Extraction container and method with controlled dispensing

Abstract

An extraction container includes a tubular member having an opening and a cap to be secured over the opening. The cap includes a porous member including a heat-responsive material therein or thereon. When the cap 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 cap over the opening. The liquid mixture is heated to a reaction temperature sufficient to lyse cells of the biological sample. When the temperature of the liquid mixture is at or near the reaction temperature, the heat-responsive material prevents dispensing the liquid from the tubular member. After the liquid mixture cools to a predetermined dispensing temperature, the heat-responsive material permits the liquid mixture including target compounds to pass through the porous member.

Description

[0001] EXTRACTION CONTAINER AND METHOD WITH CONTROLLED DISPENSING

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an extraction container having heat-responsive material that prevents dispensing liquids above a predetermined temperature.

[0004] RELATED APPLICATIONS

[0005] This application claims priority to U.S. Application No. 63 / 618,734 filed 8 January 2024, titled "Extraction Container With Controlled Dispensing" (the "'734 Application") and is related to U.S. Application No. 17 / 992,774, titled "Extraction Container", filed 22 November 2022 (the "'774 Application"). Each of the '734 and '774 Applications is incorporated by reference in its entirety herein.

[0006] BACKGROUND OF THE INVENTION

[0007] Extraction containers are used to combine a biological sample with a reagent in preparation for analysis of one or more target compounds within the biological sample. For example, the reagent may be a lysis buffer configured to lyse cells of the biological sample when the combined biological sample / lysis buffer is heated to a predetermined reaction temperature. Lysing the cells releases constituents of the cells, e.g., antigens or nucleotides, making them accessible to analysis. Typically, after combining the biological sample and liquid, e.g., after lysis, at least some of the liquid and released cell constituents are dispensed onto a test strip configured to analyze the one or more target compounds.

[0008] In some instances, an operator may inadvertently attempt to dispense the liquid / constituents when the liquid combination remains at or close to the elevated reaction temperature. If prematurely dispensed, the cells may be incompletely lysed. If the biological sample contains pathogenic cells, incompletely lysed cells may pose a danger to the operator or others. In addition, incompletely lysed cells may result in a false negative analysis of the target(s) or a quantified amount less than the true amount of target(s).

[0009] It would be advantageous to provide an extraction container that prevented dispensing the contents thereof when the contents were at an elevated temperature but would permit dispensing after the contents had cooled to a lower temperature.

[0010] SUMMARY OF THE INVENTION

[0011] In embodiments, a porous member, e.g., a filter, includes or is formed of a heat responsive material. The heat responsive material within or forming the porous member is configured to obstruct the passage of liquid and other materials through the porous member when the temperature of such liquid or other materials exceeds a predetermined dispensing temperature and to permit such passage when the temperature is at or below the dispensing temperature.

[0012] In some embodiments, an extraction container includes the porous member, e.g., a filter, having or formed of the heat 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 more biological samples, and / or a liquid mixture including one or more liquids and one more biological samples. The liquid(s) may include, e.g., a buffer such as an extraction buffer for preparing a biological sample for analysis of one or more targets therein. As used herein, targets, target compounds, and target materials may be used interchangeably. Exemplary extraction buffers include lysing buffers configured to release constituents of cells within the biological sample by lysing the cells. The biological sample(s) may include, e.g., sputum, a nasopharyngeal swab sample, an anterior nares sample, a mid-turbinate sample, a throat swab sample, a vaginal sample, blood or a blood-based sample, a stool sample, and / or a tissue sample. Such samples may be obtained from, e.g., a human or an animal such as a non-human mammal. Other sources of biological samples include environmental samples and tissues from agricultural products such as plants. Alternative samples include mineral samples, ore, and water samples.

[0013] Embodiments using such extraction container (as described herein) include a method of heating a liquid(s), biological sample(s), and / or liquid mixture to be dispensed. The methods may include forming a liquid mixture (e.g., comprising a liquid, such as a reagent, buffer, etc. as described herein, and a biological sample) within the tubular member of the extraction container and securing the cap or porous member over an opening of the tubular member, wherein (i) the cap or opening of the tubular member includes the 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 includes the heat-responsive material (as described herein). While the cap or porous member is secured overthe opening, the liquid mixture disposed within the tubular member is heated to a predetermined reaction temperature, which is higher than the dispensing temperature. When the liquid mixture is above the dispensing temperature, e.g., at the reaction temperature, the heat-responsive material obstructs or at least partially obstructs the porous member so that the liquid mixture or components thereof cannot pass or substantially cannot pass therethrough. Subsequently, the liquid mixture is cooled from the reaction temperature to a temperature at or below the dispensing temperature whereby the heat-responsive material no longer obstructs the porous member. After cooling the liquid mixture to at or below the dispensing temperature, the method may include dispensing at least some of the liquid mixture and / or target materials therein from the tubular member through the porous member of the secured cap or opening of the tubular member.

