Method and System for Sample Collection, Storage, Preparation and Detection

Abstract

A collection device for a biological sample to capture target compounds such as viruses or other pathogens or particles for testing from within the sample and move the captured target compound to a separate chamber for subsequent processing. The collection device can include an openable substance blister including capture particles located in a cup interior. Capture particles can attract and bind the target compounds from the sample. An extraction tube extracts any nucleic acid from the target compound for storage or subsequent amplification and testing to confirm presence of known microorganisms. The extraction tube can comprise a heat-deformable material and can be connected to a microfluidic cartridge for further processing of nucleic acid including, amplification and detection. The microfluidic cartridge includes valves and a plurality of chambers for amplification.

Claims

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19. A method for collecting and processing a sample comprising the steps of: collecting a sample in a cup, a substance comprising capture particles within an interior of the cup; capturing with the capture particles at least a portion of the sample; attracting the capture particles; moving the capture particles to a processing device; processing the sample in the processing device.

20. The method of claim 19 wherein the processing device is a microfluidic detection cartridge configured for carrying out thermocycling for amplification and detection of nucleic acid.

21. The method of claim 20 wherein processing the sample determines if the sample includes a pathogen in which detection of the nucleic acid determines that the pathogen is present in the sample.

22. The method of claim 21 wherein the pathogen is SARS-CoV-2 associated with disease COVID-19.

23. The method of claim 19 wherein the step of moving the capture particles to a processing device comprises moving said substance to an attachment tube received in said processing device, said attachment tube at least partially formed of a heat deformable material and further comprising the steps of: heating the heat deformable material for causing the attachment tube to contract for pushing the sample through the attachment tube to said processing device.

24. The method of claim 19 wherein the attachment tube comprises one or more reagents, or one or more reagents are added to the inner chamber, and wherein at least one of the sample and the one or more reagents comprises a liquid.

25. The method of claim 24 wherein the one or more reagents comprises: a) an enzyme, b) a thermostable enzyme, a thermophilic enzyme, a mesophilic enzyme, or a proteolytic enzyme, c) an alkaline proteinase, a serine protease, a metalloproteinase, a neutral proteinase, a threonine proteinase, an aspartate proteinase, a cysteine proteinase, or a cell-wall degrading enzyme, d) a thermostable proteinase derived from a thermophilic Bacillus species, or e) cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, Bacillus sp. EA1 proteinase, thermolysin, caldolysin, a Thermus proteinase, or a combination of any two or more thereof.

26. The method according to claim 19 wherein the substance is contained in an openable compartment within an interior of said cup and further comprising a step of: piercing with a piercing mechanism the openable compartment for opening the openable compartment to release the substance, the openable component is a blister, the capture particles of the substance comprises magnetic capture beads, wherein upon opening of the openable compartment the magnetic capture beads capture and bind to the at least one portion of the sample.

27. The method according to claim 26 further comprising the step of providing a cap, the cap configured to close an opening in the cup, said piercing mechanism is actioned by attachment of said cap to said opening, said piercing mechanism opening said blister.

28. The method according to claim 27 wherein the cap comprises a central extruding portion housing a plunger mounted movably within the central extruding portion, the plunger comprising a base and an upper surface.

29. The method according to claim 23 wherein the step of attracting the capture particles comprises attraction means for attracting the capture particles, the attraction means is a magnet; a base of the plunger is configured to contact said attraction means for moving said attraction means into said attachment tube.

30. The method according to claim 29 wherein said upper surface of said plunger is configured to seal said cup.

31. The method according to claim 29 wherein the cup comprises an upper compartment and a lower compartment, said blister positioned in said upper compartment, said attraction means positioned in or integral with said lower compartment.

32. The method according to claim 29 wherein the plunger is activated for moving said magnet into the attachment tube.

33. A sample collection and processing device comprising: a cup having an opening configured for collecting a sample within an interior of said cup; a cap, the cap configured to close the opening in the cup; and the cap comprises a central extruding portion housing a plunger mounted movably within the central extruding portion, wherein the base of the plunger is configured to move the sample to an attachment tube, said attachment tube configured for attachment to a processing device.

34. The sample collection and processing device of claim 33 wherein the attachment tube is at least partially formed of a heat deformable material.

35. The sample collection and processing device of claim 33 wherein the attachment tube comprises one or more reagents, or one or more reagents are added to the inner chamber, and wherein at least one of the sample and the one or more reagents comprises a liquid.

36. The sample collection and processing device system of claim 35 wherein the one or more reagents comprises: a) an enzyme, b) a thermostable enzyme, a thermophilic enzyme, a mesophilic enzyme, or a proteolytic enzyme, c) an alkaline proteinase, a serine protease, a metalloproteinase, a neutral proteinase, a threonine proteinase, an aspartate proteinase, a cysteine proteinase, or a cell-wall degrading enzyme, d) a thermostable proteinase derived from a thermophilic Bacillus species, or e) cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, Bacillus sp. EA1 proteinase, thermolysin, caldolysin, a Thermus proteinase, or a combination of any two or more thereof.

37. The sample collection and processing apparatus according to claim 33 wherein said processing device is a micro-fluidic cartridge configured for nucleic acid amplification.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 shows a perspective view of an embodiment of the invention.

[0037] FIG. 2 shows an angled cross-section of the embodiment of FIG. 1.

[0038] FIG. 3 shows a perspective view of an embodiment of the cap.

[0039] FIG. 4 shows a perspective cross-section view of the invention with the cap attached.

[0040] FIG. 5 shows a side, cross-section view of the cap showing the plunger.

[0041] FIG. 6 shows a cross-section perspective view of the invention with the cap attached.

[0042] FIG. 7 shows a perspective view of the invention without the cap attached.

[0043] FIG. 8 shows a birds-eye view of the invention without the cap attached.

