small vents direct the respiratory air towards the freezing surface, or make sharp right-angle turns to increase turbulence. In another embodiment, the baffles are angled at a certain angle to resemble a herringbone pattern. This pattern also directs the respiratory air towards the freezing surface, while creating pockets of high and low pressure, which causes turbulence. In this embodiment, the turbulence inducer may remain in the syringe while it is being centrifuged, and the condensed liquid will be directed towards the tip.

[0105] The workflow steps require the completion of a process for detecting virus and bacteria samples, including freeze capture of liquid frost from breath, collection and coalescence of the liquid, lysis of the combined liquid or in a detection vial, followed by direct detection without additional sample cleanup. Due to the efficient capture of liquid vapor and particles from breath, it is possible to quantify the amount of virus, bacteria or organic chemicals in a defined volume, such as 1, 2, 3, 4 or 5 exhaled breaths.

[0106] Essentially, it is possible to capture all of the liquid from the breath and process and quantitate all of the liquid samples.

[0107] A portion of the sample in the vial may be retained and stored, or archived for further processing.

[0108] In another embodiment, the sample device may pump a defined volume of ambient air and freeze liquid from the sample to collect the sample to detect viruses, bacteria, spores, nucleic acids, proteins, biomolecules or organic chemicals introduced into a defined chamber.

[0109] The inlet diameter and tube length of the collection tube of the present invention have an effect on the collection volume and resistance to the flow of the respiratory gas inlet. In some embodiments, the tube size may be based on a 1, 3, 5, 10 or 20 mL syringe or even larger syringe volume. As the syringe volume increases, the body of a commercially available syringe has a larger diameter. This may allow the diameter of the respiratory gas inlet tube to increase. This may be advantageous to reduce the resistance to blowing into the tube. In some embodiments, the diameter of the respiratory gas inlet tube is increased to reduce the space between the inlet tube and the collection tube, allowing the respiratory gas to interact with the cold wall and collect condensate.

[0110] Typical syringe sizes may be 3 mL or 5 mL. A 5 mL syringe collector has a higher collection surface area and lower resistance to respiratory gas relative to a 3 mL syringe. The time to collect 50 μL of respiratory gas condensate at refrigerated temperatures of −15° C., −20° C., −30° C., or −40° C. may typically be approximately 15 seconds to collect 50 μL of respiratory gas condensate, approximately 30 seconds to collect 100 μL of respiratory gas condensate, and approximately 60 seconds to collect 150 μL of respiratory gas condensate. Although difficult to quantify, resistance to breathing was insignificant for collections based on a 3 mL syringe collector, but was less noticeable for collections based on a 5 mL closed-end syringe collector. Larger tube collectors and diameters and lengths may allow for faster collection of larger volumes. The 10 mL syringe tube collector has a larger surface area and lower back pressure, allowing a higher volume of respiratory liquid to be collected.

[0111] The turbulence inducer placed inside the closed-end collection tube of the present invention enhances the contact of the respiratory air against the inner cold wall of the collection tube. In one set of experiments with a closed-end 5 mL syringe-based collection tube cooled to -15°C, a spiral-shaped turbulence inducer insert was tested in comparison to a straight straw insert. The turbulence inducer consisted of a hollow tube with an inner diameter of 6 mm and an outer diameter of 8 mm and a length of 65 mm. The mouth inlet was included in the design and created by 3D printing. The outer surface was a spiral-shaped baffle that extended out 2 mm and created 11 coils from the base to the tip of the tube. At the base, the tube was attached directly to the mouthpiece with an inner diameter of 12 mm and an outer diameter of 14 mm and a length of 40 mm. The attachment point between the mouthpiece and the turbulence inducer included a wedge-shaped shaped buttress that allows the turbulence inducer to be pressed firmly into place within the syringe. The tip included four triangular vents to allow the respiratory air to disperse freely through the end of the syringe.

[0112] The performance of the spiral design was compared to a straight-walled inlet straw with an internal diameter of 6 mm. The 5 mL syringe collector was tested at approximately -15°C with 15 seconds of breath, and at three different turbulence conditions. Two trials with the spiral turbulence inducer fully inserted yielded an average of approximately 60 μL of breath condensate. Two trials with a straight-walled straw fully inserted into the syringe barrel yielded an average of 30 μL. Two trials with a straight-walled straw inserted just past the opening of the syringe barrel yielded an average of 15 μL of breath condensate. Thus, yields increased with the introduction of turbulent air flow past the cold face of the collector tube.

