Method and device for collecting and dispensing breath condensate

Abstract

The device and method of the invention address the need to ensure the adequate and accurate collection and delivery of small volumes of breath condensate liquid samples to detection reagents for detection of targets including nucleic acids, RNA, DNA, pathogen(s), organic compounds, cells, virus, bacteria, fungi, protein, chemical, biomolecules, molecules or spores. Small amounts of liquid condensate are collected in a syringe barrel in the form of film, frost or droplets on the internal wall of a collection chamber. A central turbulence inducer tube is used to introduce exhaled breath into the collector. Most of the syringe barrel collection volume is dead space and air. Collected liquid volumes are dispensed out of the syringe barrel in the presence of air. This accomplished through a low dead volume design of the syringe plunger and dispensing needle. Withdrawing the turbulence inducer from the syringe engages the tip to convert the turbulence inducer to a plunger to dispense the collected liquid.

Description

[0001] Method and Device for Collecting and Dispensing Breath Condensate

[0002] Field of the Invention

[0003] The present invention relates to devices and methods for collecting and dispensing liquid breath condensate from exhaled breath.

[0004] Background of the Invention

[0005] Pathogens and other entities present in exhaled breath can be detected and identified. The collection of exhaled breath condensate can be performed with open tubes. Exhaled breath is directed into the inside of a tube. When the tube is cooled on the outside, breath will condense into liquid on the inside of the tube. Then, to recover the collected condensate liquid for processing, many tube devices use a plunger that is inserted into the tube. The condensate is pushed by the plunger to one end of the tube and liquid is collected on the other end of the tube. In the prior art, a plunger is inserted into the tube with the tube positioned so liquid does not exit either end. Then the open end of the tube is pointed up in a vertical orientation. As the plunger is inserted further into the tube, air in the tube is directed out of the end of the tube while liquid collects on the plunger face. As such, the tube is tilted upward to more or less up the vertical orientation orientated to remove air while not allowing liquid to drop out from the tube.

[0006] In prior art, the dispensing of liquid and air from a tube or syringe with a plunger is performed by pointing the tube upward or essentially, in a vertical orientation, removing air first and then the liquid. Air removal must be performed with the tube / syringe orientation pointed upward enough to prevent the loss of liquid as the liquid reaches the end of the tube.

[0007] Finally, when the air has been removed and liquid is collected at the face of the plunger in the tube, the liquid may be removed for processing. In one method, the collected liquid condensate may be removed from the tube by inserting a pipette tip into the tube and drawing up the liquid prior to transfer. In this method, the tube remains pointed up. In another method, the plunger pushes the collected liquid through a dispensing needle at the end of the tube and transfers the liquid into another vial or chamber. In this method, the tube is pointed down. Both of these methods rely on first removing air from the collection tube and then removing the collected liquid for processing.

[0008] In addition, the liquid collected at the face of the plunger in a syringe tube must be of sufficient volume to manipulate in order to push and transfer the liquid out of the tube. If the volume of the condensate liquid is insufficient, in the range of 0.5 mL and lower, then the condensed liquid has residual air present and cannot be transferred from the syringe collection tube by pushing the liquid out. Air moving with the liquid will splatter the liquid coming out of the syringe tube and liquid cannot be effectively and completely transferred to a vial or other chamber structure. Furthermore, collecting these large amounts of liquid requires a large collection tube. The collection is greater than 10 mL, in the range of 50 mL or larger. This larger volume helps in the removal of air first and then liquid later. However, collection of large volumes of breath condensate is lengthy, often requiring 10 minutes or more.

[0009] But If breath collection is rapid, i.e. collection is less than 5 minutes or is less than 1 minute, the amount of breath condensate liquid than can be collected is very small. For short collection times, the volume collected may be less than 500 pL volume.

[0010] Recovery of 500 or 400 pL of liquid with air present from a syringe breath collector is not possible without a pipette. The recovery of 300 pL and especially 200 pL and smaller volumes with air present is even more difficult. There is so little liquid, it is impossible to collect and coalesce liquid completely and displace all air at the face of a plunger.

[0011] One method that is reliable to remove these small liquid volumes in the presence of air is to use a centrifuge. After breath condensate collection, the collection tube is spun and any liquid in the tube will travel down to the end of the tube or out the end of the tube into a receiving vial. This is a very effective way to collect small volumes of breath condensate liquids, even down to 5 - 10 pL. Air is removed from the liquid by forcing liquid into the receiving tube ahead of the air. But this centrifuge process complicates the collection process with additional equipment and additional steps needed including pipetting steps.

[0012] There exists a need to improve the collection and recovery of small amounts of breath condensate liquid, especially with air present. There exists a need to ensure adequate and minimum breath condensate sample volume has been collected and transferred to a processing chamber. There exists a need to confirm that the transferred sample volume is completely mixed with the detection reagents prior to processing. There exists a need to perform these operations rapidly. There exists a need for a plunger and method of use to dispense small amounts of liquid from a large volume syringe breath condensate collector.

[0013] Summary of the Invention

[0014] In the device of the invention a syringe breath collector is used to collect breath condensate. The syringe breath collector can have a chamber volume of 30 mL, 20 mL, 10 mL, 5 mL, 3 mL, 2 mL or 1 mL. In some embodiments, the syringe breath collector can have a chamber volume of 30 mL or less. The volume of collected breath condensate can be less than 500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL or in the range of 1 - 500 pL, 2 - 400 pL, 3 - 200 pL, 4 - 150 pL, or 5 - 100 pL. The volume of collected breath condensate can be 400 pL or less. The liquid condensate collected is in the form of film, frost, or droplets with air taking the majority of the internal collection chamber volume. Most of the syringe collection volume is air and breath condensation is collected on the walls of the syringe barrel. Air may be 90 to 95% or greater volume than the collected liquid volume (including frost converted into liquid form).

[0015] In some embodiments of the invention, the breath collection device is comprised of a modified syringe. A breath introduction tube is inserted into the open end of the syringe and seated at the distal end of the syringe. Breath is exhaled and directed into the tube into the syringe device. As breath flows through the tube, the breath flow enters the tube through the centre of the tube and then flows out along the outside of the tube in the interface between the syringe barrel and outside of the tube and exits the device.

[0016] In some embodiments of the invention the centre tube is a turbulence inducer comprised of baffles on the outside of the centre inlet tube. The central tube with attached baffles forms a turbulence inducing apparatus. The baffles break the laminar flow of breath into turbulent flow causing repeated interaction of the breath with the inside wall of the syringe barrel. The cooled wall of the inside of the syringe condenses breath droplets and gaseous breath capturing the breath liquid condensate. The tube is heated from the breath and breath does not condense on the turbulence inducer tube insert and baffles. Breath condensate is captured on the cool inside wall of the syringe barrel.

[0017] After collection, the liquid is collected and dispensed. Dispensing very small liquid volumes in the presence of air is accomplished through a low dead volume design of the syringe plunger and dispensing needle. The plunger collects liquid on the edge of the plunger collector wall interface. Although some air is retained, much of the air will move toward the outlet. The plunger, plunger tip, syringe interface and needle are designed to contain very little dead volume for liquid and air, especially relative to the size of the syringe.