[0014] The step of dispensing may include applying at least some of the dispensed liquid mixture and / or components thereof to an application port (application zone) of a test strip and subjecting the liquid mixture to an assay for detecting the one or more targets. The dispensing may include inverting the tubular member with the porous member or the cap secured over the opening of the tubular member.

[0015] In embodiments of any of the foregoing method embodiments, the liquid mixture includes a liquid buffer and a biological sample. The liquid buffer may include a lysing buffer, the biological sample may include one or more cells, and the reaction temperature is a temperature sufficient to lyse the one or more cells in the liquid mixture during the step of heating. For example, the one or more cells may include bacteria such as Mycobacterium tuberculosis.

[0016] In embodiments of any of the foregoing method embodiments, the reaction temperature is a temperature between about 70°C and about 100°C. In embodiments of the method, the dispensing temperature is about 70°C or less, about 60°C or less, about 50°C or less, about 40°C or less, or about 30°C or less.

[0017] In embodiments of any of the foregoing porous member, extraction container, or method embodiments, the heat-responsive material comprises a hydrogel. For example, the hydrogel may include a polymer formed at least in part of acrylamide or the combination of acrylamide and acrylic acid. An exemplary heat-responsive hydrogel includes poly(ispropylacrylamide).

[0018] In embodiments of any of the foregoing method embodiments, the tubular member may have a total internal volume of between about 2 mL and about 50 mL, and the liquid mixture disposed therein has a total volume of between about 1 mL and 40 mL.

[0019] In embodiments of any of the foregoing porous member, extraction container, or method embodiments, the porous member, after cooling the liquid mixture to the dispensing temperature, has a porosity configured to permit the passage of liquid of the liquid mixture and target compounds released from lysed cells but to obstruct the passage of e.g. fragments of the lysed cells, e.g., the walls of the lysed cells.

[0020] In embodiments of any of the foregoing extraction container or method embodiments, the tubular member may include, prior to use thereof, a liquid disposed therein and a gas / liquid- impermeable film that seals the opening thereof. Methods of using the extraction container include removing the film thereby exposing the opening and the liquid therein.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure (Fig.) 1 illustrates an extraction container having a tubular member and a cap, according to an embodiment of the invention.

[0023] Fig. 2 illustrates an exploded view of the cap and porous member of the extraction container of Figure 1, according to an embodiment of the invention.

[0024] DETAILED DESCRIPTION

[0025] Referring to Fig. 1, an extraction container 10 includes a tubular member 12 and a cap 14. Extraction container 10 may be used to heat a biological sample, along with any liquids (e.g., buffers) or other reagents, to a reaction temperature sufficient to achieve a desired effect, such as to prepare the sample for analysis of one or more target compounds therein. For example, the reaction temperature may be sufficient to enhance solubilization / mixing of reagents or other materials, to rupture, render permeable, or lyse cells, to denature proteins, to enhance availability of target compounds, or any combination thereof. After heating, the liquid and target compounds to be analyzed may be dispensed through the cap 14. A temperature responsive material disposed within the cap 14 prevents such dispensing until the liquid / target compounds have cooled to a predetermined dispensing temperature (e.g., cooled from the reaction temperature).

[0026] Tubular member 12 has an opening 16 configured to permit liquids, reagents, and / or biological samples to be added thereto. As provided to a user or consumer, tubular member 12 may include an amount of liquid 18, e.g., a buffer such as a lysing buffer. To prevent evaporation or leakage of liquid 18, opening 16 of tubular member 12 is sealed with a removable gas / liquid-impermeable film 20. Film 20 may be composed of, e.g., a film of metal and / or polymer. During use, an operator removes film 20 thereby exposing opening 16 and liquid 18 therein.

[0027] Cap 14 includes a cover 15, an endcap 27, and a porous member 24. Cover 15 is removably securable over opening 16 of tubular member 12, e.g., by threads or a snap fitting (not shown). Porous member 24 is secured within a cavity 25 of cover 15, e.g., by compression fit. A passage 22 (see Fig. 2) extends through cover 15. When cover 15 of cap 14 is secured over opening 16, liquid or other materials (e.g., target compounds) within tubular member 12 must pass through the porous member 24 and passage 22 to exit the extraction container 10. Endcap 27 can be secured over passage 22 to prevent liquids / gases from exiting extraction container 10, e.g., during a heating step or storage. The endcap 27 may also be removed and / or removable to permit dispensing of liquids / gases.