[0113] There are several different turbulence inducer designs, all of which have been found to outperform straight walled straws. These include channel separators, open chevrons, staggered projections, and helices with irregular projections. In one design, the open chevron turbulence inducer has the same overall dimensions as the helical design, but instead of a helix extending from the outer surface, there is a series of broken chevron shapes. Each of these consists of a pair of wedges 3mm long. These wedges abut against the cold surface of the syringe to guide the respiratory air into a turbulent path while allowing the liquid to flow easily towards the tip of the syringe. There are four such rows of broken chevrons arranged from the base to the tip of the turbulence inducer, with eight broken chevrons in each row.

[0114] Each form of turbulence inducer was designed to achieve and balance two primary objectives. First, the overall dimensions should minimize the backpressure created when breathing through the device. Second, the various baffles and flanges should impede the air flow enough to create turbulence and maximize contact of the breathed gas with the cold exterior surface. In these embodiments, to minimize backpressure, the cross-sectional area of ​​the inner tube was roughly half the total cross-sectional area of ​​the syringe. This allowed the air to travel through a channel with a consistent overall width.

[0115] The helical design directs the breath air in the longest possible path along the surface of the syringe, thus maximizing the opportunity for condensation. Because the turbulence inducer does not create an airtight fit, some of the breath air was able to go beyond the helical deflector. This further encouraged the air to come into contact with the cold surface and further encouraged the breath air to condense. In some forms of the helical design, wedge-shaped protrusions redirected some of the airflow from its smooth helical path. This allowed the airflows to interact with each other and create turbulence, which also increased contact between the air and the cold surface.

[0116] In another design, vertical and horizontal baffles protruded from the outside of the tube. In each case, there was an open channel for the air to pass through, but the path was long and circuitous. The baffles were angled so that a significant portion of the air passed around them and came into direct contact with the cold wall.

[0117] After capture, collection of the liquid into a vial at the closed end can be done with a scraping plunger or with a centrifuge. As described above, in collection with a plunger where the vial at the end of the barrel is sealed, the plunger needs to allow air to escape when it is pushed down or inserted into the barrel. Using a centrifuge may eliminate the need for special plunger designs. Insertion of the collected respiratory condensate and processing it in a centrifuge will move the liquid into the collection vial and also expel the air. Scraping with a plunger is not necessary when applying centrifugal force. In some embodiments, the turbulence inducer does not need to be removed and the tube can be centrifuged to directly collect the liquid in the closed end vial.

[0118] Turbulence inducer designs can enhance liquid collection by centrifugal force. The broken chevron design is one design suitable for combining and collecting captured respiratory gas liquid in a centrifuge because it allows both turbulent air flow from the syringe tip to the opening and unimpeded water flow from the opening to the tip.

[0119] Several application specific centrifuge rotors were tested for collecting exhaled breath condensate. Three different mounts were designed and these were spun on three different rotors. In the rotors tested in these experiments, the closed end capture tubes were in the same plane. In other embodiments, the capture tubes may be angled for easier insertion into the centrifuge. One battery powered rotor rotates at approximately 500 rpm, one hand cranked rotor can rotate at approximately 1000 rpm, and one AC powered rotor rotates at 4000 rpm. The mounts for holding the syringes in place on the centrifuge rotor can have at least three basic configurations. In one configuration, two syringes were held in place 75 mm from the center of the mount, directly opposite each other and aligned with each other. In another configuration, the syringes were placed off-center, allowing them to be loaded more easily without interfering with each other. In this configuration, the base of the syringes was 5 mm from the center of the rotor in the direction the syringes were facing and offset laterally by 15 mm. In a third configuration, the syringes were mounted in a vertical stack facing in the opposite direction. The base of each syringe was set 5 mm from the axis of rotation.

[0120] Each mount was tested on a battery-powered 500 rpm rotor. 100 μL of water was dispensed along the length of the turbulence inducer before inserting it into a syringe. Ten tests were then run for 10 seconds with each mount in the straight, off-center, and vertically stacked configurations. The water collected in the vial was then measured. The vertical stack collected an average of 89.1 μL, the off-center configuration collected an average of 88.5 μL, and the straight configuration collected an average of 94.7 μL. The difference between the straight configuration and the other two configurations was statistically significant, but the difference between the vertical stack and the off-center configuration was not statistically significant.

[0121] Because of its geometry, the force at the base of the syringe is 15 times greater in the in-line arrangement than the other two, but only changes at the tip by a factor of 2. For bench-top convenience, a more compact, easier-to-load design such as a side-by-side arrangement may be preferable, but in other cases the in-line arrangement may be more effective.