[0044] FIG. 9 shows a diagrammatic view of the plunger connecting to the magnet.

[0045] FIG. 10 shows a side view of an embodiment of the invention.

[0046] FIG. 11A-E show diagrammatic views demonstrating use of the invention.

[0047] FIG. 12 shows a perspective view of an embodiment of the invention.

[0048] FIG. 13 shows a cross-section perspective view of an embodiment of the invention.

[0049] FIG. 14 shows a cross-section perspective view of an embodiment of the invention.

[0050] FIG. 15 shows a perspective view of an embodiment of the invention.

[0051] FIGS. 16A-16B are schematic diagrams of a prior art device.

[0052] FIG. 17 is a schematic diagram of a microfluidic cartridge in accordance with the teachings of the present invention.

[0053] FIG. 18 is a schematic diagram of a single amplification chamber with a Tillable unit.

[0054] FIG. 19 is a schematic diagram of a hydrophobic valve for use with the microfluidic cartridge in accordance with the teachings of the present invention.

[0055] FIG. 20 shows an outline view of an assembled extraction tube within a cartridge opening of the microfluidic cartridge.

[0056] FIG. 21 shows a disassembled configuration of the microfluidic cartridge.

[0057] FIG. 22 shows filled chamber windows during use.

[0058] FIG. 23 shows an of the microfluidic cartridge including a gripping area.

[0059] FIG. 24 shows an alternative embodiment of the hydrophobic valve.

[0060] FIG. 25 shows a front view configuration of the invention assembled and inserted into a processing device.

[0061] FIG. 26 shows a perspective front view configuration of the invention assembled and inserted into a processing device.

DETAILED DESCRIPTION

[0062] Reference will now be made in greater detail to a preferred embodiment of the invention, an example of which is illustrated in the accompanying drawings. Wherever possible, the same reference numerals will be used throughout the drawings and the description to refer to the same or like parts.

[0063] FIGS. 1-9 illustrate collection device 10 in accordance with the teachings of the present invention. Cup 12 is comprised, in certain preferred embodiments, of a tapered upper compartment 14 into which a saliva sample is collected from a patient. In a preferred embodiment, 0.25-2 mL collection is provided in upper compartment 14 to an optional fill line 15 marked on a perimeter of wall 16 of cup 12. Magnet cage 42 can be used to mark the fill-line 15.

[0064] In use, saliva sample collection is achieved by the user spitting, expelling saliva through their mouth, into opening 18 of cup 12. This is achieved by spitting directly into upper compartment 14 of cup 12. Once the required sample size is met, cap 22 is placed on cup 12 such as by the same user or a separate device handler. Cap 22 can be provided separately in separate packaging and may rest on collection cup 12 prior to use.

[0065] In the preferred embodiment, upper compartment 14 of cup 12 is tapered. Upper compartment 14 includes blister 24 filled with diluent 26. In a preferred embodiment, capture particles 28 with a means of capturing target compounds such as viruses and other pathogens or biological targets, are also stored in upper compartment 14, preferably within the diluent within the blister 24. Capture particles 28 can be magnetic beads within lyophilised or unlyophilised pellets. In use, the beads are capable of capturing virus from within a sample which is achieved within a short time frame which can preferably be within5 200 seconds, optionally within 30-60 seconds. In a particularly preferred embodiment the target compounds which the magnetic beads can capture are biological pathogens including SARS-CoV-2 and Influenza Types A and B. When mixed with the sample, the magnetic beads capture and bind the target compounds. Suitable capture particles include any coated or uncoated, lyophilised or unlyophilised magnetic beads or enrichment beads capable of compound capture.

[0066] Upper portion 30 of walls 16 of upper compartment 14 are capable of connecting with walls 31 of cap 22. In one embodiment, upper compartment 14 tapers down to a narrower diameter portion. A seal base 32 can separate the upper compartment 14 from a lower compartment 34. Above seal base 32 are flaps 36 which are closed and openable by the action of plunger 38. Preferably, flaps 36 comprise a snappable face, which, when contacted by plunger 38, snaps downwards acting to pierce seal base 32. This action opens upper compartment 14 to lower compartment 34 allowing the movement of the sample down to lower compartment 34. Seal base 32 can be formed of foil or other suitable sealing material.

[0067] A nest compartment 40 can be situated below plunger 38, when the plunger 38 is in the first, unplunged position within cap 22. When cap 22 is connected to upper compartment 12, nest 40, comprising pelleted substance 43 sits directly above flaps 36 in a closed position. Nest compartment 40 can include a plurality of claw features configured to hold a pelleted substance. Pelleted substance 43 can be, for example, a positive control or a required compound or substance to be mixed with or combined with the collected sample. In a preferred embodiment, the positive control is a bacterial phage.

[0068] Lower compartment 34 of cup 12 comprises magnet cage 42. Magnet cage 42 can house magnet 44. Magnet 44 can be a diametrically-poled magnet. Preferably, magnet 44 is moveably positioned within magnet cage 42 to allow for downward movement actuated by motion of plunger 38 through lower opening 46 of cup 12 and into attachment tube 48 for collection. In a preferred embodiment, attachment tube 48 is an extraction tube configured for carrying out nucleic acid extraction. In a preferred embodiment of use, magnet 44 attracts the magnetic capture particles 28 from the saliva sample having captured any virus or target compound. These capture particles will stay connected by magnetic attraction to magnet 44 allowing magnet 44 and capture particles 28 to be moved into attachment tube 48. The movement of the plunger 38 from an initial plunged position to a further plunged position will act to push the cage 42 and magnet 44 down through into the attachment tube 48 where it will fall freely within the attachment tube 48.

[0069] Optionally, pelleted substance 43 is not included. Seal base 32 or flaps 36 are not included and upper compartment 14 is open to lower compartment 34.

