Extraction vessel and method with controlled dispensing

Abstract

An extraction vessel includes a tubular member having an opening and a lid to be secured over the opening. The lid includes a porous member that includes a thermally responsive material therein or thereon. When the lid is secured over the opening, liquid must pass through the porous member to exit the tubular member. In use, an operator forms a liquid mixture including a lysis buffer and a biological sample within the tubular member and then secures the lid over the opening. The liquid mixture is heated to a reaction temperature sufficient to lyse cells in the biological sample. When the temperature of the liquid mixture reaches or approaches the reaction temperature, the thermally responsive material prevents dispensing of liquid from the tubular member. After the liquid mixture cools to a predetermined dispensing temperature, the thermally responsive material allows the liquid mixture including a target compound to pass through the porous member.

Description

Technical Field

[0001] The present invention relates to an extraction container having a thermally responsive material that prevents the dispensing of liquid above a predetermined temperature.

[0002] Related applications

[0003] This application claims priority to U.S. Application No. 63 / 618,734 (“'734 Application”), filed January 8, 2024, entitled “Extraction Container with Controlled Allocation”, and relates to U.S. Application No. 17 / 992,774 (“'774 Application”), filed November 22, 2022, entitled “Extraction Container”. Each of the '734 Application and the '774 Application is incorporated herein by reference in its entirety. Background Technology

[0004] Extraction containers are used to combine biological samples with reagents to prepare for analysis of one or more target compounds in the biological sample. For example, the reagent may be a lysis buffer configured to lyse the cells of the biological sample when the combined biological sample / lysis buffer is heated to a predetermined reaction temperature. Lysing the cells releases cellular components, such as antigens or nucleotides, making the cellular components readily available for analysis. Typically, after combining the biological sample with the liquid, such as after lysis, at least a portion of the liquid and the released cellular components are dispensed onto test strips configured for analysis of one or more target compounds.

[0005] In some instances, an operator may inadvertently attempt to dispense the liquid / component while the liquid composition is still at or near a high reaction temperature. If dispensed prematurely, cells may not be fully lysed. If the biological sample contains pathogenic cells, incompletely lysed cells can pose a danger to the operator or others. Furthermore, incompletely lysed cells can lead to false-negative analyses of the target or result in quantifications that are smaller than the true amount of the target.

[0006] It would be advantageous to provide an extraction container that prevents dispensing of its contents when they are at a high temperature, but allows dispensing after the contents have cooled to a lower temperature. Summary of the Invention

[0007] In implementations, porous components, such as filters, include or are formed of a thermally responsive material. The thermally responsive material within or forming the porous component is configured to: block the passage of liquids and other materials through the porous component when the temperature of the liquids and other materials exceeds a predetermined dispensing temperature, and allow the passage of liquids and other materials when the temperature reaches or falls below the dispensing temperature.

[0008] In some embodiments, the extraction container includes a porous member, such as a filter, having a thermally responsive material or formed of a thermally responsive material. For example, the porous member may be included in a cap or opening of the extraction container. The extraction container includes a tubular member configured to contain one or more liquids, one or more biological samples, and / or a liquid mixture containing one or more liquids and one or more biological samples. The liquid may include, for example, a buffer, such as an extraction buffer for preparing a biological sample to analyze one or more targets therein. As used herein, targets, target compounds, and target materials are used interchangeably. Exemplary extraction buffers include lysis buffers configured to release cellular components within the biological sample by lysing cells. Biological samples may include, for example, sputum, nasopharyngeal swab samples, anterior nasal nostril samples, middle turbinate samples, pharyngeal swab samples, vaginal samples, blood or blood-based samples, fecal samples, and / or tissue samples. Such samples may be obtained from, for example, humans or animals such as non-human mammals. Other sources of biological samples include environmental samples and tissues from agricultural products such as plants. Alternative samples include mineral samples, ores, and water samples.