[0122] In tests using exhaled breath condensate rather than manually adding droplets of water, a battery-powered motor with a side-by-side mount was less efficient, capturing less than half the exhaled breath. A rotor with a more powerful AC motor was substituted, capable of spinning at 4000 rpm, eight times the speed. This allows for 64 times the centrifugal force, which is enough to collect over 99% of the exhaled breath condensate. A comparison is shown in Table 1.

[0123] [Table 2]

[0124] Effect of cooling time for collection syringe barrels In some cases, the syringe may be placed in a cooling device for a period of time before breathing through the mouthpiece. When testing with a broken Chevron turbulence inducer in a 5 mL syringe, a comparison was made between blowing through the tube immediately after insertion, waiting 20 seconds after insertion, or waiting 1 minute after insertion. In all cases, breath air was collected for 20 seconds and the device was allowed to cool to below -20°C between tests. Tests in which breath air was collected immediately after inserting the syringe yielded an average of 61.25 μL. Tests in which the syringe was cooled for 20 seconds yielded an average of 75.17 μL, a statistically significant increase over immediate sampling. Cooling the syringe for a full minute did not increase yields versus waiting 20 seconds.

[0125] Syringe barrel size comparison In one embodiment of the present invention, a syringe barrel may be used for closed-end tube collection. The yield of exhaled air collected from a 3 mL syringe and a 5 mL syringe was compared over various periods of collection time. In each case, a helical turbulence inducer was used and the liquid was collected using a plunger. When collected for 10 seconds with the 3 mL syringe, the yield ranged from 32 μL to 46 μL, averaging 40 μL. When collected for 15 seconds with the 3 mL syringe, the yield ranged from 30 μL to 65 μL, averaging 49.5 μL. When collected for 30 seconds with the 3 mL syringe, the yield ranged from 60 μL to 84 μL, averaging 67 μL. The 5 mL syringe had a range of 42 μL to 55 μL at 10 seconds, averaging 52.6 μL. When collected from a 5 mL syringe for 15 seconds, the yield ranged from 61 μL to 75 μL, with an average of 68 μL. When collected from a 5 mL syringe for 30 seconds, the yield ranged from 60 μL to 83 μL, with an average of 64.5 μL.

[0126] Syringe Dimensions Two sizes of syringes were extensively tested: 3 mL and 5 mL. A 3 mL syringe has an internal diameter of 9 mm and a length of 70 mm. This is approximately 19.8 cm. 2 A 5 mL syringe has an internal diameter of 13 mm, a length of 65 mm, and an internal surface area of ​​26.5 cm 2 For larger syringe barrels, the sampling surface is 50, 100, and 150 cm 2 or even larger.

[0127] Centrifugation process and reagent mixing The centrifugation process, which coalesces the liquid against the end of the tube collection vial, also provides mixing capabilities. In one set of experiments, a drop of food coloring gel dye was added to the top of the collected liquid and to the bottom of the collection vial. Centrifugal force was applied for 15 seconds. In both cases, the dye mixed thoroughly with the liquid, providing uniform coloring throughout. In addition, all air was removed. This mixing method is useful for mixing solvents, master mixes, or any storage or detection buffers.

[0128] detection The collected RNA and DNA can be detected by various methods, including CRISPR-Cas-based nucleic acid detection, rolling circle amplification, helicase-dependent amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, real-time quantitative polymerase chain reaction, n-counter (Nanostring), SMART-seq cDNA (Takara), single molecule FISH (smFISH), single molecule optofluidic chip, mass spectrometry with or without amplification, and any other suitable detection method. There are many detection methods under development, any of which would be suitable for detecting and analyzing nucleic acids. The liquid from the device can be transferred to a vial or flow tube to facilitate detection and analysis. Proteins, carbohydrates, lipids and other biological materials can be detected from the collected liquid.

[0129] Sample collection, processing and detection process The sample flow process using the device of the present invention may proceed in different ways depending on the analyte and the purpose. The steps of the overall process are shown here. 1. Collect breath samples 2. Optionally analyze directly 3. Optionally Process the Sample a. Physical treatment such as heating b. Solvent treatment c. Enzyme treatment d. Chemical treatment such as labeling 4. Analyze the sample A. Direct analysis b. Optional transcription c. Optionally amplify i. Direct analysis ii. Selective labeling and analysis 5. Report the results a. Individually b. At the venue

[0130] Respiratory condensate is processed and collected by the device. Collected samples are either analyzed directly or processed and then analyzed. Post-analysis data is reported as yes / no and / or as a quantity.

[0131] Pre-analytical sample treatment is performed in different ways depending on the analyte and detection process. Treatment with organic solvents or other chemical reagents, or with heat, may be used to release or dissolve the sample and preserve the sample. In some embodiments, the sample may be treated or processed with a nucleic acid sample preparation kit. The organic solvent is water-miscible. The organic solvent is aprotic.

