Non-toxic preparation for collecting biological samples and device for capturing and eluting nucleic acids in the samples

Abstract

A formulation for collecting a biological sample of saliva or nasal secretion and capturing nucleic acids in the collected sample has a non-toxic chaotropic agent, ethanol, and a coloring agent and / or flavoring agent. The formulation can be received in the oral or nasal cavity, a sample is collected, and the non-toxic chaotropic agent(s) lyse the sample cells. The device has a sample port for receiving the sample-containing formulation and a solid membrane in fluid communication with the sample port. A first pump causes the sample-containing formulation to flow across the solid membrane and into a waste chamber. The ethanol binds nucleic acids in the lysed cells of the sample to the solid membrane. A second pump causes an eluent to flow from the eluent chamber across the membrane, eluting the captured nucleic acids from the membrane and flowing with the captured nucleic acids into an eluent reservoir.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED PATENT APPLICATIONS) This patent application claims priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 268,742, filed on March 1, 2022, and entitled “Non-Toxic Formulation For Collecting Biological Samples, And Device For Capturing And Eluting Nucleic Acids In The Samples,” which is hereby incorporated by reference in its entirety as part of this disclosure.

[0002] FIELD OF THEINVENTION The present invention relates to a device and method for isolating, concentrating, amplifying and detecting nucleic acids in a biological sample such as saliva or mucus, and more particularly to a composition comprising non-toxic sample preparation materials for collecting an oral or nasal sample, capturing nucleic acids therein, and for collecting and preparing the sample to capture nucleic acids in the biological sample and amplify the captured nucleic acids in a reaction chamber. [Background technology]

[0003] Saliva is an attractive sample type for clinical diagnostics because it is non-invasive and can be used to detect many diagnostic targets that are traditionally collected via nasal swabs, sputum, or blood samples. However, saliva poses several problems for use in microfluidic devices for molecular diagnostics (diagnostics based on RNA or DNA targets). First, saliva can vary significantly in viscosity, which can affect the fluid dynamics of the device. Second, it is difficult to standardize the volume of saliva collected and used because sample collection methods are imprecise and the amount of saliva an individual can produce and spit varies. In addition, saliva can foam, and thus bubbles can be introduced into the microfluidic channels, which can disrupt the fluid flow in the device.

[0004] To address some of these issues, saliva sample preparation (cell lysis) is typically performed outside of the device or microfluidic system, where the user adds saliva to a container outside of the system that contains sample preparation chemicals. This helps to standardize the viscosity of the sample prior to introduction into the device and helps address foaming issues, but does not address differences in the volume of saliva collected and can expose the user to potentially toxic sample preparation chemicals. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention, and / or its currently preferred embodiments, to overcome one or more of the above-mentioned shortcomings and / or disadvantages of the prior art. [Means for solving the problem]

[0006] According to a first aspect, the present invention provides a method and apparatus for detecting nucleic acids in a biological sample comprising: (i) a sample port or sample chamber for receiving therein a sample-containing mixture having a biological sample therein; (ii) a solid membrane in fluid communication with the sample port or sample chamber and configured to receive the sample-containing mixture therefrom, the solid membrane for passing the sample-containing mixture through the membrane and capturing nucleic acids in the biological sample on the membrane; and (iii) a first pump in fluid communication with the solid membrane and a waste chamber in fluid communication with the pump, the first pump causing the sample-containing mixture to flow through the solid membrane and capture nucleic acids in the biological sample on the membrane. (iv) an eluent chamber having an eluent therein; (v) an eluent reservoir in fluid communication with the solid membrane; and (vi) a second pump in fluid communication with the solid membrane and the eluent chamber, such that actuation of the second pump causes the eluent to flow from the eluent chamber across the solid membrane, eluting the captured nucleic acids from the solid membrane and flowing with the captured nucleic acids into the eluent reservoir.

[0007] In some embodiments of the invention, the first pump is a syringe including a barrel and a plunger housed within the barrel. The barrel defines a waste chamber therein. Movement of the plunger draws or pulls or pushes the sample-containing mixture across the solid membrane and into the waste chamber of the barrel.