[0028] In some embodiments, the cover 15 may not be required and the porous member 24 may simply be configured to be secured (e.g. by press-fitting) directly into the opening 16 of the tubular member 12 to form a cap therefor.

[0029] Porous member 24 includes pores 26 configured to permit the passage of liquid and / or target compounds, such as the constituents of cells released from the cells by 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 polypeptides. Typically, pores 26 of porous member 24 obstruct the passage of larger materials such as debris from dirty samples or the remnants of cell walls and internal organelles.

[0030] Porous member 24 includes a heat-responsive material 28 disposed on an external surface of porous member 24 and / or within pores 26 thereof. Heat-responsive material 28 is configured to obstruct or at least partially obstruct the passage of liquid / target materials through porous member 24 when the liquid / target materials are at a temperature greater than a predetermined dispensing temperature (as described herein). For example, heat-responsive material may be provided as a film or other configuration disposed about the porous member and at least partially around the pores, such that when in the presence of temperature greater than the predetermined dispensing temperature, the heat-responsive material can expand so as to obstruct or at least partially obstruct the pores of the porous member. When the liquid / target materials are at or a below a predetermined dispensing temperature, heat responsive material 28 permits the passage of the liquid / target materials through porous member 24. For example, once the heat-responsive material is no longer in the presence of temperature greater than the predetermined dispensing temperature, the heat-responsive material may constrict or at least partially constrict from its expanded state so as to no longer obstruct or partially obstruct the pores of the porous member.

[0031] The reaction temperature may be a temperature sufficient to enhance solubilization / mixing of reagents or other materials, to rupture, render permeable, or lyse cells, to denature proteins, to enhance availability of target compounds, or any combination thereof. As described herein, the reaction temperature can be from about 70°C to about 100°C. The dispensing temperature may be a temperature at which it is appropriate to dispense liquid / target materials from extraction container 10. At such dispensing temperature, for example, it may be expected that cell lysis is complete so that the cells are destroyed (and therefore could not be pathogenic) and target materials fully released. Because the dispensing temperature is less than the reaction temperature, liquid is also less likely to cause burns or damage a test device, e.g., test strip, when dispensed. Accordingly, the heat-responsive material as applied with the porous member can also prevent accidental escape of heated reaction mixture through the cap or opening during lysis. As described herein, the dispensing temperature is about 70°C or less, about 60°C or less, about 50°C or less, about 40°C or less, or about 30°C or less.

[0032] Heat-responsive material 28 may be, e.g., a hydrogel. For example, the hydrogel may include a polymer formed at least in part of acrylamide or the combination of acrylamide and acrylic acid. An exemplary heat-responsive hydrogel includes poly(ispropylacrylamide). An exemplary heat- responsive hydrogel is formed from a mixture of one or more monomers such as N- isopropylacrylamide and a solvent such as water. The monomer and solvent are combined at a weight ratio of about 0.08 monomer to about 0.9 water.

[0033] As alternative to or in combination with a heat-responsive material disposed on or within the pores of porous member 24, a porous member, e.g., filter, may itself be formed of a heat- responsive material that obstructs or at least partially obstructs the passage of liquid therethrough when the porous member is at a temperature greater than a predetermined dispensing temperature. For example, a porous member may be formed of a material that expands or swells above the dispensing temperature thereby occluding or at least partially occluding pores or other passages extending therethrough. When the porous member cools to or below the dispensing temperature, the pores or other passages open permitting the passage of liquid.

[0034] Extraction container 10 and components thereof including porous member 24 are typically formed of a material suitable for handling and containment of biological samples such as a polymer, e.g., polypropylene. Other suitable materials include glass, polyethersulphone, nylon, polytetrafluoroethylene. Porous members, e.g., filters, incorporating and / or formed of heat- responsive materials as disclosed herein may be used with, e.g., any of the extraction containers disclosed in the '774 Application.

[0035] The porous member of extraction container 10 has been described as performing a filtration function, e.g., separation of cell wall components and organelles from a filtrate containing target compounds of interest. In some embodiments, the primary, e.g., the sole, function of the porous member is to prevent dispensation when the liquid contained within the extraction container is above the dispensing temperature. In such methods, for example, the heating step may effect solubilization / mixing of a combination of a sample and reagents in which there are no particulates to separate from the resulting liquid mixture.