[0009] Embodiments using such extraction containers (as described herein) include methods for heating liquids, biological samples, and / or liquid mixtures to be dispensed. The method may include: forming a liquid mixture (e.g., containing liquids such as reagents, buffers, etc. (as described herein), and a biological sample) within a tubular member of the extraction container, and securing a cap or porous member over an opening in the tubular member, wherein (i) the cap or opening of the tubular member includes a porous member disposed therein, such that when the opening is secured with the cap or porous member, liquid must pass through the porous member to exit the tubular member, and (ii) the porous member comprises a thermally responsive material (as described herein). When the cap or porous member is secured over the opening, the liquid mixture disposed within the tubular member is heated to a predetermined reaction temperature, which is above the dispensing temperature. When the liquid mixture is above the dispensing temperature, for example at the reaction temperature, the thermally responsive material blocks or at least partially blocks the porous member, preventing or substantially preventing the liquid mixture or its components from passing through the porous member. Subsequently, the liquid mixture is cooled from the reaction temperature to a temperature at or below the dispensing temperature, whereby the thermally responsive material no longer blocks the porous member. After cooling the liquid mixture to a temperature at or below the dispensing temperature, the method may include dispensing at least a portion of the liquid mixture and / or target material therein from the tubular member through a porous member with a fixed cap or opening.

[0010] The dispensing step may include: applying at least a portion of the dispensed liquid mixture and / or its components to the application port (application area) of the test strip, and measuring the liquid mixture to detect one or more targets. Dispensing may include inverting a tubular member with a porous member or cap fixed above the opening of the tubular member.

[0011] In any of the foregoing method embodiments, the liquid mixture comprises a liquid buffer and a biological sample. The liquid buffer may include a lysis buffer, the biological sample may include one or more cells, and the reaction temperature is sufficient to lyse one or more cells in the liquid mixture during the heating step. For example, the one or more cells may include bacteria, such as Mycobacterium tuberculosis.

[0012] In any of the foregoing embodiments of the method, the reaction temperature is between about 70°C and about 100°C. In the embodiments of the method, the dispensing temperature is about 70°C or lower, about 60°C or lower, about 50°C or lower, about 40°C or lower, or about 30°C or lower.

[0013] In any of the foregoing embodiments of the porous component, extraction container, or method, the thermally responsive material comprises a hydrogel. For example, the hydrogel may comprise a polymer at least partially formed of acrylamide or a combination of acrylamide and acrylic acid. An exemplary thermally responsive hydrogel includes poly(isopropylacrylamide).

[0014] In any of the foregoing method embodiments, the tubular member may have a total internal volume between about 2 mL and about 50 mL, and the liquid mixture disposed in the tubular member has a total volume between about 1 mL and 40 mL.

[0015] In any of the foregoing embodiments of the porous component, extraction container, or method, after the liquid mixture is cooled to the dispensing temperature, the porous component has a porosity configured to allow the liquid of the liquid mixture and the target compound released from the lysed cells to pass through, but to block, for example, fragments of the lysed cells, such as the walls of the lysed cells, from passing through.

[0016] In any of the foregoing extraction container or method embodiments, the tubular member may, prior to its use, include a liquid disposed therein and a gas / liquid impermeable membrane sealing its opening. The method of using the extraction container includes removing the membrane to expose the opening and the liquid therein. Attached Figure Description

[0017] Figure 1 shows an extraction container with a tubular member and a cap according to an embodiment of the present invention.

[0018] Figure 2 shows an exploded view of the lid and porous component of the extraction container in Figure 1 according to an embodiment of the present invention. Detailed Implementation

[0019] Referring to Figure 1, the extraction container 10 includes a tubular member 12 and a cap 14. The extraction container 10 can be used to heat a biological sample, along with any liquid (e.g., buffer) or other reagent, to a reaction temperature sufficient to achieve a desired effect, such as preparing a sample for analysis of one or more target compounds. For example, the reaction temperature may be sufficient to promote the dissolution / mixing of reagents or other materials, cause cell rupture, become permeable or lysedated, denature proteins, improve the availability of the target compound, or any combination thereof. After heating, the liquid and the target compound to be analyzed can be dispensed through the cap 14. A temperature-responsive material disposed within the cap 14 prevents such dispensing until the liquid / target compound has cooled to a predetermined dispensing temperature (e.g., cooled down from the reaction temperature).