[0132] In some embodiments, nucleic acid reverse transcription may be performed directly or after sample processing. In some embodiments, nucleic acid amplification may be performed directly after addition of amplification reagents, after reagent processing, and / or after reverse transcription. Amplification may be performed by thermal cycling or isothermally. Detection may be a two-step detection, for example, CRISPR or Illumina respiratory panels. In some embodiments, the analyte may be labeled and detected. Labeling may be performed as part of the amplification process.

[0133] The sample matrix is ​​most often water and the analytes are collected as part of the process of collecting the water. The organic material that is collected is soluble or at least compatible with the water sample matrix.

[0134] Analytes that may be detected include RNA, DNA, proteins, carbohydrates, lipids, sugars, inorganic or organic molecules. The diseases being tested for may be associated with viruses, bacteria, fungi, cancer and other biological diseases.

[0135] Testing can be done in many different types of venues to ensure safety. Venues in which the invention may be used include entertainment venues such as concerts, theme parks, movie theaters, transportation facilities such as airports, ferries, trains and buses, high-risk population centers such as hospitals, nursing homes, ICUs and neonatal care facilities, and other venues such as schools, community centers, churches, shopping malls, public meetings, assemblies and businesses.

[0136] In one embodiment of the invention, dry ice was added to the top of the Peltier cooler in the device shown in FIG. 3. In another embodiment of the invention, dry ice took the place of the Peltier cooler in the device shown in FIG. 3. A Peltier cooled device can typically cool from ambient temperature to -20°C in roughly 2-5 minutes, whereas dry ice alone will cool the device to -60°C or lower in less than a minute. In one experiment, a 20 second breath sample raised the temperature of the Peltier cooled device by 4-12°C, whereas in the dry ice cooled device, the sample raised the temperature by 10-20°C, but in both cases the device returned to its base temperature within about 30 seconds. Both were tested with ethanol to promote thermal contact between the EBC collector and the device cooler, and the dry ice cooled device collected an average of 95 μL in 20 seconds, whereas the Peltier cooled device collected 80 μL in 20 seconds. Some data for dry ice cooling with and without ethanol is shown below in Table 2.

[0137] [Table 3] EXAMPLES

[0138] EXAMPLES

[0139] A 40 mm square thermoelectric cooler, Peltier module TEC1-12706, was placed hot side down on a 70 mm square aluminum heat sink equipped with a cooling fan. An aluminum 20 × 20 × 15 mm freezing cube with a 9.7 mm hole was placed on the cold side of the Peltier module. A 20 mm long circular aluminum tube vial, with dimensions of 9.5 mm outer diameter with a central hole with an inner diameter of 7.5 mm, was placed in the freezing cube in a straight upright position. A craft paper straw approximately 20 cm (7.75 in) long with an outer diameter of 6 mm was placed in the vial in a straight position. A 3D printed plastic attachment with vent holes maintained the straw in a straight position. With this device, respiratory air can be exhaled through the straw and into the inner wall of the cooled vial. Breath vapor and liquid particulates collected on the inside walls of the vial, while cleaned breath air escaped in the opposite direction to the top of the vial. Figure 1 shows the configuration of the collection device of the present invention used in this experiment.

[0140] In one set of experiments, 12V was applied to the Peltier cooler for 60 seconds. One breath was exhaled through a straw, which took 8-12 seconds. The vial was removed and centrifuged for 10 seconds. 5μL of liquid was combined and collected with the tip of a 20μL pipette. Some liquid remained in the vial. The collected liquid was placed in a 0.2mL PCR tube with 15μL of master mix, solvent lysate and primers derived to detect the desired virus. RT-qPCR with a Chai Bio (Santa Clara, CA) 16-well instrument was run for 40 minutes to detect the presence of the virus.

[0141] In another experiment, cooling was applied for 120 seconds and 1.5 exhaled breaths were collected. This experiment was performed four times. The vials were centrifuged each time the liquid was combined and collected. In two of the experiments, 15 μL of exhaled breath liquid was collected. In the other two experiments, a sample size of 20 μL of breath was collected. EXAMPLES

[0142] Two stacked Peltier modules were mounted on an aluminum heat sink with a cooling fan at the bottom, with an 8 cm tall copper tube with an 11 mm inner diameter attached to a copper plate at the top. The tube was insulated with foam and 2 mL of ethanol was added to the tube. A 3 mL disposable Luer-lock syringe, 7 cm long and 1 cm in diameter, was sealed with a cap and placed into the tube, allowing the ethanol to rise around the syringe to the rim of the tube.