[0018] Most collected breath liquid in the presence of air is transferred from the breath condensate collector. Very little air and liquid is trapped in the plunger and syringe barrel bottom interface. Surprisingly, the dispensing method of the invention can be performed with the syringe barrel and dispensing needle can in any orientation including pointing down or mostly down without losing liquid. The plunger may be inserted into the syringe barrel breath collection with dispensing end pointed up or down without losing liquid. The plunger may be pushed into the syringe barrel breath collector with the dispensing end pointed up or down without losing liquid. Thus, a vial can be positioned directly below the outlet of the dispensing needle. It is not necessary to remove air from the collected liquid by pointing the collection device mostly up as in prior art when inserting and depressing the plunger.

[0019] In the device and method of the invention, small liquid sample volumes (500 pL, 400 pL 300 pL, 200 pL 100 pL, 50 pL or less) can be directly transferred from a breath collection syringe to the detection reagents, eliminating the need for a centrifuge.

[0020] In some embodiments of the invention, a turbulence inducer that has been inserted into the syringe collector may be partially withdrawn from the collector without removing or disturbing collected condensed liquid condensate. Then the end of the inducer may engage a plunger end or flare out to become a plunger end. A plunger flare out can be accomplished in different ways. In one embodiment of the invention, the turbulence inducer plastic or rubber end engages a circular ring as the inducer is withdrawn from the syringe barrel. This ring pushes the plastic or rubber end to flare out so that the end of the turbulence inducer can now act as a syringe plunger. Then as the turbulence inducer plunger end is inserted and depressed into the syringe collector body, breath condensate liquid is collected and dispensed as described. The breath condensate liquid is directed to a vial, volume measuring vial, tube, chamber or device. The device may be a detecting or measuring device.

[0021] In some embodiments of the invention, a known and / or specified volume is obtained and directed. In the device and method of the invention, confirmation is obtained that the specified volume has been transferred. The confirmation may be performed visually, by sensor or mechanically. In some embodiments of the mechanical invention, the volume measurement is performed optically. In some embodiments of the mechanical invention, the volume measurement is performed electrically or electronically. Any amount of liquid exceeding a specified volume can be directed to an overflow or to an absorbent. The measurement and confirmation of the transfer of a known volume ensures that a minimum amount of breath condensate liquid is available and provided for processing and detection. This provides a positive control mechanism to confirm the introduction of an adequate breath condensate sample volume for processing. Confirmation of sample volume helps avoid a false negative result. In addition, the proper and full mixing of the breath condensate sample with detection reagents provides another control to eliminate a false negative result. The device and method of the invention may be used to verify that a specified breath condensate has been collected and has been transferred to the detection chamber with detection reagents, if needed, and may verify the collected and transferred sample is thoroughly mixed with the detection reagents.

[0022] In some embodiments of the invention, the volume of the condensate liquid or total reaction mixture may be measured by comparing liquid height to a volume mark in a tube, vial or chamber. In some embodiments of the invention, the volume of the condensate liquid or the total reaction mixture may be measured by flowing the liquid through a tube and measuring the mass or volume of the liquid passing by a sensor in the tube.

[0023] Detection of a target or targets present in the breath may be performed using isothermal or thermal cycling nucleic acid amplification. Detection may be by optical, fluorescence, spectrometry, electrochemical or any transducer capable of detecting nucleic acid, protein, or other biomolecule. Detection may include nucleic acid amplification. Detection may be of the target nucleic acid, protein, carbohydrate, or other chemical related to a target.

[0024] In some embodiments of the invention, the method of the invention has the following steps:

[0025] 1. Breath condensate liquid possibly containing a target is collected with a small syringe barrel collector.

[0026] 2. A plunger is inserted and air and collected liquid are pushed out of the syringe collector. The dispensing needle can be level or pointed down. However, in the device and method of the invention plunger insertion and movement and liquid dispensing can be performed in any orientation.

[0027] 3. Liquid dispensing is performed with air present in the collection chamber. There is no two-step process needed of first removing air from the chamber and then liquid. However, as the final drops are dispensed from the chamber mostly liquid is dispensed.

[0028] 4. The dead volume configuration of the device results in very little trapped air and liquid not dispensed.

[0029] 5. A plunger that is converted from a turbulence inducer may be used. This is performed by withdrawing the turbulence inducer from the syringe barrel to engage and convert the distal end of the turbulence inducer to a plunger tip.

[0030] 6. Detection reagents including lysing reagent(s) are optionally added.

[0031] 7. The liquid may be further transferred into another vial, tube or chamber. Pressure or gravity may be used. 8. The liquid volume may be optionally measured optically or electronically.

[0032] 9. Optionally, excess liquid may be removed. Wicking, gravity, vacuum or other methods may be used.

[0033] 10. Detection reagents may be added to the liquid either before or after volume measurement. One or more of the detection reagents may be lyophilized. The volume of the detection reagents may be known, and the volume of the breath condensate may be measured and confirmed.

[0034] 11. Optionally the sample or detection reagents contains dye or similar reagent. The dye or similar reagent may be lyophilized.

[0035] 12. The sample and reagents are mixed. Optionally measure uniformity of mixing.

[0036] 13. The sample can be processed for detection of a target including but not limited to pathogen(s), virus, cell, spore, fungi protein, lipid, carbohydrate, biological or inorganic entity or biomolecule.

[0037] Definitions

[0038] Exhaled breath condensate liquid: Exhaled breath condensate liquid (or frost) collected from exhaled breath. The terms exhaled breath condensate liquid, breath condensate liquid, breath liquid, exhaled breath and breath condensate are used interchangeably in this document.

[0039] Breath condensate volume. Volume of breath condensate liquid either collected, captured or dispensed. In the methods and device of the invention, a majority, most or all of the breath condensate collected or captured is dispensed, even in the presence of air.

[0040] Dead volume or dead space: Dead volume or dead space refers to space within a syringe collector and optionally a dispensing needle when a plunger is depressed that can lead to retained liquid and air and consequently, the loss of liquid that is not dispensed. Because air is present and not removed before the dispensing process is started, retained volume could hold liquid and / or air.

[0041] Dead volume or dead space can contain liquid and / or air.

[0042] Breath collection tube: A breath collection tube is a tube designed for the collection of breath condensate and dispensing breath condensate. In some embodiments of this invention, the tube is a syringe tube collector into which a plunger can be inserted after breath condensate collection. Syringe: A syringe is defined to be a liquid handing device designed for drawing in and expelling liquids. However, a syringe is not used this way in this invention. In the context of this invention, a syringe can be used as a breath liquid collector. In this application, the syringe can include a breath inlet turbulence inducer inserted into the syringe barrel.

[0043] Tube or syringe dispensing: Liquid from breath condensate is dispensed from the syringe.

[0044] In the invention, the dispensing of liquid and air through the lower end of the syringe barrel is performed with one single stroke or operation. The tube or syringe can be oriented horizontally, vertically or any orientation in between without loss of any breath condensate liquid. The tube or syringe can be in any orientation when dispensing the breath condensate liquid into a detection chamber or collection vial or chamber.