[0020] The tubular member 12 has an opening 16 configured to allow the addition of liquids, reagents, and / or biological samples. When provided to a user or consumer, the tubular member 12 may include a quantity of liquid 18, such as a buffer solution, like a lysis buffer. To prevent evaporation or leakage of the liquid 18, the opening 16 of the tubular member 12 is sealed by a removable gas / liquid impermeable membrane 20. The membrane 20 may be composed of, for example, a metal and / or polymer membrane. During use, the operator removes the membrane 20, thereby exposing the opening 16 and the liquid 18 therein.

[0021] The cap 14 includes a cover 15, an end cap 27, and a porous member 24. The cover 15 is detachably secured over the opening 16 of the tubular member 12, for example by a threaded or snap-fit ​​engagement (not shown). The porous member 24 is secured within the cavity 25 of the cover 15, for example by a press fit. A channel 22 (see FIG. 2) extends through the cover 15. When the cover 15 of the cap 14 is secured over the opening 16, liquid or other material (e.g., target compound) within the tubular member 12 must pass through the porous member 24 and the channel 22 to exit the extraction container 10. The end cap 27 may be secured over the channel 22 to prevent liquid / gas from leaving the extraction container 10, for example, during heating steps or storage. The end cap 27 may also be removable and / or removable to allow dispensing of liquid / gas.

[0022] In some implementations, cover 15 may not be necessary, and porous member 24 may be simply configured to be directly fixed (e.g., by press fit) in opening 16 of tubular member 12 to form cover.

[0023] The porous component 24 includes pores 26 configured to allow the passage of liquids and / or target compounds, such as cellular components released from cells via lysis. Exemplary target compounds include biological materials and / or molecules of interest. For example, target materials may include proteins, enzymes, polynucleotides, and / or antigens, such as proteins or peptides. Typically, the pores 26 of the porous component 24 block the passage of larger materials, such as debris from dirty samples or remnants of cell walls and internal organelles.

[0024] The porous member 24 includes a thermally responsive material 28 disposed on its outer surface and / or within its pores 26. The thermally responsive material 28 is configured to block or at least partially block the passage of the liquid / target material through the porous member 24 when the liquid / target material is at a temperature above a predetermined dispensing temperature (as described herein). For example, the thermally responsive material may be provided as a membrane or other structure disposed around the porous member and at least partially around the pores, such that when at a temperature above the predetermined dispensing temperature, the thermally responsive material can expand, thereby blocking or at least partially blocking the pores of the porous member. When the liquid / target material is at or below the predetermined dispensing temperature, the thermally responsive material 28 allows the liquid / target material to pass through the porous member 24. For example, once the thermally responsive material is no longer at a temperature above the predetermined dispensing temperature, the thermally responsive material may contract or at least partially contract from its expanded state, thereby no longer blocking or partially blocking the pores of the porous member.

[0025] The reaction temperature can be sufficient to promote the dissolution / mixing of reagents or other materials, cause cell rupture, make cells permeable or lysed, denature proteins, improve the availability of the target compound, or any combination thereof. As described herein, the reaction temperature can be from about 70°C to about 100°C. The dispensing temperature can be a temperature suitable for dispensing liquid / target material from extraction container 10. At such dispensing temperatures, for example, complete cell lysis can be expected, resulting in cell destruction (and therefore, an inability to be pathogenic) and complete release of the target material. Because the dispensing temperature is below the reaction temperature, the liquid is also less likely to cause burns or damage to the test device, such as the test strip, during dispensing. Therefore, the thermally responsive material used in conjunction with the porous component also prevents the heated reaction mixture from accidentally escaping through the cap or opening during lysis. As described herein, the dispensing temperature is about 70°C or lower, about 60°C or lower, about 50°C or lower, about 40°C or lower, or about 30°C or lower.