[0143] The Peltier module cooled the tube to approximately -40 °C. The 3D printed plastic device and turbulence inducer for inducing respiratory air was inserted into the syringe and respiratory air was induced through it for 15, 20, 25 and 30 seconds. After each test, the syringe was removed from the tube and carefully dried. The plastic respiratory air induction tube was removed and the plunger was partially inserted into the syringe. The cap was then removed and replaced with a 200 µL PCR tube, which does not form an airtight seal. The plunger was depressed sufficiently and the syringe was spun in a manual centrifuge to elute all the liquid. In these five experiments, a range of 40-90 µL of liquid was collected over a range of 2.5 µL / s to 3.6 µL / s. EXAMPLES

[0144] A 2.2 cm diameter, 8.8 cm long aluminum tube with an internal diameter of 1 cm was cooled on dry ice to -40 °C. One end was plugged and 2 mL of ethanol was added to the tube to aid thermal contact. A 7 cm long, 1 cm diameter, 3 mL disposable luer lock syringe was sealed with a cap and placed into the tube, allowing ethanol to rise around the syringe to the edge of the tube. A 3D printed plastic device for directing respiratory gas and inducing turbulent flow was inserted into the syringe and respiratory gas was directed through it for 10 seconds. This experiment was repeated six times, with liquid yields ranging from 25 to 44 µL or 2.5 to 4.4 µL / s. EXAMPLES

[0145] In another set of experiments, two different metal tubes were compared. 3 mL disposable Luer-lock syringe barrels, 7 cm long and 1 cm in diameter, were sealed with caps and placed one inside a copper tube and the other inside an aluminum tube, both cooled to -30°C to -32°C. A 3D-printed plastic device for directing respiratory gas and inducing turbulence was inserted into the syringes, and respiratory gas was directed through them for 15, 20, 25 and 30 seconds, using one exhaled breath all the way. The volumes of liquid collected from the frost are shown in Table 3.

[0146] [Table 4]

[0147] The results also show that frost collection in the device of the present invention is rapid at first and then tapers off as the frost is collected. In this experiment, enough liquid for analysis was collected in 15 seconds in both cases. EXAMPLES

[0148] VosCryo Device and Process VosCryo is an example of an inventive device for collection of exhaled breath liquid particles and vapors. Sampling often takes less than a minute or even less than 30 seconds to generate sufficient exhaled breath condensate (EBC) for analysis. When the test subject breathes through the mouthpiece, the breath is directed to strike the cooled surfaces inside the syringe barrel and collection tube. Droplets and vapor from the breath condense on the cold surfaces. After collection, the condensate may be collected by scraping, draining or centrifugation, with centrifugation allowing for rapid collection into a collection vial. This process consistently results in more than 50 μL of EBC sample ready for analysis using PCR, RT-PCR, RT-LAMP, RPA microbial culture, mass spectrometry or other analytical tools. RPA (Recombinase Polymerase Amplification) is similar to LAMP and uses isothermal amplification, but at lower temperatures, e.g., 30°C to 40°C.

[0149] For collection, a 3 mL syringe barrel with a cap or vial attached to a Luer end fitting was placed closed end down into a vertical copper tube. The vertical copper tube was fixed perpendicular to a copper plate that was cooled by a 24 V thermoelectric Peltier tip (40 × 40 × 4.7 mm, DigiKey3.5 A 2223-CP354047-ND). The copper tube had an inner diameter of 15.88 mm (5 / 8 in), an outer diameter of 14.29 mm (9 / 16 in), and a length of 8.89 cm (3.5 in) (height above the copper base plate). The base plate into which the tube fitted was 3.1 mm (1 / 8 in) thick and 3.81 cm (1.5 in) on a side. The Peltier tip was attached to the underside of the copper base plate with Artic Silver thermal paste. A voltage was applied such that cold temperature was transferred to the copper plate. Excess heat was removed from the hot side of the Peltier chip by an aluminum heat sink with radiator fins and a 90 CFM fan to blow air across the radiator fins. The Peltier used 3.8A and the fan added 0.31A for a total of 4.1A. Thermal contact between the cold copper tube and the syringe inserted into the tube can be increased by adding alcohol to the inside of the copper tube before inserting the closed-end syringe barrel.

[0150] After collection, in some embodiments, a syringe plunger may be placed into the syringe to scrape the liquid and solidify it. In this case, the closed end of the tube is opened to allow air to escape, or the plunger is modified so that air escapes along with the plunger when it is placed into the syringe. In some embodiments, a centrifuge is used to collect the liquid in a vial at the end of the tube.