[0070] Below the magnet cage 42, the interior of the cup wall 49 can be coated in a wiping means 50 able to remove any sample from the magnet 44 or magnet cage 42 before the magnet and/or magnet cage is pushed down into the subsequent connected attachment tube 42 for storage or for extraction of nucleic acid from capture particles. Wiping means 50 can be a wiper coating of a plurality of separate materials or one continuous coating.

[0071] Optionally, in one embodiment, pelleted substance 43 is not included. Seal base 32 or flaps 36 are not included and upper compartment 14 is open to lower compartment 34. In this embodiment, below magnet cage 42, interior of cup wall 49 can be coated in wiping means 50.

[0072] Rim 54 of lower opening 46 of cup 12 includes a collar 56 to connect to an attachment tube 48 for storage or extraction or suitable processes. In a preferred embodiment, the connection of attachment tube 48 to cup 12 is completed during manufacturing stage, in an alternative embodiment attachment, attachment tube 48 may be provided to a user unconnected for the user themselves to connect. Collar 56 is a Luer connection taper or suitable medical standard for connecting interfaces.

[0073] Cap 22 comprises an exterior circular base 60 with central extruding portion 62 as shown in the FIGS. 1-8, in which plunger 38 is situated in a first unplunged position. Plunger 38 is not accessible to the user as it is housed and hidden within interior walls of extruding portion 62 of cap 22. This prevents accidental or premature activation by the user. In a preferred embodiment, plunger 38 is circular in diameter. In use, when cap 22 is placed and locked in position on cup 12, base 39 of plunger 38 rests just above closed flaps 36 as seen in FIG. 2 and FIG. 4. In a preferred embodiment, plunger 38 is cylindrical in diameter to enable smooth movement within the tapered narrow portion 82 of lower compartment 34 of the cup and acts to create an airtight seal when fully plunged the full length to be in the fully plunged position. The air-tight seal is formed between the outer perimeter 84 of plunger 38 and the inner perimeter of walls of the lower compartment 34, as exemplified in FIG. 9.

[0074] Plunger 38 preferably comprises a liquid-sealing portion at its top which acts to seal upper compartment and prevent spillage out of cup 12. Liquid-sealing portion can be a rubber layer.

[0075] In use, after collection of saliva sample within upper compartment 14, cap 22 is closed and secured onto upper opening 18 of cup 12 by a closure mechanism to be actioned by the user. In a preferred embodiment, cap 22 is not re-openable or unattachable once securely attached. The user-actioned, closure mechanism brings piercing mechanism 70 shown in FIG. 3, located within the interior of cap 22, into contact with the surface of the blister 24 filled with diluent 26 and capture particles 28 with a means of capturing virus causing piercing or tearing of blister 24 and triggering diluent 26 and particles with a means of capturing virus 28 to empty out of blister 24 and mix with the collected saliva sample within upper compartment 14. In the preferred embodiment, piercing mechanism 70 is a bladed edge and closure mechanism is a twist-action.

[0076] Blister 24 sits within upper compartment 14 as shown in FIGS. 1, 2 and 6. Blister 24 can be comprised of any suitable material known in the art. In use, the opening of blister 24 allows the sample to be mixed together with diluent 26 stored within blister 24 and with capture particles 28 with means of capturing target compound. Together, the entire mixed substance (mixture 1) flows down to the base of upper compartment 14, in use, due to gravity, when the device is in the upright position. In an embodiment, this movement of mixture 1 to the base of upper compartment 14 causes mixture 1 to submerge and come into contact with pelleted substance 43, optionally a lyophilised pellet, situated in nest compartment 40 directly below plunger 38 within cap 22, when the plunger is in the first, un-plunged position. In a preferred embodiment, pelleted substance 43 is a positive control in the form of a bacterial phage. When cap 22 is secured to cup 12, pelleted substance 43 within nest compartment 40 rests just above the base of upper compartment 14. Pelleted substance 43 will be mixed with mixture 1 to form mixture 2. When pelleted substance is a lyophilised pellet, this will be dissolved by mixture 1.

[0077] At this point, in a preferred method of use, the collection device 10 can be motioned in a swirling motion manually by the user or in an automated shaking, swirling or other appropriate motion device, to facilitate the dissolving of pelleted substance 43 to create mixture 2 whereby capture particles 28 with a means of capturing virus, capture the pelleted positive control bacterial phage as well as any virus within the sample. Mixture 2 includes capture particles 28, having captured the positive control bacterial phage as well as any target compound within the sample.

[0078] After the appropriate length of time and with the appropriate motion to allow for adequate mixing, mixture 2 sits directly below the base face of plunger 38 and directly above seal base 32 comprised of flaps 36 which divides upper compartment 14 from the lower compartment 34. Collection device 10 is manually connected to a detection cartridge 200 and the entire assembly 111 is inserted into detection control device 400 (FIGS. 26, 27). Plunger 38 is first activated by the detection control device. In a preferred embodiment of use, plunger 38, hidden within the mechanism of cap 22 is actuated by an instrument (not shown) for a determined amount of time after the twisting closure action of cap 22, allowing adequate time for the mixing of the diluent 26 stored in blister 24, with the sample and the compounds stored within nest compartment 40 at the base of upper compartment 14.