[0008] In some embodiments of the invention, the second pump is movable between a non-actuated position and an actuated position. The eluent chamber includes a frangible or breakable wall that is breakable by movement of the second pump between the non-actuated position and the actuated position, and the eluent is pumped from the eluent chamber across the solid membrane and into the eluent reservoir. In some such embodiments, the second pump is a plunger. Movement of the plunger from the non-actuated position to the actuated position breaks the frangible or breakable wall of the eluent chamber and pushes or draws the eluent across the solid membrane and into the eluent reservoir. In other embodiments, the second pump is a syringe that includes a barrel and a plunger housed within the barrel. Movement of the plunger pushes or draws the eluent across the solid membrane and into the eluent reservoir.

[0009] Some embodiments of the invention further include a first valve fluidly connected between the sample port or sample chamber and the solid membrane and configured to allow the sample-containing mixture to flow in a direction from the sample port or sample chamber to the solid membrane, but to prevent liquid flow in the opposite direction.

[0010] Some embodiments of the invention further include a second valve fluidly connected between the solid membrane and the eluent reservoir and configured to allow fluid flow in a direction from the solid membrane into the eluent reservoir but prevent liquid flow in the opposite direction.

[0011] Some embodiments of the invention further include at least one reaction chamber and at least one conduit, e.g., a capillary conduit, fluidly connected between the eluent reservoir and the reaction chamber, the capillary conduit(s) being configured such that the eluent together with the captured nucleic acid can flow by capillary action through the capillary conduit(s) and into the reaction chamber(s).

[0012] According to another aspect, the invention provides a method for detecting nucleic acids in a biological sample comprising: (i) a first means for receiving therein a sample-containing mixture having a biological sample therein; (ii) a second means in fluid communication with the first means for receiving the sample-containing mixture therefrom, passing the sample-containing mixture through a second means, and capturing nucleic acids in the biological sample on the second means; (iii) a third means for receiving and retaining the sample-containing mixture after passing through the second means; (iv) a fourth means in fluid communication with the second means and the third means for pumping the sample-containing mixture across the second means and into the third means; and (v) a fourth means having an eluent therein and for capturing the sample-containing mixture on the second means. (vi) sixth means, in fluid communication with the solid membrane, for receiving and collecting the eluent together with the captured nucleic acids from the biological sample therein; and (vii) seventh means, in fluid communication with the second means and the fifth means, for pumping and flowing the eluent from the fifth means across the second means to elute the captured nucleic acids from the second means and into the sixth means together with the captured nucleic acids.

[0013] In some embodiments of the invention, the first means is a sample port or sample chamber, the second means is a solid membrane, the third means is a waste chamber, the fourth means is a first pump, the fifth means is an eluent chamber, the sixth means is an eluent reservoir, and the seventh means is a second pump.

[0014] In another aspect, the present invention relates to a formulation for collecting a biological sample of saliva or nasal secretions and capturing nucleic acids in the collected biological sample on a solid membrane. The formulation comprises: (i) one or more non-toxic chaotropic agents, (ii) ethanol, and (iii) a coloring agent and / or flavoring agent. The formulation can be received in the oral or nasal cavity from which the biological sample of saliva or nasal secretions is collected. The one or more non-toxic chaotropic agents optionally lyse cells of the biological sample, and the ethanol binds nucleic acids in the lysed cells of the biological sample to the solid membrane.

[0015] Some embodiments of the invention comprise about 0.1% to about 40% w / v of a non-toxic chaotropic agent and about 5% to about 30% w / v of ethanol. In some such embodiments, the non-toxic chaotropic agent is selected from the group comprising the following, individually or in any combination thereof: (i) about 5% to about 30% w / v of urea, about 0.1% to about 3% w / v of sodium lauryl sulfate, and about 2% to about 40% w / v of ammonium trichloroacetate.

[0016] In some embodiments of the invention, the formulation is provided in combination with a long chain fatty alcohol wash configured to flow over the solid membrane following the formulation, substantially displacing residual ethanol of the formulation on the solid membrane.

[0017] One advantage of the present invention, and / or its embodiments, is that it can provide a solution to the saliva collection problems encountered in the prior art. Another advantage is that the system allows the use of mouthwash or nasal rinse that the user can swish in the mouth or spray into the nose before introducing the saliva or nasal secretions into a microfluidic or other nucleic acid isolation and purification system. Yet another advantage is that the mouthwash or nasal rinse contains sample preparation chemicals that are non-toxic, thus allowing partial and / or complete sample preparation (e.g., cell lysis) before introduction into a microfluidic or other nucleic acid isolation and purification system or into an external sample collection cup. Further advantages of the invention and / or its embodiments are that they can standardize the volume of collected samples, eliminate or substantially eliminate viscosity and foaming issues, and eliminate, substantially eliminate, or minimize the user's exposure to toxic chemicals. Yet another advantage is that the invention can be extended to nasal rinses for easier collection of nasal samples.