[0151] In this example, the device and kit components for collecting 50 breath samples were supplied as follows: Packing List: Quantity Equipment :(1) 1. Cooling device Power supply and power cord 2. Low-force dual-channel centrifuge Side mounted on / off switch Adapter for fitting sample tubes Safety hinged cover Direct Power Cord The operation was at 4000 rpm and 10 seconds.

[0152] Disposable Sampling Packets(50) 1.3.5mL syringe barrel 2. Turbulence inducer with a mouthpiece inserted into a syringe barrel 3. Collection vial (attached to a syringe barrel) 4. Plastic mouthpiece cover (removed before use)

[0153] Initial setup of the device: 1. Plug the power cord into a wall outlet and power source. 2. Once the cooling unit is plugged into a power source, the unit will begin cooling immediately. Add 3.4 mL of alcohol (ethanol or isopropanol) to the copper tubing of the device. This aids in the heat transfer as well as sterilization. 4. Allow the apparatus to initially cool for approximately 6 minutes before collecting the first sample.

[0154] Sample Collection: 1. Tear open the sealed sampling packet and hold the syringe by the plastic sleeve and place it into the copper tubing of the apparatus. 2. Allow the syringe barrel with the mouth insert to cool for at least 10 seconds before collecting the breath sample. 3. Have the test subject remove the sleeve and breathe firmly through the mouthpiece for 20 seconds (approximately 2 deep breaths). 4. Typically, 20 seconds and two complete breaths are adequate to collect a 50 μL sample. 5. Children may require more breaths in the same time frame. 6. Multiple breaths or longer sampling times will increase sample yield. 7. To compensate for evaporation, add 0.5 mL of alcohol to the copper tube for every 5 samples.

[0155] Important guidance for sample insufflation: 1. When providing a breath sample, blow in firmly until you feel uncomfortable. 2. Blowing into the device is similar to blowing up a balloon or blowing into a windmill to make it spin. 3. Make sure you empty your lungs completely with each breath. 4. You can only exhale through the mouthpiece. To inhale, you can either remove your lips from the mouthpiece or inhale through your nose. 5. Moderate rhythmic breathing results in lower volume yields.

[0156] Factors that affect respiratory collection yield: Temperature - Under normal ambient conditions of 20°C, the VosCryo collector cools to -20°C within 6 minutes and continues to cool more slowly to -40°C. The instrument collects larger sample volumes as the collection temperature decreases, ideally when the temperature of the copper assembly drops below -20°C. The instrument is recommended to be used between -20°C and -40°C. Frost will form on the lip of the copper well within this temperature range. Under warmer ambient conditions, the collection temperature will increase and collection times will need to be increased. In another configuration of the VosCryo instrument, cooling dropped below -20°C within 2 minutes. Cooling continued to approximately -30°C after 10 minutes, eventually reaching -40°C after 15 minutes.

[0157] Time-EBC collected volumes increase with sampling time. The following volumes were acquired at starting temperatures of -20°C to -28°C, exhaling one complete breath for 10 seconds each. Sampling Time Minimum Expected Volume 10 seconds 40μL 20 seconds 70μL 30 seconds 100μL 40 seconds 120μL

[0158] Breath rate-collection rate is related to the number of breaths sampled. One complete breath every 10 seconds is recommended for sample collection. Note that the collected volume generally increases with more breaths regardless of the breathing rate, but is no longer linear after approximately 50 μL is collected.

[0159] Lung volume-physiological differences between test subjects can lead to variations in the volume of breath air and therefore the volume of EBCs collected. If an adequate sample is not collected from a given test subject, the sampling process is simply repeated to collect a larger sample. As warm breath air is drawn into the device, a small rise in the temperature of the cooled copper can be detected. The amount of energy can be quantified and correlated to the amount of breath air drawn. In this manner, a green light can indicate that the device is cool enough to begin drawing a breath sample. Then, as breath drawing proceeds, a yellow light may be indicated to indicate a slight rise in the temperature of the device. A red light can signal that the sample draw may be terminated and that a sufficient specified sample volume has been collected. The appearance of the red light can be correlated to time, EBC volume, amount of temperature rise and / or amount of electrical energy required to counteract the warming of the device due to the volume of warm breath air drawn. In this manner, sampling can be standardized between individuals. Either way, with clear breathing instructions, a minimum sample time will generate an adequate volume.