[0026] The thermally responsive material 28 may be, for example, a hydrogel. For instance, the hydrogel may comprise a polymer at least partially formed of acrylamide or a combination of acrylamide and acrylic acid. An exemplary thermally responsive hydrogel includes poly(isopropylacrylamide). An exemplary thermally responsive hydrogel is formed from a mixture of one or more monomers such as N-isopropylacrylamide and a solvent such as water. The monomers and solvent are combined at a weight ratio of about 0.08 monomers to about 0.9 water.

[0027] As an alternative to or in combination with a thermally responsive material disposed on or within the pores of the porous member 24, the porous member, such as a filter, may itself be formed of a thermally responsive material that blocks or at least partially blocks liquid from passing through the porous member when the porous member is at a temperature above a predetermined distribution temperature. For example, the porous member may be formed of a material that expands or swells above the distribution temperature, thereby blocking or at least partially blocking pores or other channels extending through the porous member. When the porous member cools to or below the distribution temperature, the pores or other channels open, allowing liquid to pass through.

[0028] Extraction container 10 and its components, including porous members 24, are typically formed of materials suitable for handling and containing biological samples, such as polymers, for example, polypropylene. Other suitable materials include glass, polyethersulfone, nylon, and polytetrafluoroethylene. Porous members, such as filters, containing thermally responsive materials and / or formed of thermally responsive materials, as disclosed herein, can be used with any extraction container disclosed, for example, in application '774.

[0029] The porous component of extraction container 10 has been described as performing a filtration function, for example, separating cell wall components and organelles from a filtrate containing a target compound of interest. In some embodiments, the primary, and perhaps only, function of the porous component is to prevent dispensing when the liquid contained within the extraction container is above the dispensing temperature. In such methods, for example, a heating step can achieve the dissolution / mixing of the sample and reagent combination, wherein the resulting liquid mixture does not contain particles that need to be separated.

[0030] Example

[0031] Example 1: Preparation of the extraction container

[0032] The extraction container is prepared as follows, comprising a lid with a porous component having a thermally responsive material:

[0033] Combine 1.84 g of deionized water with 0.16 g of poly(N-isopropylacrylamide) in a 2-dram glass vial. The polymer-to-water ratio is 0.08 g polymer / 0.92 g water. Cap the vial and rotate it on a vortex mixer for approximately one hour until the polymer is completely dissolved in the water. After dissolution, add six filters to the liquid mixture and recap the vial. Invert the vial every 15 minutes for 2 hours until the liquid mixture permeates through the filters. After 2 hours, remove the filters from the vial and place them on a clean surface. Insert each filter into the cap of the extraction vial by pressing the cap opening onto the filter until the filter is secured inside the cap. Allow each cap with its secured filter to dry for 16 hours.

[0034] Example 2: Verification of Controlled Assignment (I)

[0035] Add approximately 1 ml of water to each extraction container prepared in Example 1. Secure the cap to the opening of each container and then heat the container to a temperature of 95°C for approximately 5 minutes. As the temperature cools, invert each extraction container and squeeze it with the operator's fingers as if to dispense it. The extraction container does not allow liquid to pass through the porous structure (and therefore does not allow water to be dispensed) until the temperature cools to approximately 30°C. After cooling, water can be dispensed from the extraction container through the filter of the secured cap.

[0036] Example 3: Verification of Controlled Assignment (II)

[0037] The experiment of Example 2 was repeated, except that the liquid in each extraction container was subjected to a heating cycle typically used to lyse cells such as Mycobacterium tuberculosis. The extraction container was not allowed to pass liquid through the porous structure (and therefore water was not allowed to be dispensed) until the temperature cooled to approximately 30°C. After cooling, water could be dispensed from the extraction container through a filter with a fixed cap.