[0018] Other objects and advantages of the present invention, and / or embodiments thereof, will become more readily apparent in view of the following detailed description of the embodiments and the accompanying drawings. [Brief description of the drawings]

[0019] [Figure 1A] FIG. 1 is a schematic diagram of one embodiment of a microfluidic system of the present invention, generally designated by reference numeral 10, including a microfluidic device 12 for receiving a sample mixture 16 through an inlet port 14. The sample mixture 16 includes a biological sample, such as saliva or nasal secretion (e.g., mucus), a non-toxic chaotropic agent for cell lysis, ethanol, and a colorant and / or flavoring agent. The sample mixture 16 is introduced into the inlet port 14 of the microfluidic device 12 by a transfer device, such as from a syringe 18A, a funnel 18B, or a collection vessel 18C, through a sample introduction tube 18D. The inlet port 14 is in fluid communication with a capture membrane 20 (e.g., a glassy or solid membrane), and a syringe or other pump 22 is actuated to draw or pull the sample mixture 16 across or through the solid membrane 20, where RNA and / or DNA in the sample is captured by or collected on the membrane, and the remainder of the sample mixture is drawn into the syringe 22 as waste. An elution blister or other pump 24 containing eluent 26 (e.g., water (marked in blue)) is then depressed or actuated, breaking the blister and releasing and pumping the eluent across the solid membrane 20, removing the captured RNA and / or DNA on the membrane, and carrying the captured RNA and / or DNA into an eluent reservoir 28, where the eluent with the captured RNA and / or DNA can be collected and pooled. A plurality of capillary tubes 30, 30 are connected in fluid communication between the reservoir 28 and a plurality of reaction chambers 32, 32 and a negative control chamber 40, and are configured to transfer the eluent with the captured RNA and / or DNA from the reservoir into the reaction chambers and negative control chambers by capillary action. [Figure 1B]1B is a schematic diagram of syringe 18A, funnel 18B and sample collection container 18C of FIG. 1A, each connected or connectable in fluid communication with sample introduction tube 18D, preferably by a lockable or locking connector 50. [Diagram 2] FIG. 2 is a schematic diagram of an alternative embodiment of the microfluidic system 10 of FIG. 1, in which the microfluidic device 12 includes an elution syringe or other pump 24′ pre-filled with eluent 26 (e.g., water (lined in blue)) instead of the elution blister 24 of FIG. 1. When the elution syringe 24′ is actuated, the eluent 26 is expelled and pumped across the solid membrane 20, removing the captured RNA and / or DNA on the membrane and carrying the captured RNA and / or DNA into an eluent reservoir 28, where the eluent 26 with the captured RNA and / or DNA is collected or pooled and then transferred by capillary action into a reaction chamber, 32,32. [Diagram 3] 2 is a schematic diagram of the microfluidic system 10 of FIG. 1 highlighting in rectangle 34 features or portions of the system focused on sample collection, RNA / DNA isolation and purification. [Figure 4] FIG. 2 is a schematic diagram of the microfluidic system 10 of FIG. 1 showing a first step in the use of the device 12, in which a user collects a biological sample and introduces a sample mixture 16 into the microfluidic device by one of three options 18A, 18B or 18C. [Diagram 5] FIG. 2 is a schematic diagram of the microfluidic system 10 of FIG. 1 showing a second step in the use of the device 10, in which the patient or other user pulls on the empty syringe 22 of the microfluidic device 10, thus drawing the sample mixture 16 (marked with a green line) across the capture membrane 20 and into the barrel of the syringe 22, which acts as a waste container. [Figure 6]FIG. 2 is a schematic diagram of the microfluidic system 10 of FIG. 1 showing a third step in the use of the device 12, in which the patient or other user continues to pull on the plunger of the syringe 22 of the device 10, which pulls dodecanol (or other long chain fatty alcohol) 36 (shown in red) across the membrane 20 and / or air across the membrane 20, removing substantially all of the residual ethanol from the membrane. [Figure 7A] 1 showing a fourth step in the use of device 12, where a patient or other user presses down on elution blister 24, which forces elution buffer 26 across membrane 20 and elutes captured DNA and RNA from membrane 20 into capture reservoir 28 (eluent with captured DNA and RNA is lined in blue). Fluid flow is controlled by one-way valves 38, 38 (shown as arrows lined in red) fluidically connected (i) between inlet port 14 and the inlet side of