[0036] EXAMPLES

[0037] Example 1: Preparation of Extraction Container

[0038] An extraction container including a cap with a porous member having a heat-responsive material was prepared as follows:

[0039] 1.84 g of deionized water and 0.16 g poly(N-isopropylacrylamide) were combined in a 2-dram glass vial. The ratio of polymer to water was 0.08 g polymer to 0.92 g water. The vial was capped and rotated on a rotator for approximately one hour until the polymer was completely dissolved in the water. Once dissolved, 6 filters were added to the liquid mixture and the vial recapped. The vial was inverted every 15 minutes over a 2 hour period until the liquid mixture had permeated the filters. After 2 hours, the filters were removed from the vial and placed on a clean surface. Each filter was inserted into the cap of an extraction vial by pressing the opening of a cap onto a filter until the filter was secured therein. The caps each with a filter secured therein, were allowed to dry for 16 hours.

[0040] Example 2: Demonstration of Controlled Dispensing (I)

[0041] Approximately 1 ml water was added to each extraction container prepared in Example 1. The cap was secured over the opening of each container, which was then heated to a temperature of 95 °C for a period of about 5 minutes. As the temperature cooled, each extraction container was inverted and the container squeezed with an operator's fingers as if to dispense therefrom. The extraction containers did not permit liquid to pass through the porous member (and therefore did not permit the water to be dispensed) until the temperature had cooled to a temperature of about 30 °C. After cooling, water could be dispensed from the extraction container through the filter of the secured cap.

[0042] Example 3: Demonstration of Controlled Dispensing (II)

[0043] The experiment of Example 2 was repeated except that, the liquid within each extraction container was subjected to a heating cycle typically used for lysing cells to be analyzed, e.g., cells of Mycobacterium tuberculosis. The extraction containers did not permit liquid to pass through the porous member (and therefore did not permit the water to be dispensed) until the temperature had cooled to a temperature of about 30 °C. After cooling, water could be dispensed from the extraction container through the filter of the secured cap.

[0044] EQUIVALENTS

[0045] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed, is:

1. A method of heating a liquid mixture to be dispensed, comprising: forming a liquid mixture within a tubular member; securing a cap over an opening of the tubular member, wherein (i) the cap comprises a porous member disposed therein such that, when the opening is secured with 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 heat-responsive material; while the cap is secured over the opening, heating the liquid mixture disposed within the tubular member to a predetermined reaction temperature that is greater than a predetermined dispensing temperature; when the liquid mixture is above the predetermined dispensing temperature, obstructing the porous member with the heat-responsive material so that the liquid mixture or components thereof cannot pass therethrough; and cooling the liquid mixture from the predetermined reaction temperature to the predetermined dispensing temperature or lower, whereby the heat-responsive material no longer obstructs the porous member.

2. The method of claim 1, further comprising, after cooling the liquid mixture to or below the predetermined dispensing temperature, dispensing at least some of the liquid mixture and / or target materials therein from the tubular member through the porous member of the secured cap.

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

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

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

7. The method of any of the foregoing 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 dispensing temperature is about 70 °C or less, about 60 °C or less, about 50 °C or less, about 40 °C or less, or about 30 °C or less.

9. The method of any of the foregoing claims, wherein the heat-responsive material comprises a hydrogel.

10. The method of claim 9, wherein the hydrogel comprises a hydrogel formed at least in part of poly(ispropylacrylamide).

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

12. The method of any of the foregoing claims, wherein the porous member, after cooling the liquid mixture to the predetermined dispensing temperature, has a porosity configured to permit the passage of liquid of the liquid mixture and target compounds released from lysed cells but to obstruct the passage of the walls of the lysed cells.

13. An extraction container, the container comprising: a tubular member comprising a lysing buffer disposed therein and an opening, wherein the lysing buffer is configured to lyse cells of a biological sample that has been combined with the lysing buffer to form a liquid mixture and heated to a predetermined reaction temperature, the predetermined reaction temperature being greater than a predetermined dispensing temperature; a cap configured to be secured about the opening of the tubular member, wherein the cap comprises a porous member, such that when the cap is secured to the tubular member, liquid of the liquid mixture or components thereof disposed within the tubular member pass through the porous member to exit the tubular member, and further wherein the porous member comprises or is formed of a heat-responsive material configured to (i) obstruct the passage of liquid of the liquid mixture and / or components of the liquid mixture through the porous member when the liquid is above the predetermined dispensing temperature, and (ii) permit the passage of liquid of the liquid mixture and / or components of the liquid mixture through the porous member when the liquid has cooled to the predetermined dispensing temperature.

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