[0038] equivalent

[0039] The invention may be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be regarded in all respects as illustrative and not as limiting of the invention described herein. Consequently, the scope of the invention is indicated by the appended claims, not by the foregoing description, and all variations falling within the equivalent meaning and scope of the claims are intended to be included within the scope of the invention.

Claims

1. A method for heating a liquid mixture to be dispensed, the method comprising: A liquid mixture is formed within the tubular component; The cap is fixed over the opening of the tubular member, wherein (i) the cap includes a porous member disposed therein, such that when the opening is fixed by the cap, the liquid mixture must pass through the porous member to exit the tubular member, and (ii) the porous member comprises or is formed of a thermally responsive material. When the cap is fixed over the opening, the liquid mixture disposed within the tubular member is heated to a predetermined reaction temperature, which is higher than a predetermined dispensing temperature; When the liquid mixture is above the predetermined dispensing temperature, the thermally responsive material blocks the porous member, preventing the liquid mixture or its components from passing through the porous member; and The liquid mixture is cooled from the predetermined reaction temperature to the predetermined dispensing temperature or lower, thereby the thermally responsive material no longer blocks the porous component.

2. The method according to claim 1, further comprising: After the liquid mixture is cooled to or below the predetermined dispensing temperature, at least some of the liquid mixture and / or the target material therein is dispensed from the tubular member through the porous member with a fixed cap.

3. The method of claim 2, wherein the dispensing comprises applying at least some of the dispensed liquid mixture and / or its components to the application port of the test strip.

4. The method of claim 2 or 3, wherein the dispensing comprises inverting the tubular member together with the fixed cap.

5. The method according to any one of the preceding claims, wherein the liquid mixture comprises a liquid buffer and a biological sample.

6. The method of claim 5, wherein the liquid buffer comprises a lysis buffer, the biological sample comprises one or more cells, and the predetermined reaction temperature is sufficient to lyse the one or more cells in the liquid mixture during the heating step.

7. The method according to any one of the preceding claims, wherein the predetermined reaction temperature is a temperature between about 70°C and about 100°C.

8. The method of claim 7, wherein the predetermined distribution temperature is about 70°C or lower, about 60°C or lower, about 50°C or lower, about 40°C or lower, or about 30°C or lower.

9. The method according to any one of the preceding claims, wherein the thermally responsive material comprises a hydrogel.

10. The method of claim 9, wherein the hydrogel comprises at least a portion of a hydrogel formed of poly(isopropylacrylamide).

11. The method according to any one of the preceding claims, wherein the tubular member has a total internal volume between about 2 mL and 50 mL, and the liquid mixture disposed in the tubular member has a total volume between about 1 mL and 40 mL.

12. The method according to any one of the preceding claims, wherein after cooling the liquid mixture to the predetermined dispensing temperature, the porous member has porosity configured to allow the liquid in the liquid mixture and the target compound released from the lysed cells to pass through, but blocks the walls of the lysed cells from passing through.

13. An extraction container, the container comprising: A tubular member comprising: a lysis buffer disposed therein; and an opening, wherein the lysis buffer is configured to lyse cells in a biological sample, the biological sample having been combined with the lysis buffer to form a liquid mixture and heated to a predetermined reaction temperature above a predetermined dispensing temperature; A cover, configured to be fixed around the opening of the tubular member, wherein the cover includes a porous member such that when the cover is fixed to the tubular member, liquid in the liquid mixture or its components disposed within the tubular member exits the tubular member through the porous member, and further wherein the porous member comprises or is formed of a thermally responsive material configured to: (i) block the liquid from passing through the porous member when the liquid mixture and / or liquid in its components are above the predetermined dispensing temperature, and (ii) allow the liquid to pass through the porous member when the liquid mixture and / or liquid in its components have cooled to the predetermined dispensing temperature.

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