[0079] In a preferred embodiment, the plunger mechanism is activated by an instrument (not shown) within detection control device 400, which actuates a rod, pushing the plunger 38 in a downward motion through its internal tunnel, from a first unplunged position, into an initial plunged position into the upper compartment 14, until connecting with seal base 32 comprising flaps 36. Base 39 of plunger 38 comprises a flat surface that can connect with the magnet upper face. Flaps 36 facilitate the base 39 of plunger 38 in breaking though seal 32, by providing a sharpened edge to pierce seal base 32 as described below. Plunger 38 can be activated in two stages with a pause of sufficient, variable time to allow any required substance reactions to occur. In a preferred embodiment, the first plunger movement from a first unplunged position to an initial plunged position, is actuated after 30-60 seconds to allow adequate initial mixing for mixture 1 and for any positive control and for target compound capture to occur to create mixture 2. In a first stage, plunger 38 is moved sufficiently to an initial plunged position to connect with flaps 36 which comprise a sharp edge. The sharp edges may act to tear open seal base 32, at the base of upper compartment 14, causing mixture 2 to move into lower compartment 34 of cup 12 where magnet 44 is housed, optionally within magnet cage 42.

[0080] In use, after seal base 32 is opened, plunger 38 motion can be paused at this stage, stopping just above the surface of magnet 44 to allow adequate time for capture particles 28 with a means of capturing viruses or other biological target compounds within the sample, to reach magnet 44. Magnet 44 sits within lower compartment 34, optionally within magnet cage 42.

[0081] In the second stage of its action, from an initial plunged position to a further plunged position, plunger 38 pushes down further along an interior of cup 12, connecting with the top of magnet 44 or optionally magnet cage 42 housing it. In the further plunged position, the plunger 38 pushes magnet 44 and optionally magnet cage 42 down through to the bottom narrowest tapered opening 82. In being pushed downwards, magnet 44 drops through, into connected attachment tube 48 below, after passing through wiping means 50 contained within the bottom of the opening to ensure no saliva is brought through with magnet 44. Wiping means 50 captures any excess sample and acts to wipe down magnet 44 or magnet cage 42 before it enters attachment tube 48. The magnet or magnet cage entering attachment tube will act to introduce any attached capture particles into the attachment tube 48 for subsequent nucleic acid extraction.

[0082] In the further plunged position, base 39 of plunger 38 remains in narrow opening 82. Outer rim 84 around the circumference of base 39 of plunger 38 acts to seal lower compartment 34 of cup 12 from attachment tube 48 below, as exemplified in FIG. 9. This creates an air-tight, liquid-tight seal required for the subsequent extraction to work successfully. Magnet 44, magnet cage 42 and means of capturing virus 28 are therefore transported through, whilst any saliva sample is retained within cup 12.

[0083] In one embodiment, attachment tube 48 can be an extraction tube such as the PDQex tube manufactured by MicroGEM or to MicroGEM standards and as described in U.S. Pat. No. 10,758,900 or WO16144192 A1 hereby incorporated by reference into this application. In a preferred embodiment, the bottom of cup 12 is connected to a top of the extraction tube at the manufacturing stage via a Luer taper interface with protruding lines and is supplied pre-assembled. The PDQex extraction tube comprises a heat-deformable material, such that an inner chamber, into which the capture particles 28 with the captured virus is deposited, can, when heated, adopt a second configuration having a chamber volume less than the chamber volume of a first configuration, thereby expelling at least a part of the processed sample through a second opening from the device, optionally into a microfluidic cartridge.

[0084] In an alternative embodiment, pelleted substance 43, preferably a lyophilised positive control pellet, is stored to the side of plunger 38 rather than at the base of the upper compartment 14. In this embodiment, capture particles 28 can be mixed with the sample and positive control pellet by a twist mechanism of cap 22. Subsequent actions include a user sliding the entire attachment tube 48 into the detection cartridge device for analysis.

[0085] It is an intent of the invention that collection device 10 is used to efficiently collect a saliva sample, enrich the saliva sample by capturing any target compound and be used with a connectable attachment tube 48 to extract nucleic acid from any captured target compound for subsequent analysis and testing within a portable testing device which may provide results of the analysis, all within a rapid time frame, suitable for use in any field setting outside of the lab. The entire process may be carried out in one setting, by non-medical professionals in a time frame which is drastically shorter than the current laboratory sample collection, testing and analysis process.

[0086] FIGS. 10-15 illustrate an alternative but similar functioning embodiment of the present invention. Cup 112 comprises tapered upper compartment 114 with extending walls 118 into which a sample is collected, such as a saliva sample from a patient. Upper compartment 114 can include blister 124 filled with diluent or similar solution 126 and optionally capture particles. Upper portion 130 of walls 118 of upper compartment 114 are capable of connecting to cap 122. In this embodiment, upper compartment 114 is not physically separated from lower, tapered compartment 134 and therefore collected saliva or other sample is able to flow down to the base of lower compartment 134 of cup 112. Lower compartment 134 of cup 112 can further comprise magnet 144, magnet cage 142 and guiding feature 115. FIG. 10 shows an exterior side view of cup 112 and a connected attachment tube 148. The outer exterior of guiding feature 115 can be viewed through the walls 134 of the lower compartment. Guiding feature 115 can act as a sample fill line guide for the user. In some embodiments guiding feature 115 can be coloured conspicuously so as to clearly been seen through the walls 134 of cup 112. Guiding feature 115 can simultaneously act as an internal guide to ensure the plunger 138 meets magnet 144 in the correct alignment and keeps magnet 144 positioned securely in transit.

[0087] FIG. 14 shows an internal cutaway view of cup 112 with cap 122 connected. Lower compartment 134 can be seen, comprising magnet 144 and magnet cage 142 held within guiding feature 115. Magnet 144 can be a diametrically-poled magnet. Preferably, magnet 144 is moveably positioned within magnet cage 142 to allow for downward movement actuated by motion of plunger 138, similarly to the embodiment shown in FIGS. 1-9. In a preferred embodiment of use, magnet 144 will attract magnetic capture particles from the sample, having captured any target compound. These capture particles will stay connected by magnetic attraction to magnet 144 allowing magnet 144 and capture particles to be moved into attached attachment tube 148. The downward movement of plunger 138 will act to push magnet cage 142 and magnet 144 down through into the attachment tube 148 where magnet 144 and magnet cage 142 will fall freely within the attachment tube 148. This action will take place after enough time has elapsed to allow sufficient attraction of magnetic beads to magnet 144.