membrane 20, and (ii) between the outlet side of membrane 20 and capture reservoir 28. Valves 38, 38 prevent liquid from flowing back from capture reservoir 28 and / or reaction chamber 32, 32 into membrane 20, as well as preventing liquid and / or other material from flowing back through inlet port 14 into transfer / collection device(s) 18A, 18B, 18C and / or 18D. Once the captured RNA and DNA enter capture reservoir 28, reaction chamber 32, 32 fills via capillary action by capillary conduit 30, 30 extending in fluid communication between capture reservoir 28 and each individual reaction chamber 32, 32 and negative control chamber 40, where elution fluid dissolves reaction chemicals previously stored as lyophilized beads in the reaction chamber. Depression of elution blister 24 also activates device heating element 42 in thermal communication with reaction chamber 32, 32 and negative control chamber 40, heating the chamber to the required temperature for reaction. [Figure 7B]FIG. 7B is a cross-sectional view of the syringe barrel of the device of FIG. 7A showing waste dodecanol 36 (marked in red) collected on top of the waste sample mixture 16 (marked in green) within the barrel of syringe 22. [Figure 8] 2 showing a fourth step in the use of the device 12, in which a patient or other user depresses or otherwise actuates an elution syringe 24', which forces an elution buffer 26 across the membrane 20 and elutes the captured DNA and RNA from the membrane and into a capture reservoir 28 (where the elution buffer with the captured DNA and RNA is lined in blue). Fluid flow is controlled by one-way valves 38, 38 (shown as arrows lined in red) fluidically connected (i) between the inlet port 14 and the inlet side of the membrane 20, and (ii) between the outlet side of the membrane 20 and the capture reservoir 28. Valves 38,38 prevent liquid from flowing back from capture reservoir 28 and / or reaction chamber 32,32 into membrane 20, as well as preventing any material from flowing back through inlet port 14 into transfer / collection device(s) 18A, 18B, 18C and / or 18D. Once RNA and DNA enter capture reservoir 28, reaction chamber 32,32 and negative control chamber 40 are filled via capillary action by capillary conduit 30,30, which dissolves reaction chemicals previously stored as lyophilized beads in the chambers. In FIG. 8, the previously stored reaction chemicals are shown as circles in each empty reaction chamber 32,32, while in FIG. 9 below, the reaction chemicals are not shown because at that stage it is dissolved in the elution buffer with the captured DNA and RNA. Depression of the plunger of the elution syringe 24' also activates the heating element 42 of the apparatus 10 for heating the reaction chambers 32, 32 and the negative control chamber 40 to the required temperature for the reaction. [Figure 9]9 is a schematic diagram of the microfluidic system 10 of FIG. 1 showing a fifth step of use of the device 12, in which the reaction chambers 32,32 are heated by the heating element 42 of the device to initiate an amplification reaction, in which the target RNA / DNA, if present, is amplified. Positive amplification is detected by user visualization, e.g., color change, through the transparent / semi-transparent windows 44,44 of the reaction chambers 32,32. For example, as shown in FIG. 9, the positive amplification in each reaction chamber 32,32 is lined with a green color and can be seen through the transparent / semi-transparent windows 44,44 of the reaction chambers 32,32, while the negative control in the negative control chamber 40 is lined with a blue color and can be seen through the negative control chamber transparent / semi-transparent window 44. As can be appreciated by one of ordinary skill in the relevant art based on the teachings herein, positive amplification can be detected in any of many different ways now known or that will later become known, for example, by a change in fluorescence, turbidity, or by digital image capture via an embedded CCD camera or other sensor, or an external device such as a smartphone. [Figure 10] FIG. 11 is a perspective view of another apparatus, generally designated 112A, for sample collection, RNA / DNA isolation and purification, embodying the present invention. [Figure 11] FIG. 11 is a side view of the apparatus of FIG. 10 with its lower manifold 152 shown in dashed lines and its interior shown. [Figure 12] FIG. 1 is a plan view of a multiplex amplification chip or device 112B that can be used in conjunction with device 112A to amplify RNA / DNA isolated and purified in device 112A and to detect target RNA / DNA therein. [Figure 13A] 12 , showing in cross-hatched lines a single vent membrane component 149 covering multiple vent ports 148, 148 fluidly connected to