[0045] Turbulence inducer: Breath inlet tube for insertion into a syringe and breath dispersion device to force breath against the walls of the syringe to induce efficient breath condensation. In some embodiments of the invention, the turbulence inducer has an end that can be modified to transform the turbulence inducer into a syringe plunger.

[0046] Syringe or tube volume: Syringe volume refers to internal chamber volume where breath condensate can be collected. Large syringe volumes are defined to be 50 mL or more. Small syringe volumes are defined to be 30 mL or less.

[0047] Plunger: A plunger is a component of a syringe normally used to create a vacuum for aspirating or dispensing fluids. In this invention the plunger is used to dispense breath condensate liquid and air.

[0048] Needle: A needle is a thin, pointed, and hollow tube typically used for piercing or puncturing, commonly used in syringes for the transfer of fluids or samples. In this invention, the needle is a dispensing needle used to dispense collected breath condensate liquid and air.

[0049] Dispensing needle interface connection: The dispensing needle interface connection refers to the point of attachment or connection between the dispensing needle and the syringe or collection device plunger ensuring proper liquid dispensing.

[0050] Syringe luer connection: The syringe luer connection is a standardized system used for connecting needles or other accessories to a syringe. Minimum breath condensate sample volume: The minimum amount of breath condensate volume required for a valid diagnostic measurement. A minimum prevents underestimation of the total target (including virus, cell, protein, molecule or pathogens) shed per unit of time or misstatement of the target detection limit.

[0051] Maximum breath condensate volume: The maximum amount of breath condensate volume required for a valid diagnostic measurement to not overestimate the total target including, virus, cell, protein or pathogen shed per unit of time.

[0052] Shed rate: Number of exhaled targets including virus, cell, protein or pathogens detected per unit of breath condensate collection time. An accurate shed rate assumes that all or most of the breath condensate liquid was collected and that all or most of the collected liquid volume was transferred to the detection reagents. If the volume of liquid is too low, it is likely that an inadequate volume of breath condensate sample liquid was collected.

[0053] Absolute shed number or detection: Number of exhaled target including virus, cell, protein or pathogens detected from a breath condensate sample.

[0054] Target: In this invention, a target is a virus, bacteria, cell, protein, spore, fungi, organic molecule, inorganic molecule, biomolecule or any biological entity that causes disease or is indicative of disease and is exhaled in breath.

[0055] Short sample collection time: Any breath condensate collection time that is short, such as less than 5 minutes, less than 4 minutes, less than 3 minutes but usually less than 2 minutes or less than 1 minute.

[0056] Detection reagents: Any reagents that are used to lyse or detect a target. Reagents may include a dye. Detection reagent(s) may be in liquid form, may be lyophilized or may be in both forms. Detection reagents include any reagent that combines with a target to make it detectable.

[0057] Dye: Substance that is used to measure and ensure the breath condensate sample is mixed thoroughly and uniformly with the detection reagents. In some embodiments, the dye reagent may be inert to the detection process. In other embodiments, the dye may play an additional role in the detection process. Dye reagents may be anything that can be measured or detected. The dye may be detected by visible, UV, fluorescent light. The dye may be detected visually or by instrument. Direct addition of sample volume to detection reagents: Method where the breath condensate sample is added directly from the collection device to the detection reagents.

[0058] Isothermal detection: Any detection method where the mixture of breath condensate sample and detection reagent temperature is held constant for a portion or most of the detection time.

[0059] Thermal cycling detection: Detection method where the mixture of breath condensate sample and detection reagents is cycled at temperatures such as PCR to promote amplification and detection of nucleic acid.

[0060] Visual confirmation of volume: Confirmation by eye that the desired volume of breath condensate was added to the detection reagents.

[0061] Instrument sensor confirmation of volume: Confirmation by instrument that the desired volume of breath condensate was added to the detection reagents. Volume may be measured by comparing liquid to a volume mark or may be measured by a flow and volume measurement through a tube and sensor.

[0062] Master mix or detection reagents: A mixture that is used to amplify nucleic acid from the exhaled target including virus, cell or pathogen. Detection reagents may be comprised of a master mix and optionally other reagents. Detection reagents include reagents that combine with the target to make the target detectable.

[0063] Lysing reagent: Solvent(s) or chemical(s) used to lyse the walls of a target including virus, cell or pathogen contained in a breath condensate liquid. Use of the lysing reagent releases nucleic acids or other molecules of interest.

[0064] Lyophilized reagents. Dried detection reagents optionally including dyes. The dried reagents may be reconstituted with sample liquid and / or buffer, or solvent / water.

[0065] Collection chamber: Chamber for collection of breath condensate liquid including frost.

[0066] Mixing chamber: Chamber where breath condensate liquid and detection reagents are mixed. Detection chamber: Chamber where breath condensate liquid samples are detected and measured for the presence of target including pathogens, virus, cells, spores, or other biomolecules.

[0067] Sample liquid: Any liquid sample condensed from breath that contains one or more of the target including nucleic acids, RNA, DNA, pathogen(s) organic compounds, cells, virus, bacteria, protein, bio, organic and inorganic molecules, fungi or spores to be detected and measured.

[0068] Target: The target is in the sample that is collected by the device and method of the invention. The target may be analysed, detected or processed. The target may be one or more of the following nucleic acids, RNA, DNA, pathogen(s) organic compounds, cells, virus, bacteria, fungi or spores to be detected, processed or measured.

[0069] Brief Description of the Fiaures

[0070] Embodiments of the invention are discussed in more detail below with reference to the accompanying drawings, in which:

[0071] FIGS. 1A - 1C illustrate stages of operation of a low dead volume breath condensate dispenser;

[0072] FIG. 2 is a schematic diagram of a breath condensate collecting and dispensing device that provides a combination of a turbulence inducer and a syringe plunger; and

[0073] FIGS. 3A - 3E illustrate stages of the capture, collection, delivery and mixing of breath condensate liquid that can use the device of FIG. 2.

[0074] Detailed Description of the Invention

[0075] Breath condensation collection devices are designed to collect and deliver specified and adequate amounts of liquid for the downstream processing requirements. Tubes have been used to collect breath condensate. Breath collection designs based on a tube can work well; however, the recovery of the liquid condensate from the tube can be complicated or difficult requiring specific orientation of the tube. This is because tube collectors contain significant air after collection of breath condensate liquid. Tube designs can be 50 mL or more volume that collect 1- 10 mL or more of breath condensate liquid. Some successful tube designs are ones that use a plunger to move liquid inside of the collection tube. However, these designs require holding the tube vertically and pushing the plunger up into the liquid collection. Air is pushed out of the tube while liquid is moved to the face of the plunger as it moved through the tube. Breath condensate liquid is collected and consolidated on the face of the plunger. Air must be removed first and then liquid is collected and recovered. The end of the collection tube may be open, and the breath condensate liquid may be recovered by pouring or pipetting the liquid out of the collection tube.