[0088] In an embodiment, the connection of the saliva cup 112 to a subsequent attachment tube 148 and microfluidic cartridge or other suitable testing cartridge 200, is completed during a manufacturing stage to provide a fully assembled device 111 as shown in FIG. 12 and assembled device 222 shown in FIG. 20. In an alternative embodiment cup 112 is provided unconnected but configured for subsequent attachment to an attachment tube 148. In a further alternative embodiment, cup 112 is pre-assembled to only extraction tube 148 for subsequent insertion into cartridge 200, as shown in FIG. 11E and FIG. 13.

[0089] In use, after collection of saliva or other sample within upper compartment 114 as shown in FIG. 11A, cap 122 is closed and secured onto and over walls 118 of cup 112 as shown in FIG. 11B. Secure closure can be achieved by a twist mechanism to be actioned by the user, as shown in FIG. 11C.

[0090] The user-actioned, twist closure mechanism brings piercing mechanism 170 shown located within the interior of cap 122, into contact with the surface of blister 124 filled with diluent 126 as shown in FIG. 13. This movement causes opening of blister 124 for example by piercing or tearing of a foil portion of blister 124 and triggering diluent 126 and capture particles within to empty out the blister compartment and mix with the collected saliva sample within cup 112. Once blister 124 is pierced open, the collected sample is mixed together with diluent 126 and means of capturing any pathogen or virus or other target compound to form an enhanced or enriched mixture. In one embodiment, the target compound is the virus is SARS-CoV-2 associated with the disease COVID-19.

[0091] In use in this embodiment, no positive control is present within the nest compartment, and alternatively there is no nest compartment present within cup 112. In use, in this embodiment, a suitable positive control can instead be introduced downstream. The enriched mixture comprises the user's sample together with diluent 126 and magnetic capture particles. As shown in FIG. 11D, shaking or twisting, or swirling action by the user happens following the attachment of cap 122, to facilitate sample mixing with the diluent, compound capture, and attraction of magnetic beads to the magnet. In the present embodiment, this action can be taken for a certain time period between about 5-200 seconds or may not be required. Alternatively, cup 112 can be placed in a twisting or swirling mechanical device such as those typically found in a laboratory, to enable the appropriate motion to occur

[0092] Following the action taken as shown in FIG. 11E to assemble cup 112 together with a suitable detection cartridge 200, forming assembled device 111, the magnetic capture particles located within the enhanced mixture can be attracted towards magnet 144. In this embodiment, the entire assembled device 111 can be inserted into a detection control device (FIGS. 26 and 27) which can control the plunger action, required heating controls, amplification, detection and analysis.

[0093] Similarly to the previous embodiment, the detection/control device (exemplified in FIGS. 26 and 27) may actuate a rod (not shown), causing the plunger 138 to be pushed in a downward motion, through its internal tunnel within cap 122, from a first unplunged position, into a first plunged position sufficiently to connect with the top of magnet 144 or optionally magnet cage 142 housing it. Guiding feature 115 can act to guide the plunger to align accurately with the top of the magnet. This subsequent movement of plunger 138 from the first plunged position to a further plunged position acts to push magnet 144 and magnet cage 142 down through to the bottom narrowest tapered opening and into an extraction tube 148 or suitable attachment tube for collection or further processing.

[0094] Outer rim portion 198 may be included around the outer circumference of the magnet cage 142 to retain the sample within cup 112 as shown in FIG. 14.

[0095] Preferably attachment tube 148 is an extraction tube. In one embodiment, extraction tube 148 can be a PDQex tube manufactured by MicroGEM or to MicroGEM specification or alternative suitable lysis tube. The PDQex comprising a heat-deformable material and functions as previously described. Suitable extraction reagent can be included within the extraction tube. These reagents can be thermostable enzymes such as those referenced in U.S. Pat. No. 10,758,900, this patent is hereby incorporated by reference in its entirety into this application. In various embodiments the one or more reagents comprises an enzyme. In various embodiments the enzyme is selected from the group comprising a thermostable enzyme, a thermophilic enzyme, a mesophilic enzyme, a proteolytic enzyme, an alkaline proteinase, a serine protease, a metalloproteinase, a neutral proteinase, a threonine proteinase, an aspartate proteinase, a cysteine proteinase, a cell-wall degrading enzyme, and a combination of any two or more thereof. In various embodiments the one or more reagents comprises cellulase, hemicellulase, pectinase, glucouronidase, glucanase, chitinase, laminarinase, lyticase, lysozyme, subtilisin, proteinase K, trypsin, Bacillus sp. EA1 proteinase, thermolysin, caldolysin, a pectate lyase, polygalacturonase, lysozyme, a lysin, a lytic enzyme, a Thermus proteinase, or a combination of any two or more thereof. In a preferred embodiment, the bottom of cup 112 is connected to the top of the PDQeX extraction tube at the manufacturing stage via a Luer taper interface with protruding lines.

[0096] In an alternative embodiment, the sample to be collected may include other bodily fluids and tissue including but not limited to blood, urine, skin cells, mucus, plasma and serum, faecal matter, cultured cells, vaginal fluid, semen or sputum as well as plant tissue, microorganisms, water or soil or, edible fluids such as beverages and anything that might be needed to test for the presence of a certain target compound or nucleic acid. . The fill line, perimeter size and shape can be adjusted to suit the sample requirement.

[0097] In further alternative embodiments of the invention, the diluent blister is stored in a compartment within the cap or is directly beneath the plunger, whereby the closing mechanism of the lid causes the bursting of the blister due to pressure to releases the diluent or other substance. The blister may be formed of a laminate pouch with a heat seared rim to be squeezed open by pressure from the cap closure mechanism or any material suitable for tearing, ripping or squeezing open with the action of the lid. The diluent blister may be held in place via a compartment or holding means.

