How Breath is Collected
The proprietary VosCryo™ breath collection device works by condensing exhaled breath using a frozen sleeve. Warm breath is condensed at the interface between the internal barrel and frozen cooling sleeve, ready to be collected or dispensed for testing. When compared to other breath collection devices, the VosCryo™ breath collector is not only faster but is much easier to use. The VosCryo™ system collects 50 µL of EBC in 20 secs.
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Prior to use, the whole device is stored in the freezer until the sleeve is frozen. It can be stored in an ice chest or cooler for up to 8 hours before use.
The breath pathway of the VosCryo™ breath collector is inert and nontoxic, composed of PLA and polypropylene.
Breath inlet tube
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Directs breath to base of a syringe, reverses flow along chilled barrel surface
Small syringe barrel
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Chilled below freezing, droplets, aerosols and vapor condense on contact
Frozen cooling sleeve
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Flexible tubing holds layer of frozen fluid in thermal contact with syringe barrel
Dispenser tip
• Collected volume allows condensed breath to be transferred efficiently with plunger
Cap/vial
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Keeps syringe sealed until time to transfer breath liquid
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Ensures reverse flow of air
Plunger
• Gathers condensed breath to deposit wherever needed
The composition of Exhaled Breath Condensate (EBC)
EBC is mostly composed of water, but does include other components such as proteins, lipids and other molecules. At VosBio™, EBC has been used to sample viral particles that have been shed and exhaled by a person that may be infectious with SARS-CoV-2 or other respiratory diseases.
Droplet size and water vapor
Exhaled breath includes gaseous water vapor and tiny droplets ranging from less than 1 μM to over 1,000 μM. These droplets can be differentiated based on size, with those below 5 μM considered aerosols, and those above 5 μM considered droplets. The World Health Organization now refers to these collectively as respiratory particles. The majority of the water in breath is gaseous when it is exhaled, although it rapidly condenses on contact with cold surfaces such as the inside of the VosCryo™ collection chamber. Large and small respiratory particles also coalesce on the cold surface and combine with the condensed water vapor.
Particles of different sizes originate in different areas of the respiratory tract through different mechanisms, and therefore may have different compositions or analytes of interest. The VosCryo™ breath collector captures all droplets together, allowing any components of the sample to be analyzed.
Addressing saliva contamination
The VosCryo™ breath collector does not accumulate excess saliva. Users breathe through the tube at a comfortable angle, approximately horizontal. This prevents saliva from dripping into the sample due to gravity. Sample times are very short, typically under a minute. This prevents saliva from accumulating over time, while breath condensate is rapidly collected. Finally, the VosCryo™ system's patented reverse-flow collection allows breath to condense efficiently over a wide surface area, while any saliva is retained in the mouthpiece.
Compatible with any detection method
Because EBC is composed of mostly water, this makes for a much cleaner clinical specimen when compared to nasal swabs, nasopharyngeal swabs, bronchoalveolar lavage, and oropharyngeal swabs. This allows for sample collection to be used directly without the need of prepping the sample for analysis. EBC is suitable for molecular detection such as PCR and LAMP, as well as mass spectrometry, ELISA, and any other detector monitoring for any biomarker in the sample.
For research use only.
LAMP
VosBio™ has developed optimized molecular detection assays using Loop-mediated isothermal AMPlification (LAMP). The LAMP reaction takes place at a constant temperature using a combination of 6 oligonucleotides that bind to the target sequence at 8 binding sites. This technology allows for detection of nucleic acids in a way that is rapid, selective, and sensitive.
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Because of LAMP’s speed and sensitivity, it is well suited to be paired with rapid Exhaled Breath Condensate (EBC) collection. VosBio’s SARS-CoV-2 assay has demonstrated the ability to detect 20 copies of virus in a 50 μL sample of EBC consistently under 30 minutes, and 200 copies within 15 minutes.
Because the reaction takes places at a constant temperature, it can be carried out in a relatively simple instrument, making it useful for testing in the field and at the point of need.
PCR
VosBio™ has developed molecular detection assays using Polymerase Chain Reaction (PCR). PCR takes place while temperatures cycle repeatedly using 2 – 3 oligonucleotides that bind to the target sequence. Two of these oligonucleotides, known as primers, are necessary to exponentially replicate the target sequence, and an optional fluorescently tagged nucleotide called a probe can be used for real-time detection.
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PCR can be extremely sensitive, able to detect a single copy of DNA or RNA if it is present. Due to the controlled thermal-cycling, real-time fluorescent data can be used to quantify the initial amount of target nucleic acid, as the number of copies doubles every cycle. This allows not only sensitive, but quantitative detection of nucleic acid targets. PCR is the gold standard of molecular detection.
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PCR is an excellent approach for research with Exhaled Breath Condensate to detect and quantify any nucleic acid of interest in the breath. This includes respiratory pathogens and the microbiome of the lungs, or human DNA or RNA which may be exhaled under some circumstances.
Primer Design and Libraries
Designing primers for LAMP and PCR requires careful attention to several factors. The binding sequences must be complementary only to their binding sites, with an appropriate melting temperature and binding energy. Primers must not be complementary with one another, and binding sites should be spaced appropriately. VosBio™ has developed a method for predicting the most efficient primer sets by optimizing several parameters in parallel to identify the best primer candidates. Our approach can generate primer sets for any length of target sequence, and results may be sorted based on any parameter of interest.
As we continually refine our process for identifying top primers for a given target, we are building up a library of primer sets suitable for LAMP and PCR amplification of various common respiratory viruses. Please contact us for primer design assistance.
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