[0076] In some designs, the collection tube may be a (large) syringe barrel comparable to the size of the collector tube. These collectors operate in the same manner. After collection of the breath condensate liquid, the syringe is positioned vertically or at least mostly up from horizontal. A plunger is inserted into the syringe. As the plunger is pushed further into the tube, liquid collects on the plunger face. Air is expelled as a first step while keeping the orientation of the syringe point at least a little above level so that liquid is not lost out the end of the tube.

[0077] With the air removed and liquid consolidated, the syringe is positioned to point down so that liquid breath sample may be pushed out the end of the syringe collector through a needle into a receiving vial or chamber. Finally, the liquid is pipetted from this vial or chamber into a separate device for processing (that may include target detection.)

[0078] In a previous patent application, we developed a smaller syringe collection device in the range of 30 mL and smaller, (typically 1 - 10 mL syringe size) was described. The device was able to collect only smaller volumes usually less than 400 pL, less than 200 or less than 100 pL. Once the capacity of the syringe is exceeded, no more condensate can be collected.

[0079] But it proved to be impossible to recover the collected liquid from the device. Upon insertion of a plunger usually zero or at most only a couple pL of spattered liquid could be removed from the syringe. Collection and consolidation of the spattered liquid was inconsistent and useless for processing. In most cases no liquid could be removed. This was true even when it was attempted to remove air first as in previous technology and methods. The only method that successfully removed liquid was to place a vial at the end of the syringe barrel with collected breath condensate and then apply centrifugal force to the vial. With this centrifuge force process, the liquid was removed from the syringe collector and recovered in the bottom vial. The centrifuge force process was very efficient and volumes as low as 10 or 5 pL could be recovered. Even though the centrifuge process is efficient, it is tedious and requires extra equipment.

[0080] A 5 mL commercial syringe configured into a breath condensate collector used in previous work did not allow dispensing samples of 50 pL or even 100 pL of liquid. In any case, syringes are designed to dispense liquid and not a combination of liquid and air. And we could not dispense small volumes of liquid with air present. Also, increasing the syringe size to 10 or 20 mL to collect more breath condensate liquid volume did not help in the dispensing problem. The problem of dispensing liquid in the presence of air increased with syringe volume even though more liquid was available to dispense.

[0081] Reducing dead volume in syringes has been important and different designs have been published, mostly with 1 mL syringes for injecting insulin and similar drugs. However, the prior art only discusses lowering the dead volume of syringes that contain only liquid, not a combination of condensed liquid and air. They were not designed to dispense air and liquid. The combination of dispensing small amounts of liquid with air present appears to present a new problem.

[0082] The device and method of the invention does not involve dispensing small volumes of liquid multiple times from a large volume of liquid held in the syringe. Dispensing is performed in a single downward stroke of the plunger.

[0083] Nevertheless, the design of the plunger, syringe barrel face and possible other factors such as the dispensing needle were the most probable areas to study to attempt to remedy the problem. As it turned out, there are several sources of dead volume that had to be redesigned in the device and method of the invention. These include the dead volume in the dispensing needle, dead volume in the dispensing needle connection to the syringe, dead volume in the interface of the plunger and the bottom face of the syringe barrel and dead volume in the interface of the plunger and the dispensing needle syringe side port.

[0084] Redesign of any one of these parameters could allow improved and acceptable performance. Depending on the detection method volume requirements, re-designing these different parameters was found to provide an acceptable amount of delivered liquid.

[0085] The optimized syringe capture devices of the invention have redesigned all parameters. We have found that all of these parameters are important for dispensing the absolute smallest volume amounts from the syringe breath capture device. However, reducing the dead volume in one or more of dead volume spaces allowed the delivery of acceptable amount of liquid condensate. In some embodiments of the invention, the lowest amount of liquid dispensed is 1 - 5 pL.

[0086] The following sections discuss dead volume for liquid and air in specific areas of the device. Also discussed is the progress of changes of dead volume for liquid and air as the plunger is depressed and its shape changes. 1 Removal of dead volume in the interface shape of plunger and the wall face and the bottom face of the syringe barrel

[0087] In normal syringe design, the shape of the plunger will match the bottom of the syringe barrel. In some syringes, the centre of the syringe barrel has a depression leading to the needle or needle port. As the plunger is inserted into the syringe barrel, the bottom of the plunger will meet the bottom at the syringe barrel face at more or less the same point in time. This design results in possible dead volume along the outside edges of the plunger where it touches the wall of the syringe barrel. This dead volume is small and inconseguential for normal aspiration and expulsion operation of a syringe. But in the device and method of the invention, the loss of 10 pL or more is a large loss of liguid, especially for condensate liguid samples that are collected in amounts of 200 pL breath condensate or less. In some embodiments, 100 pL or less, or 50 pL or less, or 20 pL or less. Loss from inadeguate or improper design can result in the loss of 5 - 50% of breath condensate sample liguid.

[0088] In the method of the invention, the plunger insertion process continues, the plunger interface travels progressively to the centre of the syringe bottom face and the dispensing needle interface. This progression of movement may allow air to be pushed ahead of the liguid, or behind the liguid, thus possibly trapping air which in turn could trap liguid.

[0089] FIGS. 1A - 1C show a device plunger 10 in various position with respect to a syringe barrel interface that can be adopted in embodiments of the invention. The syringe barrel 12 has an upper end and a lower end. FIG. 1A depicts plunger 10 with plunger bottom 17 and central protrusion 18 positioned in syringe barrel 12 that has a syringe bottom face 14. Below syringe barrel 12 is dispensing needle 20, which is in fluid communication with inside of the syringe barrel through a syringe barrel interface at the bottom face 14. When the syringe is used to capture breath condensate, breath condensate liguid is present on the inside wall of the syringe. As the syringe plunger is depressed, condensate liguid is pushed along the inside wall of the syringe barrel down the syringe toward syringe bottom face 14. In the syringe and plunger design of the invention, when plunger 10 is pushed into and through the barrel, the outer circumference of plunger bottom 17 first meets a peripheral region 16 (e.g. a side face) of the bottom of the syringe. As shown in FIG. 1 B, as plunger 10 continues to be pushed down toward a central region of the syringe bottom face 14, a contact area between the plunger 10 and the syringe bottom face 14 expands radially inwards in a progressive manner towards the syringe barrel interface. As shown in FIG. 1C, when plunger 10 is fully inserted, the central protrusion 18 of plunger 10 meets the centre of the bottom of the syringe barrel. With this progressive motion, the volume of trapped liguid is minimized. The liquid is progressively pushed from the concentric outside of the syringe barrel inside wall toward centre needle / syringe interface 19 where the top of needle 20 is located. The plunger is fully depressed and the complete inside of the face including the needle / syringe interface is filled. In this way, very little or no dead volume is retained and collected breath condensate liquid can be dispensed. Needle 20 will have some dead volume. The amount of dead volume depends on the inside diameter and length of needle 20 and the dead volume of the needle syringe interface.