[0098] In further alternative embodiments the nest compartment, below the plunger, within the cap may house various suitable compounds to be mixed with the collected sample. This compound can be in the form of a lyophilised pellet or others suitably enriched or modified compounds for enriching the sample.

[0099] In a further alternative embodiment, the magnet or magnet cage is attached to the base of the plunger and is brought down into the cup with the lowering action of the plunger from the first un-plunged position to the initial and further plunged positions. The magnetic capture particles will be attracted to the magnet in the same way after having been mixed with the sample. A second stage of action of the plunger can then push the magnet down into the attachment tube 148. A barrier layer can be situated beneath magnet cage 142 to prevent the magnet entry into attachment tube 148 prematurely. The barrier layer can comprise foil or suitable material, which can be broken through when the magnet or magnet cage gets pushed by the plunger, into the attachment tube. The barrier layer can serve the purpose of sealing any required substances such as but not limited to reagents and buffer, within the attachment tube 148.

[0100] In an alternative embodiment, the wipers at the base of the cup can be any suitable material capable of removing the sample from the magnet or magnet cage or may not be a necessary requirement depending on the sample collected. Suitable materials include but are not limited to bristles, rubber wipers and/or felt strip surrounding the outer rim of the exit opening and acting to prevent unnecessary sample entering the attachment tube.

[0101] In a further optional embodiment, the plunger may be activated by the user or device handler for example where the user is a medically trained professional with instructions provided on the required time delay with first and second plunging actions.

[0102] Optional means for achieving adequate mixing of the capture particles with the sample in a suitable amount of time may include: written, visual or audible instructions for the user to motion the cup for a required amount of time; adding a chemical agent to cause effervescence within the sample included but not limited to effervescent tablet formations,

[0103] Further examples of use include collection of multiple users' samples within one cup. This pooling of samples can be done by users depositing as mentioned previously, individual saliva sample into the same cup in succession. Alternatively, additional saliva collecting tubes can be used to individually transfer a saliva sample from a user to the single cup. The cup can be varied in size to accommodate multiple sample types and quantities of sample. The device will work essentially in the same way as previously described for pooling of samples. A separate device can be used to transfer multiple individual samples to the cup compartment. The separate device can be configured to securely interact with the cup compartment.

[0104] In certain embodiments, the cup can be configured to fit, communicate or interact with additional devices including but not limited to: devices to facilitate and/or automate saliva collection and discharge into the cup including mouthpieces, swabs and saliva production catalysts, features to indicate adequate sample collection including built-in measurements and sensors, features to facilitate sample collection including holders, features to divide the sample into separate portions including separate chambers within the compartments, features to amend the shape or diameter of the cup to allow communication with varying sized upstream processing devices.

[0105] In further alternative embodiments, the attachment tube is a vessel for storage, visual analysis, further enrichment, purification or other suitable downstream processing.

[0106] FIG. 17 shows microfluidic cartridge 200 configured for connection to extraction tube 240 (FIGS. 20, 23). In a preferred embodiment, extraction tube 240 is configured to provide a sample to be amplified via a polymerase chain reaction (PCR) within microfluidic cartridge 200 and analyzed via subsequent detection and analysis processes such as a detection laser and fluorometer. Microfluidic cartridge 200 is configured with cartridge opening 212 for insertion of extraction tube 240 by a user or insertion during a manufacturing assembly phase to be provided to a user pre-assembled. FIG. 20 shows assembled extraction tube 240 within cartridge opening 212. Cartridge opening 212 for extraction lies vertically within the same plane as microfluidic cartridge 200. The plane can be horizontal.

[0107] Microfluidic cartridge 200 is configured for insertion into control detection device 400, as shown in FIGS. 26 and 27.

[0108] Microfluidic cartridge 200 comprises a planar surface with two faces 203, 204 and outer front edge 205. A network of microfluidic channels 214 connects extraction tube 240 to a plurality of chambers 220 for amplification. The plurality of chambers 220 are stacked vertically within the planar surface of microfluidic cartridge 200. Optionally, chambers 220 can be stacked horizontally or diagonally. In a preferred embodiment, the amplification for which the cartridge is configured, is a polymerase chain reaction (PCR). In a preferred embodiment, there are eight separate chambers 220 used for PCR. In use, each of the plurality of chambers 220 comprise fillable area 224 which is pre-loaded with necessary PCR reagents. In a preferred embodiment, the pre-loaded amplification reagents may include a Mastermix, enzymes, primers and probes including nucleotides for the amplification of nucleic acid from a sample.

[0109] Cartridge opening 212 is configured within microfluidic cartridge 200 to allow for insertion of extraction tube 240 which can contain pre-filled reagents to prepare a sample to be analyzed. Connection interface 213 situated at the lower end of opening 212, provides a connection to extraction tube 240. In a preferred embodiment, connection interface 213 is a barb fitting. Alternatively, connection interface 213 can be any adequate connection to provide a standardized level of secure join of extraction tube 240 including but not limited to a Luer taper fitting. Cartridge opening 212 is shaped to fit the entirety of tube 240. In a preferred embodiment, once the extraction tube 240 is inserted into cartridge opening 212 of cartridge 200, it is irremovably fixed in place. FIG. 20 shows the assembled configuration with extraction tube 240 inserted into microfluidic cartridge 200. Collection cup 112 can be attached to extraction tube 240 to be situated above the upper edge of microfluidic cartridge 200, within cartridge opening 212.