[0160] Sample Liquid Collection and Processing: 1. Remove the syringe from the apparatus and place directly into the centrifuge. Identify and note the sample number in the centrifuge. 2. Ensure the centrifuge is balanced by loading two samples, or one sample and one unused syringe. 3. Close the lid and ensure the green power switch is in the ON position. 4. Hold the black switch forward to run the centrifuge for 10 seconds. 5. Twist the vial off the syringe tip. The collected liquid may be removed via pipette or may be stored in the included cap. 6. Reagents including acetonitrile and reverse transcriptase, control reagents, and amplification reagents may be added to the tube before or after centrifugation, which will combine and mix the reagents.

[0161] COVID 2 standard for LAMP detection after acetonitrile treatment: 1.10-20% acetonitrile dissolved samples are prepared with a final acetonitrile detection concentration of 1-2%. Materials: NAT-rol COVID positive control (50 cp / μL), COVID LAMP primers (E and N), acetonitrile, 2x LAMP mix, nuclease-free water, 50x fluorescent dye, 50x guanidine hydrochloride (New England Biolabs). 2. Prepare 50 μL of 10% acetonitrile in vial sample. Add 5 μL of acetonitrile to 45 μL of sample and mix thoroughly by pipetting. Make a 10% acetonitrile control by adding 5 μL of acetonitrile to 45 μL of water. 3. Add master mix, primers, incubate at 65°C and detect. Positives are bright pink.

[0162] Samples can be analyzed using LAMP, PCR or other analytical tools. EBC treated samples were analyzed by RT-PCR and RT-LAMP. Possible methods include PCR, RT-PCR, RT-LAMP, microbial culture, mass spectrometry or other analytical tools including digitized nucleic acid amplification methods. Amplification methods may be performed by thermal cycling or isothermal. Some isothermal amplification methods include NASBA (Nucleic Acid Sequence-Based Amplification, a method used to amplify RNA), LAMP (Loop-Mediated Isothermal Amplification, a single-tube technique for the amplification of DNA. It uses 4-6 primers, and the primers form a loop structure to facilitate subsequent rounds of amplification), HAD (Helicase-Dependent Amplification, which utilizes the double-stranded DNA unwinding activity of helicase to separate the strands for in vitro DNA amplification at a constant temperature), RCA (Rolling Cycle Amplification, which starts with a circular DNA template and a short DNA or RNA primer to form a long single-stranded molecule), MDA (Multiple Displacement Amplification, a technique that begins when multiple random primers anneal to a DNA template and a polymerase amplifies the DNA at a constant temperature), and RPA (Recombinase Polymerase Amplification, a low-temperature DNA and RNA amplification technique).

Claims

1. 1. A method for detecting a target in a biological sample obtained from a user's exhaled breath, comprising: The method includes using a device for collecting biological samples from breath, the device comprising: a tube (32) adapted to allow the user to pass breath through the device; a collection chamber in fluid communication with the tube and having a collection surface; a cooling element (40) capable of cooling the collection surface to a temperature below the freezing point of water; and a turbulence inducer (26) comprising structure disposed in or around an outer surface of the tube to induce turbulence in the flow of breath to enhance contact between the collection surface and the user's exhaled breath; The method comprises: providing a biological sample by passing breath through the tube as the user's breath condenses or freezes on the capture surface of the collection chamber; analyzing a volume of said biological sample to detect the presence of said target; A method comprising:

2. 1. A device for collecting a biological sample from a user's breath, comprising: a tube (32) adapted to allow the user to exhale their breathing into the device; a collection chamber in fluid communication with the tube, the collection chamber having a capture surface; a cooling element (40) capable of cooling said capture surface to a temperature below the freezing point of water; a turbulence inducer (26) comprising structure disposed in or around the outer surface of the tube for causing turbulence in the flow of respiratory air to enhance contact between the capture surface and the user's exhaled air; The device wherein the biological sample from the user's exhaled breath condenses or freezes on the capture surface of the collection chamber.

3. 2. The method of claim 1, wherein the tube has a first end through which the user exhales into the device, the collection chamber is a vial having an internal capture surface, the vial is disposed over the second end of the tube, and the flow of exhaled air is reversed around the interior wall of the vial, causing the biological sample to condense or freeze on the capture surface.

4. 4. The method of claim 1 or claim 3, wherein the biological sample is a frozen sample, a combination of a liquid sample and a frozen sample, or a liquid sample.

5. 4. The method of claim 1 or claim 3, wherein the collection chamber has a volume of 0.5 to 50 μL.

6. The method of claim 1 or claim 3, wherein the tubes are used in a vertical configuration.

7. 4. The method of claim 1 or claim 3, wherein the tube is used in a horizontal configuration to allow capture of respiratory gases without capturing saliva or drool.