[0090] The turbulence inducer has a proximal end and a distal end. In some embodiments of the invention, a turbulence inducer may be designed to include a plunger function. This design concept is shown in FIG. 2 and FIGS. 3A to 3E. In FIG. 2, exhaled breath turbulence inducer 22 having breath inlet 24 at the proximal end and the distal end is situated inside syringe barrel 12. In this embodiment of the invention, the end of turbulence inducer 22 includes bottom feature 28 that will become a plunger end as described below. FIG. 2 also shows air filter 32 that may be included in embodiments of the invention. This allows air to escape as the plunger is depressed but does not allow liquid to exit thus forcing liquid to be dispensed out of dispensing needle 20. Dispensing needle 20 at the end of the syringe is placed inside of attached receiving vial 34.

[0091] The operation of the device in FIG. 2 is shown in FIGS. 3A to 3E. In FIG. 3A, breath condensate liquid 36 is collected on the side of the syringe barrel with the help of the turbulence inducer. Then FIG. 3B shows that after collection, the turbulence inducer is retracted or drawn out from the collection chamber. At the apex of withdrawal, the end of the turbulence inducer transforms to turbulence inducer plunger 38. In some embodiments of the invention, the turbulence inducer plunger 38 may flare out upon retraction to become a scraping device. In other embodiments of the invention, the turbulence inducer plunger 38 may engage with a plunger scraper (not shown) at the apex of motion. Then, as the turbulence inducer plunger is depressed or reinserted into the device, the inducer will scrape the sides of the syringe barrel, collecting the condensed liquid. FIG. 3C depicts the turbulence inducer with flared out plunger end moving down through the syringe barrel. The turbulence inducer / plunger will act to scrape and collect condensed breath condensate frost or liquid 40. FIG. 3D shows collected liquid 42 deposited into a vial. If other reagents are in the vial, may be mixed with the breath condensate liquid using a back-and-forth plunger motion in the device as shown in FIG. 3E. Mixed reagent 44 may be deposited back into the vial or elsewhere. 2 _ Dead volume in the interface of the plunger and centre of the barrel and needle port area

[0092] In some prior art syringes, the centre of the syringe barrel contains a depression to meet the plunger. Often the plunger protrudes to fill this space. But as mentioned previously, the protrusion can cause dead volume to form at the outer plunger face at the barrel face centre In the device and method of the invention, a plunger will fill this space completely or near completely pushing out the liquid condensate sample completely into the dispensing needle port area. The same requirement holds as the rest of the plunger and barrel bottom design. This space must not fill until the outside edge of the plunger pushes all of the liquid away from the wall and into the inlet of the dispensing needle.

[0093] Dead volumes at the end of the syringe barrel may be of little consequence for recovery of breath condensate liquid if the amount collected is 400 pL, 500 pL or more. Some large syringes have dispensing tubes at their ends. However, in smaller volumes, significant fractions of the breath condensate liquid will be left in the device and wasted and cannot be processed. The volume of unrecoverable breath condensate is caused by the luer needle dead volume and the needle dead volume. For a luer syringe connection, the dead volume for the inside of the fitting is 92 pL. For some 1 mL syringes, a modified plunger may be used that displaces some of this dead volume and reduces the dead volume to 59 pL. Larger syringes do not account for or control this dead volume. It is not an issue for conventional syringe operation.

[0094] In some embodiments of the invention, the interface of the needle with the syringe barrel does not contain excess dead volume. In many cases, this is caused by the internal volume of a luer connection of the dispensing needle with the syringe. In some commercially available needles, attempts have been made to fill this internal luer connection space with a circular plastic attachment to the needle. However, the filling is incomplete, and this configuration can trap air and liquid. It is expected since the luer fitting tapers to smaller diameter with the needle connection end. There is dead volume inside of the luer taper. The diameter inside the luer taper also decreases as the taper proceeds to the distal (needle) end. Therefore, any space-filling plastic having a given diameter will fill the distal end but cannot fill the entire internal volume of the luer fitting, leaving large unfilled dead volumes.

[0095] In the device and method of the invention, the dead volume is reduced by using a dispensing needle design that eliminates the inside of the luer connection. Replaceable needles have large dead volumes in a syringe breath collector ranging in dead volume of 60 - 90 pL. The dead volume is present in both the needle and in the (luer) connection. In some embodiments of the invention, a luer connection may be used but the needle can be designed to bypass the luer internal dead volume. By eliminating the luer inside design, the entire internal dead volume due to the luer connection is eliminated. In some embodiments, elimination or reduction of dead volumes is accomplished by dropping the dispensing needle inside the syringe through an internal luer connection and sealing the needle at the top. In some embodiments of the invention, the syringe is moulded to allow the insertion of a press fit, welded, or glued dispensing needle. In some embodiments of the invention, the outside of the luer connection is retained to allow vial or other attachments though a taper luer or luer lock connection. In some embodiments of the invention, the syringe capture device is plastic. In some embodiments of the invention, the plastic syringe capture devices are disposable. In some embodiments of the invention, other materials such as metal or glass may be used for the syringe collector and dispenser.

[0096] 3. Dead volume in the dispensing needle

[0097] As the syringe size is increased, the needles used with these larger syringes will increase in diameter and length. Large syringes, 3 mL and greater, use large needles. This is logical because small needle diameters would provide a resistance to flow to be able to draw liquid into the syringe or expel liquid from the syringe. It is counter intuitive to use a large syringe to manipulate small liquid volumes by reducing the needle size. In the device and method of the invention the dead volume of the dispensing needle 20 pL, 10 pL, 5 pL or less. This is accomplished by having a small inside diameter dispensing needle. In some embodiments of the invention, the diameter of the dispensing needle is altered, along the length of the dispensing needle. The taper of the needle? The needle can be tapered.

[0098] In the device and method of the invention, a breath condensate collection system is needed that can capture, collect beath condensate and then dispense small volumes of liquid. But to capture large volumes of breath condensate requires a large cooling surface of 5 - 10 cm2or more. In the device and method of the invention, this translates into a large tube, making collection of liquid possible for small volumes of liquid that are 500 pL, 400 pL, 200 pL or smaller.

[0099] As stated above, 400 pL or less of breath condensate liquid can be collected by the breath condensate collection device. After collection, the plunger is inserted, pushed into the syringe barrel, and the breath condensate liquid is dispensed, In some embodiments off the invention, after dispensing of liquid, less than 100 pL of breath condensate liquid remains in the collection device. In some embodiments off the invention, after dispensing of liquid, less than 50 pL of breath condensate liquid remains in the collection device. In some embodiments off the invention, after dispensing of liquid, less than 20 pL of breath condensate liquid remains in the collection device. In some embodiments off the invention, after dispensing of liquid, less than 10 pL of breath condensate liquid remains in the collection device.

[0100] In some embodiments off the invention, after dispensing of liquid, less than 100 pL of breath condensate liquid remains in the collection device. In some embodiments off the invention, after dispensing of liquid, less than 50 pL of breath condensate liquid remains in the collection device. In some embodiments off the invention, after dispensing of liquid, less than 20 pL of breath condensate liquid remains in the collection device. In some embodiments off the invention, after dispensing of liquid, less than 10 pL of breath condensate liquid remains in the collection device.