[0110] Area 217 of microfluidic cartridge 200 comprises a flat surface area where microfluidic cartridge 200 can be held or gripped by a user as shown in FIG. 23 for the required use including insertion into detection device 400 such as that shown in FIGS. 26 and 27. In a preferred embodiment, area 217 is configured for adequate grip by a user and may comprise a rough surface, ridges or another suitable surface.

[0111] The microfluidic channels 214 branch out into a tree structure each leading from connection interface 213 of extraction tube 240 to one of the plurality of chambers 220. Inlet valve 216 sits within microfluidic cartridge 200 close to the interface connection 213 between extraction tube 240 and microfluidic channel 214 for carrying the sample to the chambers 220. In use, inlet valve 216 prevents any back flow of the sample back into extraction tube 240. Inlet valve 216 can be ring-shaped and specifically, can be identical to subsequently mentioned ring-shaped valve 236. For the sake of clarity, “inlet valve” will be hereinafter be used to refer inlet valve 216 and “ring-shaped valve” to refer to ring-shaped valve 236.

[0112] Microfluidic channels 214 separately lead away from each chamber 220 to hydrophobic junction valve 230 within microfluidic cartridge 200. In a preferred embodiment, hydrophobic junction valve 230 comprises junction 231 covered by a hydrophobic membrane 232. FIG. 19 shows a preferred embodiment of the structural arrangement of hydrophobic junction valve 230. In this arrangement, a plurality of individual units 234 within junction 231 are arranged, each unit 234 with further connection to ring-shaped valve 236. Ring-shaped valve 236 is not covered by hydrophobic membrane 232.

[0113] In a preferred embodiment, junction 231 comprises eight units 234 laid out in a rectangular two by four configuration. Each chamber 220 is fluidically connected to an individual unit 234 of junction 231.

[0114] In use, liquid will be pushed into channels 214 of the detection cartridge through connection interface 213 and through inlet valve 216. Inlet valve 216 is initially in the open position. Liquid will fill each of chambers 220 and will flow to fill individual valve units 234 within junction valve 231.

[0115] Hydrophobic membrane 232 lies above valve units 234. Liquid is unable to pass through hydrophobic membrane 232. Air may pass through the hydrophobic membrane where it will reach the ring-shaped valve 236 where it may escape to the atmosphere. When all eight individual valve units 234 are filled, ring-shaped valve 236 is closed.

[0116] FIG. 19 shows junction 231 of hydrophobic junction valve 230 in more detail. The closure of both the inlet-valve 216 and ring-shaped valve 236 can be controlled by device control system 400 which is exemplified in FIGS. 25 and 26.

[0117] In use, hydrophobic junction valve 230 is initially in a position open to the atmosphere. The control device 400 will action the closure of hydrophobic junction valve 230 once liquid has filled all eight units 234. When both the hydrophobic junction valve 230 and inlet valve 216 are closed, a sealed environment is created which can be a pressurized liquid environment, so the valves act to limit the amount of vapor that can build up within the sealed environment. The entire microfluidic cartridge 200 is coated in an adhesive film which seals the microfluidic channels 214.

[0118] FIG. 18 shows an image of a single chamber 220 with fillable unit 224 where the sample will flow to and where PCR amplification occurs with the pre-filled reagents. The filled chamber windows 224 are shown in use in FIG. 22 where they can be seen partially and fully filled during a period of use. Chambers windows 224 are transparent to detect results.

[0119] Microfluidic cartridge 200 can comprise central aperture portion 250 configured to act as an area for mixing where necessary with additional reagents. In some embodiments the nucleic acid sample and PCR reagents can be prevented from mixing until reaching central mixing portion 250. In various embodiments, an additional device can be inserted within central portion 250 to facilitate mixing via an appropriate mechanism including but not limited to vibration or magnetic “on-chip” mixing.

[0120] In use, microfluidic cartridge 200 is inserted within a detection control device 400. Control device 400 can include heaters for thermocycling which can sit on both sides of microfluidic cartridge 200 to provide the necessary heating and cooling for PCR or alternative processing. A fluorometer within control device 400 can be used to detect fluorescence within chambers 220. In use, fluorescence detection can be done via the outer front edge 205 of microfluidic cartridge 200 or via the planar faces of the chambers 220.

[0121] Detection control device 400 can be operated by a user who is inexperienced in the amplification and detection processes involved, as the process is automated by the device control software programming. Suitable software coding can be used to program the detection device and automated hardware, including control of the valve closure and opening, thermocycling and sample preparation within the extraction tube. The detection control device 400 may comprise a suitable output interface and can be connected to central database via a wireless connection.

[0122] In a preferred embodiment, microfluidic cartridge 220 is a single-use cartridge designed to be removed from detection device 400 and disposed of after analysis in the device. Any suitable material is possible for the construction including plastic, polymeric material and glass. Disposal after use must be done in a biohazardous waste.

[0123] In use, the sample collection, nucleic acid extraction and amplification is completed within the device with no interference. The nucleic acid extracted is suitable for direct amplification.

[0124] In an alternated embodiment, the layout of hydrophobic junction valve 230 can be arranged as in FIG. 24 which shows an alternative layout of hydrophobic junction valve 230a.