8. The method of claim 1 or claim 3, wherein the tubes are used in a configuration between horizontal and vertical.

9. The method of claim 1 or claim 3, wherein the turbulence inducer is selected from an insert disposed within the pipe.

10. 10. The method of claim 9, wherein the collection chamber is a syringe barrel, the tube fits over the barrel of the syringe, and the turbulence inducer fits around an exterior surface of the tube.

11. 4. The method of claim 1 or claim 3, wherein the turbulence inducer comprises structure on an inner wall of the tube sufficient to induce turbulence in respiratory gas passing thereover.

12. 4. The method of claim 1 or claim 3, wherein the tube is open at a first end to allow the user to breathe into the device, and the tube has an end wall toward a second end to deflect the user's breath onto the capture surface to increase contact between the capture surface and the user's breath.

13. The method of claim 1 or claim 3, wherein the collection chamber and / or the tubing are removable to facilitate processing of the biological sample or to provide a multi-use device through replacement of the collection chamber and / or the tubing.

14. 4. The method of claim 1 or claim 3, wherein the method or device has a sampling time of 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds or less for the user to breathe into the device.

15. 4. The method of claim 1 or claim 3, wherein the biological sample is captured in no more than six exhaled breaths, optionally in one or two exhaled breaths.

16. The method of claim 1 or claim 3, wherein the biological sample is a liquid sample, a particulate sample, or a vapor sample of the user's breath.

17. 4. The method of claim 1 or claim 3, wherein the collection chamber is a removable vial.

18. The method of claim 1 or claim 3, wherein the cooling element uses thermoelectric cooling to cool the capture surface.

19. The method of claim 1 or claim 3, wherein the cooling element is a Peltier device.

20. 4. The method of claim 1 or claim 3, wherein the capture surface is cooled to -10°C or below, optionally the capture surface is cooled to -20°C or below, optionally the capture surface is cooled to -40°C or below.

21. The capture surface is 50 cm ２ 4. The method of claim 1 or claim 3, wherein the surface area is less than 100 nm.

22. 4. The method of claim 1 or claim 3, wherein the device comprises a removable shield to protect the capture surface from ambient air until respiratory collection begins.

23. 4. The method of claim 1 or claim 3, wherein the device is capable of collecting samples at a rate of up to 2 μL / s, and optionally up to 4 μL / s.

24. 4. The method of claim 1 or claim 3, wherein the cooling element is switchable to allow a frozen biological sample to be thawed for analysis.

25. The method of claim 1 or claim 3, wherein the biological sample comprises nucleic acids.

26. The method of claim 1 or claim 3, wherein the nucleic acid is RNA or the nucleic acid is DNA.

27. The method of claim 1 or claim 3, wherein the biological sample comprises a virus, a bacteria, a yeast, a tissue cell, or an organic molecule.

28. The method of claim 1 or claim 3, wherein the biological sample comprises a virus.

29. 29. The method of claim 28, wherein the virus is selected from the group consisting of COVID-19 (caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)), acquired immune deficiency syndrome (AIDS, caused by human immunodeficiency virus (HIV)), herpes, chickenpox, measles, influenza, herpes simplex, varicella zoster virus (VZV), respiratory syncytial virus (RSV), Epstein-Barr virus, cytomegalovirus (CMV), coronavirus, rotavirus, hepatitis, genital warts (human papillomavirus or HPV), and BK virus.

30. 28. The method of claim 27, wherein the bacteria is selected from the group Mycobacterium tuberculosis (TB) or Staphylococcus.

31. 4. The method of claim 1 or claim 3, wherein the final liquid volume of the captured biological sample is 250 μL, 125 μL, 100 μL, 80 μL, 60 μL, 40 μL, 20 μL or less.

32. 4. The method of claim 1 or claim 3, further comprising analyzing the biological sample by LAMP, RT-PCR, LC, LC-MS, GC, GC-MS, MS, IR, UV, FTIR or NMR.

33. A method as described in claim 1 or claim 3, wherein the collection chamber is a closed-end tube (34) or terminates within a vial (30).

34. The method of claim 1, further comprising the step of allowing the frozen biological sample to thaw to form a liquid biological sample for analysis.

35. The method of claim 34, further comprising a step of processing the frozen or liquid biological sample.

36. 10. Use of a device according to claim 2 for collecting a biological sample from a user's breath and detecting a target in the biological sample.

37. A kit comprising a device according to claim 2, comprising a plurality of disposable elements of said device and / or reagents for processing said biological sample.

38. 38. The kit of claim 37, wherein the disposable elements comprise the collection tube, the turbulence inducer, the collection chamber, and optionally a plastic mouthpiece cover.

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