[0101] Another reason small dead volume needles have not been used in normal syringe operation is because the syringes are designed to aspirate liquid as well as dispense liquid. Aspiration requires low back pressure to allow liquid to be drawn up into the syringe chamber.

[0102] In the device and method of the invention, the dispensing needle may be a tube. The tube or needle may be blunt or piercing. The dispensing needle may be plastic, glass, fused silica, ceramic, paper or metal.

[0103] In some embodiments of the device and method, the collection syringe inside diameter is the same as the inside diameter of the dispensing needle connected to the collector. In some embodiments of the invention, the syringe barrel is a capillary or tube and also serves as the dispensing needle.

[0104] Further discussion of the device and method of the invention

[0105] As stated before, larger volume syringes may be needed to collect larger volumes of breath condensate. But the large volumes are contained in syringes and dispensing from these syringes must have a low resistance to flow. The resistance to flow must be low and therefore the volume of the dispensing needle must be appropriate for the size of the syringe. In the syringe capture device of the invention, large syringes are used but small volumes of liquid are collected. Syringe internal volumes may be < 1 mL, < 2 mL, < 3 mL, < 4 mL, < 5 mL, < 6 mL, < 7 mL, < 8 mL, < 9 mL, < 10 mL, < 15 mL, or < 20 mL. Small volumes of breath condensate are collected relative to the volume of the syringe. Unless 400 pL or more of breath condensate are collected, the configuration of the plunger and needle in the large volume syringe contain a minimum dead volume space that is large relative to the volume of breath condensate collected.

[0106] In some embodiments of the device and method of the invention, the collection volumes of the breath condensate liquid are 500 pL, 300 pL, 200 pL, 100 pL, 50 pL or 25 pL. In other embodiments, the collection volume is at least 5 pL, at least 10 pL, at least 20 pL, at least 30 pL, at least 40 pL, at least 50 pL, at least 60 pL, at least 70 pL, at least 80 pL, or at least 90 pL.

[0107] In some embodiments of the device and method of the invention, most but not all of the collection volumes of the breath condensate liquid are dispensed. The dispensing volume is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90% of collection volume. In one embodiment, a 5 mL syringe collected 60 pL breath condensate liquid. In this embodiment, the dispensing volume is at least 30 pL. In some embodiments, a 2 - 5 mL syringe can collect at least 75 pL and dispenses at least 50 pL of breath condensate liquid. In some embodiments, a 2 - 10 mL syringe collects at least 100 pL and the dispensing volume is at least 50 pL.

[0108] In some embodiments of the invention, a 1 - 10 mL breath condensate syringe device collects at least 200 pL of breath condensate liquid. After collection, a plunger collects and dispenses at least 75% of collected breath condensate liquid. After collection, at least 50% of the collected breath condensate liquid is dispensed. In some embodiments of the invention, a 1 - 10 mL breath condensate syringe device collects at least 100 pL of breath condensate liquid. After collection, a plunger collects and dispenses at least 75% of collected breath condensate liquid. After collection, at least 50% of the collected breath condensate liquid is dispensed.

[0109] In some embodiments of the invention, a 2 - 8 mL breath condensate syringe device collects at least 200 pL of breath condensate liquid. After collection, a plunger collects and dispenses at least 75% of collected breath condensate liquid. After collection, at least 50% of the collected breath condensate liquid is dispensed. In some embodiments of the invention, a 2 - 18 mL breath condensate syringe device collects at least 100 pL of breath condensate liquid. After collection, a plunger collects and dispenses at least 75% of collected breath condensate liquid. After collection, at least 50% of the collected breath condensate liquid is dispensed.

[0110] In some embodiments of the invention, a 2 - 6 mL breath condensate syringe device collects at least 200 pL of breath condensate liquid. After collection, a plunger collects and dispenses at least 75% of collected breath condensate liquid. After collection, at least 50% of the collected breath condensate liquid is dispensed. In some embodiments of the invention, a 2 - 6 mL breath condensate syringe device collects at least 100 pL of breath condensate liquid. After collection, a plunger collects and dispenses at least 75% of collected breath condensate liquid. After collection, at least 50% of the collected breath condensate liquid is dispensed.

[0111] The syringe tube and associated plunger cross sectional shape of the invention may be any shape including round, square, polygonal, etc. A round cross section is the most convenient and most commonly used shape. The shape of the tube may be any configuration that allows the insertion and movement of a plunger.

[0112] In some embodiments of the invention a turbulence inducer is inserted into the centre of the syringe and the outside of the syringe barrel is cooled. Breath is passed through the turbulence inducer so that the liquid from breath is condensed on the inner wall of the syringe. Then the turbulence inducer is withdrawn from the syringe barrel. Then as described above, a syringe plunger is inserted into the syringe barrel to collect and deposit the liquid breath condensate.

[0113] In some embodiments of the invention, a plunger tip is fitted to the end of the turbulence inducer. The turbulence inducer plunger end is smaller diameter than the syringe barrel. In this way, the condensate is not removed by the turbulence inducer when it is withdrawn from the syringe barrel. In some embodiments of the invention the turbulence inducer is reinserted into the syringe barrel after breath collection. Through the re-insertion process, the turbulence inducer plunger end diameter is increased outward so that as the inducer plunger travels back into the barrel, the breath condensate liquid is coalesced. As the inducer plunger reaches the bottom of the barrel, the dead volume is reduced as described herein. There is little or no dead volume, and the breath condensate can be removed from the syringe and directed to the detection reagents. In the device and method of the invention, small liquid sample volumes of 300 pL, 200 pL or less are collected from exhaled breath in a syringe barrel. After collection, most of the collection chamber space is taken up by air. Air may take up 90 to 95% more volume than liquid. Dispensing very small liquid volumes in the presence of air is accomplished through a low dead volume design of the syringe plunger and dispensing needle. The plunger, plunger tip, syringe interface and needle are designed to contain very little dead volume, especially relative to the size of the syringe. In the invention, a plunger is moved through the syringe barrel. However, on the face of the plunger, there is air and liquid. As the plunger moves to the end of the barrel a mixture of air and water is dispensed from the syringe. When the plunger is fully depressed, a small amount of air and liquid is trapped in the plunger and syringe barrel bottom interface. Surprisingly, the dispensing can be operated with the syringe barrel and dispensing needle in any orientation including vertical, pointing downward without losing liquid in the presence of air.

[0114] In one embodiment, the amount of liquid collected can be expressed as the percent recovery liquid contained in the syringe of a specified size. For any given syringe there is a collected total volume of breath condensate liquid and a recovered total volume that is a fraction of the collected volume. The collected volume can be qualitatively estimated to be 5% of the syringe volume. This calculated value is considered to be the maximum capacity and so for syringe volumes of 30 mL - 100 pL the collected volume of breath condensate is in the range of 1.5 mL - 5 pL or for syringes volumes of 30 mL, 10 mL, 5 mL, 3 mL, 2 mL, 1 mL or 0.1 mL, the collected volumes are 1.5 mL, 500 pL, 250 pL, 150 pL, 100 pL, 50 pL, or 5 pL, respectively.