[0125] In a preferred embodiment, a method of using the microfluidic device of the present invention comprises the following steps: [0126] i. User/s deposits saliva sample in collection cup 12 or cup 112 which is connected to extraction tube 240. [0127] ii. User connects cap 22 or cap 122 to cup 12 of cup 112 causing sealed cup and blister 24 or blister 224 to burst and release capture particles 28 such as magnetic capture beads with a means of capturing target compound virus. [0128] iii. User initiates mixing of cup contents, at this stage, pelleted substance 43, if present, can dissolve into the sample. [0129] iv. User inserts cup 12 of cup 112 and connected extraction tube 240 into cartridge 210 into aperture 212, forming the cartridge assembly 111, the cartridge assembly 111 is then inserted into housing 401 of detection control device 400. [0130] v. Once the cartridge assembly 111 is inserted into housing 401 of detection control device 400, the subsequent processes are all automated, controlled by the detection device. [0131] vi. The magnetic capture beads, having captured virus from a sample are attracted by the magnet within the cup and moved through by the magnet to extraction tube 240, containing extraction reagent. This magnet movement is achieved by action of plunger 38 or plunger 138 within cup 12. A piston within control device 400 may action the plunger controlled by timing software withing the device 400. [0132] vii. Extraction tube 240 is heated sufficiently to activate the extraction reagent which may be a thermostable enzyme, pre-filled within extraction tube 240. [0133] viii. Heating causes extraction tube 240 which is at least partially formed from a heat-deformable material, to contract. The results of this action causes a pinch-valve within the cartridge to open, allowing the liquid through into cartridge microfluidic channels 214. [0134] ix. A polymer filter sits above the interface within microfluidic cartridge 200. The liquid sample travels through this filter. [0135] x. The heat-deformable material of the extraction tube continues to contract and effectively acts as a pump, the pressure pushing air behind the sample. The sample is forced through cartridge microfluidic channels 214 and up to the vertically stacked chambers 220. [0136] xi. The sample is pushed through the polymer filter, and inlet valve 216 which sits close to the tube and cartridge interface 213. The inlet valve prevents sample flowing back through to extraction tube 240. [0137] xii. The contraction from the deforming of extraction tube 240 and reduced internal volume, retains residual pressure which prevents any sample from falling back into extraction tube 240 due to gravity. [0138] xiii. Cartridge channels 214 in microfluidic cartridge 200 branch off, leading to separate amplification chambers 220 stacked vertically. [0139] xiv. Chambers 220 which can be used for PCR, fill in order depending on which cartridge channel 214 has the least resistance to fluid flow. [0140] xv. The liquid continues to flow from PCR hydrophobic junction valve 230 within the cartridge. [0141] xvi. Hydrophobic junction valve 230 provides hydrophobic membrane 232 which does not allow any liquid though, only air. [0142] xvii. When liquid has filled PCR chambers 220 and units 234 of the hydrophobic junction valve, closure of ring-shaped valves 230 and inlet valve 216 occurs. Valve closure is automated by the device and can be automated based on detection of liquid or time-automated. [0143] xviii. Chambers 220 within the cartridge will be heated and cooled by the thermocycling and heaters within the detection control device 400 and this will cause the amount of liquid in each chamber 220 to expand and contract. [0144] xix. A hydrophobic sheet can sit above hydrophobic junction valve 230 which acts to prevent biohazardous vapor produced during the heating stage to escape microfluidic cartridge 200. [0145] xx. At the point of valve closure, cartridge channels 214 are entirely full of liquid and thermocycling can begin. [0146] xxi. The heating and cooling source of chambers 220 can be situated on either side of microfluidic cartridge 200 and the detection viewed by user or device via the edge of the cartridge face. [0147] xxii. In the preferred embodiment, a single detector will scan all chambers, alongside the heating and cooling. The detector can be a fluorometer. [0148] xxiii. The device algorithm and software within control device 400 provides the result based on the detection and analysis. [0149] xxiv. The device may indicate a positive or negative presence of target biological compounds, pathogens or virus such as SARS-CoV-2 associated with disease COVID-19 and Influenza Types A and B [0150] xxv. The result may be displayed on a device user interface or may be transmitted via a data receiver. [0151] xxvi. After detection is complete, microfluidic cartridge 200 is configured to be removed from detection device and disposed of in biohazardous waste.

[0152] It will be appreciated that other suitable amplification techniques can be used within chambers 220 with alternative thermocycling provided by the detection device 400.

[0153] In one embodiment, cup 12 or cup 112 is pre-inserted into microfluidic cartridge 200, prior to sample collection by the user. In this embodiment the user will be provided the saliva cup pre-inserted into microfluidic cartridge 200.

[0154] In an alternative embodiment, valve 230a is arranged in a layout as shown in FIG. 24. In this embodiment, a hydrophobic membrane is also situated above at least a portion of valve 230a, which stops the fluidic flow. The arrangement in 230a creates a region of high-resistance, stopping the liquid, until increased pressure from all eight channels being filled forces the liquid against the hydrophobic membrane. In this embodiment, both an inlet valve and valve 230a will be closed at the same time to begin PCR thermocycling.

[0155] Microfluidic cartridge 200 can be produced in any suitable materials, the material may be partially or fully transparent. In alternative embodiments chambers 220 can be stacked diagonally or horizontally. The entire microfluidic cartridge 200 can be used within a horizontal orientation instead of vertical or at any suitable degree or angle, meaning the stacked chambers 220 and extraction tube interface will be in the horizontal plane or resulting plane associated with the degree or angle of use. In this embodiment, the device will function in the same way as previously described.

[0156] In various alternative embodiments, the sample to be collected and analyzed may be saliva, blood, urine, tissue, plant tissue or any biological matter from one or more users or sources. If the sample is from multiple sources, for example the saliva from multiple individuals can be pooled together within the cup and processed and analyzed simultaneously, the device will work in the same way, with a positive result indicating that any individual from the sample pool may have the positive result and each individual from the pool being re-tested on an individual basis for identification purposes.

[0157] In various other alternative embodiments, the device is configured to prevent biohazardous waste and vapor from exiting the device.

[0158] In a further alternative embodiment, opening 212 of the cartridge may extend to surround the entirety of collection cup 12 or cup 112.

[0159] The disclosed apparatus and systems may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features

[0160] It is to be understood that the above described embodiments are illustrative of only a few of the many possible specific embodiments, which can represent applications of the principles of the invention. Numerous and varied other arrangements can be readily devised in accordance with these principles by those skilled in the art without departing from the spirit and scope of the invention.