[0115] In general, the dead volume of a syringe will increase as the size or volume of the syringe increases. This is due to all volumes and all aspects of a syringe getting larger as the syringe volume gets proportionally larger. In this consideration, dead volume in a syringe can be described as a function of the total volume. Recovery of breath condensate sample liquid can be expressed as greater than 50% or for syringe volumes of 30 mL, 10 mL, 5 mL, 3 mL, 2 mL, 1 mL or 0.1 mL. The recovery volume is 750 pL, 250 pL, 125 pL, 75 pL, 50 pL, 25 pL, or 2.5 pL, respectively. These capture volumes are possible for syringes of these sizes provided there is no limit on the capture time, air breath volume, etc. The rest of the captured breath condensate liquid is lost and not recovered. Losses occur because of the inability of the device to coalesce the liquid effectively and direct the collected liquid effectively to a receptacle. Losses of these volumes are insignificant in normal syringe operation. In the device and method of the invention, recovery of collected breath condensate liquid can be 75% or greater or 50% or greater. In some embodiments of the invention, the recovery can be greater than 75%, or for syringe volumes of 30 mL, 10 mL, 5 mL, 3 mL, 2 mL, 1 mL or 0.1 mL, the recovery volume can be greater than 1.125 mL, 375 pL, 187.5 pL, 112.5 pL, 37.5 pL, or 3.75 pL, respectively.

[0116] In some embodiments, recovery of 50% or more of the collected volumes is possible with the device and method of the invention. Stated another way, for the method and device of the invention the recovery volume breath condensate liquid is at least 50% of the collected volume. The collected volume is defined as 5% of the syringe volume where the syringe volume is in the range of 30 - 0.1 mL.

[0117] In other embodiments, recovery of 75% or more of the collected volumes is possible with the device and method of the invention. Stated another way, for the method and device of the invention the recovery volume breath condensate liquid is at least 75% of the collected volume. The collected volume is defined as 5% of the syringe volume where the syringe volume is in the range of 30 - 0.1 mL.

[0118] Loss of recoverable liquid sample can be due to dead volumes within the device or the inability to coalesce liquid in the presence of air. Another way to describe the invention is to examine the shape and operation of the collection syringe and then to determine the necessary configuration and performance to reduce or eliminate dead volume and increase the recovery of the breath condensate liquid. But the lowering of dead volume involves examining the process of breath condensate recovery and looking at syringe design in a new manner.

[0119] Thus, in one embodiment of the invention the recovered total volume is greater than 50% of the collected volume. The collected volume is defined as 5% of the syringe volume or 5% multiplied by the syringe volume. In one embodiment of the invention, for a 2 mL syringe, if 100 pL of breath condensate liquid is collected, then at least 50% or 50 pL will be recovered for processing. In another embodiment of the invention, for a 2 mL syringe, if 100 pL of breath condensate liquid is collected, then at least 75% or 75 pL will be recovered for processing. In yet another embodiment of the invention, for a 2 mL syringe, if 100 pL of breath condensate liquid is collected, then at least 90% or 90 pL will be recovered for processing.

[0120] In one embodiment of the invention, the collection syringe volumes are in the range of 1 mL

[0121] - 30 mL, 2 mL - 20 mL, 3 mL - 10 mL, or 4 mL - 7 mL. In one embodiment of the invention, the device and method will recover greater than 50 - 90% of the collected breath condensate liquid. In another embodiment, the device and method will recover greater than 65 - 95% of the collected breath condensate liquid. In a third embodiment of the invention, the device and method will recover greater than 75- 95% of the collected breath condensate liquid.

[0122] To summarize, solving the problem of collecting and coalescing breath inside a syringe and dispensing very small amounts of liquid is challenging and the solution is not obvious. The volume of breath condensate liquid dispensed can be viewed as a function of the syringe volume. The capture capacity of a syringe with outside cooling for capturing breath condensate liquid is 5% of syringe volume. Thus, using a syringe for capturing breath condensate will utilize only a very small portion of the syringe capacity. There are losses when trying to recover the captured breath condensate liquid. Dead volumes increase with syringe volume and the recoverable liquid volume can be expressed as a fraction of the captured volume. Decreasing the syringe size can allow recovery of smaller amounts of breath condensate liquid. However, decreasing the syringe size will decrease the amount breath condensate liquid that can be collected. So, the problem is difficult to solve from a syringe capacity and dead volume perspective. In addition, dispensing liquid in the presence of large volumes of air adds to the problem of collection and efficient recovery of very small volumes of liquids. With these large dead volumes and the presence of air, only a portion (if any) of the condensate liquid will be able to exit the end of the syringe chamber through the needle dispenser. If the portion of condensate recovered is only 5 - 50 %, downstream processing of the condensate liquid is likely not possible.

[0123] It could be argued that as syringe volume increases, the dead volumes become relatively smaller and thus dead volume becomes of no consequence relative to the volume capacity of the larger syringes. However, syringes are designed so that dispensing is easy and proportional to the volume that is being dispensed. This means the back pressure liquid of the liquid flow must decrease as the syringe volume increases. Various strategies? are used to decrease liquid flow backpressure including increasing the needle dispenser diameter, shortening the length, etc. All of these measures increase the dead volume.

[0124] In some embodiments of the invention, a turbulence inducer is also a plunger.

[0125] This patent application also addresses the critical need for accurate measurement of delivered breath condensate samples for the detection of targets in exhaled breath. After collection of the breath condensate, the device will coalesce, collect and transfer the condensate liquid. The breath condensate liquid can be transferred directly with a pipette into a vial, tube or chamber. In some embodiments of the invention, the vial, tube or chamber is marked so that volume measurement can be performed visually. In other embodiments, volume measurement can be performed with a sensor or detector to ensure the minimum sample liquid volume required for processing has been collected. The key aspects of this invention include the direct transfer of small breath condensate samples from a collection device to a vial or chamber and measurement of the volume. Excess liquid may be diverted to prevent overloading. These innovations may be essential, especially for sample volumes in the range of 1 - 400 L, 2 - 300 L, 1 - 200 pL, and 1 - 100 pL. Having inadequate or excessive sample volume could compromise diagnostic accuracy. The fact that the volume measurement ensures a specified volume provides a positive control that sample was collected and delivered for detection.

[0126] In some embodiments of the invention the vial may be comprised of a check valve or air filter as shown in Figure 2. This feature allows air to escape as a plunger is depressed but does not allow liquid to escape. In this embodiment, the vial may be the detection vial or may be an external protection vial. In the protection vial mode for example, the receiving vial can be cooled and (salt) water or other coolant will not enter the receiving vial. But sample can enter from the dispensing syringe and / or dispensing needle.

[0127] In some embodiments of the invention, the vial may be comprised of or contain lyophilized or liquid reagents. In some embodiments of the invention, the reagents may be detection and / or lysing reagents. In some embodiments of the invention, the device plunger may pierce a pillow containing reagents and release acetonitrile, organic solvent or other lysing and / or detection reagents at the end of the dispensing stroke.

[0128] In some embodiments of the invention, the vial end or syringe collection end may be pierceable to allow draining or flowing of the sample and / or detection and lysing reagents into a detection chamber. The piercing end may be by needle. A needle pierced end may be resealable if or when the needle is removed from the vial. The detection device may perform isothermal or thermal cycling amplification. Detection may be by optical, fluorescence, spectrometry, electrochemical or any transducer capable of detecting nucleic acid. Detection may include nucleic acid amplification. Detection may be of the target including nucleic acids, proteins, carbohydrates, spores, or other chemicals related to the target.

[0129] In some embodiments of the invention, multiple detection chambers may be used for lysing, detection, multiplex detection, etc. The collected sample may be transferred directly to master mix or detection reagents either before or after volume measurement. In some embodiments, the volume measurement can be performed before or after the addition of lysing reagents. Lysing reagents may be part of the master mix.

[0130] In some embodiments of the invention there is direct transfer of a small, known sample volume from a breath collection syringe to the detection reagents. Sample volumes can be less than 400 pL, less than 300 pL, less than 200 pL, less than 100 pL or less than 50 pL. In some embodiments of the invention, excess sample over the measured minimum amount is not removed and / or measured.

[0131] In some embodiments of the invention, the sampling time and / or the sampling volume collected is used to calculate the target detection limit that can be detected from a target shedding individual. In some embodiments of the invention, the sampling time and / or the sampling volume collected is used to calculate the target shed rate from an individual.

[0132] In order for the analytical diagnostic results to be meaningful and relevant, confirmation is needed that a minimum or sufficient volume sample was collected, transferred and processed. Insufficient sample could result in a diagnostic test result that was falsely negative.

[0133] Another requirement is that the sample was properly and fully mixed with the detection reagents. Improper or incomplete mixing of breath condensate sample with detection reagents could result in a diagnostic test result that was falsely negative.

[0134] In some embodiments of the invention, a dye is added to any or all of the processing reagents including a lysing reagent, master mix, sample buffer or sample. Mixing is confirmed by uniform distribution of dye throughout the sample and reagent mixture. Uniformity may be checked visually or by an instrument, usually a spectrometer. Mixing evaluation can be undertaken in a variety of ways, which typically rely on flow visualization methods. The most common way to visualize mixing and study mixing efficiency is through mixing a dyed liquid with a clear liquid and examining the colour of the product stream over time using eyes, a camera, spectrometer or other sensor. For example, the mixing of a blue stream and a yellow stream creates a purely green stream when fully mixed. Fluorescent particles or fluids also can be used in conjunction with sensors to characterize the mixing. In some embodiments of the invention the master mix, detection reagents and / or optional dye are lyophilized or supplied as dried reagents. Volume measurement and mixing can be performed under these circumstances.

[0135] There are a number of possible mixing procedures. Some embodiments are simple manual shaking or mechanical spinning, shaking or vortexing. Passive mixing elements may be incorporated into microfluidic devices. Simple diffusion may be employed although this is at the expense of time. The two streams may be mixed together within a single channel. The diffusion between the two fluids across the interface leads to a uniform mixture at the molecular level. The transport of fluid in this case is a result of Brownian motion along the concentration gradient between the two fluids. Since the interface between the two fluids is long, the diffusion occurs at an accelerated rate across the interface. This process can be sped up with heat; however, passive mixing can still be slow.

[0136] Additional active mixing methods can be used. Micro stirrers can be fabricated for use in microfluidic devices. This type of active mixing enhancement uses a rotating magnetic field, which causes a microbar to rotate within a fluid system, enhancing the mixing in the vicinity of the bar.

[0137] Another unique way to promote mixing in microfluidic devices may be the use of acoustic waves. Surface acoustic waves are a type of elastic energy along the surface of the fluid, which can induce acoustic streaming through the fluid when excited. Diffusion coefficients typically increase with increasing temperature. Electroosmosis is a commonly used pumping method for microfluidic devices. This type of pumping utilizes electrodes within the channels to create an electric field.

Claims

Claims1 . A device for collecting and dispensing breath condensate liquid, the device comprising: a syringe barrel having a volume of 30 mL or less; a turbulence inducer retractably mounted in the syringe barrel, wherein the turbulence inducer has a proximal end and a distal end, wherein a breath inlet structure is attached to the proximal end of the turbulence inducer, and wherein the distal end of the turbulence inducer is configurable as a plunger; and a dispensing needle disposed at a needle-syringe interface at a lower end of the syringe barrel.

2. The device of claim 1 , wherein the syringe barrel and the dispensing needle are comprised of a capillary or tube.

3. A method of dispensing breath condensate liquid, the method comprising:(a) introducing breath into the breath inlet structure of the device of claim 1 or claim 2 with the turbulence inducer disposed within the syringe barrel, wherein the breath condensate liquid is collected on an inside wall of the syringe barrel, and wherein the volume of the breath condensate liquid is 400 pL or less;(b) retracting the turbulence inducer from the syringe barrel to an upper position in which the distal end of the turbulence inducer is converted to the plunger; and(c) depressing the turbulence inducer into the syringe barrel, whereby the plunger scrapes the walls of the syringe barrel as the plunger moves toward a lower end of the syringe barrel so that a combination of air and breath condensate liquid is dispensed from the dispensing needle at the lower end of the syringe barrel.

4. The method of claim 3, wherein the air and breath condensate liquid are dispensed into a detection vial, tube or chamber.

5. The method of claim 4, wherein the vial, tube or chamber contains detection reagents.

6. The method of claim 4 or claim 5, wherein the vial, tube or chamber contains a dye and wherein the breath condensate liquid and the dye are mixed, and wherein the uniformity of the mixed solution is measured.

7. The method of claim 5 or claim 6, wherein the detection reagents or the dye are lyophilized.

8. The method of any one of claims 3 to 7, wherein, after step (c), the breath condensate liquid remaining in the syringe barrel is less than 100 pL.

9. The method of claim 8, wherein after step (c), the breath condensate liquid remaining in the syringe barrel is less than 50 pL.

10. The method of claim 9, wherein after step (c), the breath condensate liquid remaining in the syringe barrel is less than 20 pL.11 . The method of claim 10, wherein after step (c), the breath condensate liquid remaining in the syringe barrel is less than 10 pL.

12. The method of any one of claims 3 to 11 further comprising: measuring the volume of the dispensed liquid optically or electronically; and optionally, if the measured volume exceeds a threshold, removing an excess volume.

13. The method of any one of claims 3 to 12, wherein, in step (c), greater than 50% of the collected breath condensate liquid is dispensed.

14. The method of claim any one of claims 3 to 13, wherein, during step (c), the plunger contacts a peripheral region of a bottom face of the syringe barrel but does not contact either a central region of the bottom face or the syringe-needle interface.

15. The method of claim any one of claims 3 to 13, wherein, during step (c), the plunger contacts both a peripheral region of a bottom face of the syringe barrel and a central region of the bottom face but does not contact the syringe-needle interface.

16. The method of claim any one of claims 3 to 13, wherein, during step (c), the plunger contacts all of a peripheral region of a bottom face of the syringe barrel, a central region of the bottom face, and the syringe-needle interface.

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