individual reaction chambers 132, 132 and negative control chamber 140 for venting the chambers therethrough. [Figure 13B]FIG. 13 is another plan view of the multiple amplification device 112B of FIG. 12 showing with cross-hatched lines a one-piece optically transparent material 144 covering each reaction chamber 132, 132 and negative control chamber 140, thereby forming a transparent / semi-transparent viewing window above each reaction chamber or negative control chamber through which the interior of each chamber can be seen. [Figure 14] FIG. 13 is a cross-sectional view of the multiplex amplification device 112B of FIG. 12 prior to heating to initiate the amplification reaction. [Figure 15] FIG. 13 is another cross-sectional view of the multiplex amplification device 112B of FIG. 12 prior to heating to initiate the amplification reaction, showing pre-stored lyo-beads or other reaction chemicals in one of the reaction chambers 132, 132, and wax pellets 172 positioned in individual fluid conduits 130 fluidly connected between the eluent reservoir 128 and the individual reaction chambers 132, preventing fluid communication therethrough prior to heating for the amplification reaction. [Figure 16] FIG. 12 is a cross-sectional view of the multiplex amplification device 112B, showing the device after amplification, when the lyophilized beads or other reaction chemicals have dissolved and the wax pellets have melted, and includes a heater 142, e.g., an electric or chemical heater, thermally coupled to the reaction chambers 132, 132, and an excitation light emitting diode ("LED") 174 or other light source that fluoresces a positive indicator such that when viewed by an operator's eye 176, the fluorescence indicates positive detection of the target DNA / RNA. [Figure 17] FIG. 12 is a cross-sectional view of another version of the multiplex amplification device 112B of FIG. 12, showing the device after amplification, when the lyophilized beads or other reaction chemicals have dissolved and the wax pellet has melted, and includes an optical sensor 174 configured to measure the emission wavelength and emission intensity (e.g., color and / or power) within the reaction chambers 132, 132, sense positive detection of target DNA / RNA, and transmit a signal indicative thereof to, for example, a smartphone or other display (not shown). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In a currently preferred embodiment, the sample collection formulation 16 is provided in the form of a mouthwash or a nasal spray or flush. The sample collection formulation 16 includes the following components: 1) Non-toxic chaotropic agents for cell lysis, used individually or in combination. Such agents include, but are not limited to, urea used in artificial saliva, sodium lauryl sulfate used in toothpaste, and / or ammonium trichloroacetate used to treat lesions on the skin and mucous membranes. The amount of urea is preferably within the range of about 5% to about 30% w / v, more preferably within the range of about 10% to about 25% w / v, and even more preferably within the range of about 15% to about 20% w / v. The amount of sodium lauryl sulfate is preferably within the range of about 0.1% to about 3% w / v, more preferably within the range of about 0.2% to about 1.5% w / v, and even more preferably within the range of about 0.4% to about 0.7% w / v. The amount of ammonium trichloroacetate is preferably within the range of about 2% to about 40% w / v, more preferably within the range of about 4% to about 20% w / v, and even more preferably within the range of about 8% to about 10% w / v. Based on the teachings herein, as can be recognized by those skilled in the relevant technical field, some applications may not require cell lysis, for example, when floating nucleic acid is present. However, such applications may nevertheless require a non-toxic chaotropic agent to chaotrope cells and promote the binding of nucleic acid to solid membranes or other nucleic acid capture devices. 2) Ethanol to promote capture of RNA and DNA, the amount of ethanol is preferably in the range of about 5% to about 30% w / v, more preferably in the range of about 10% to about 25% w / v, and even more preferably in the range of about 15% to about 20% w / v. 3) Coloring and / or flavoring agents.

[0021] Below is a representative formulation: 1) Approximately 20% w / v ethanol, 2) about 0.1% w / v sodium lauryl sulfate; 3) Ammonium trichloroacetate, approximately 20% w / v; 4) about 0.042% w / v menthol, and 5) Water.

[0022] The apparatus and method may also employ a long-chain fatty alcohol wash. Ethanol is an inhibitor of many amplification reactions. In the systems disclosed herein, ethanol is used to bind nucleic acids to the glassy or solid membrane 20 surface. Residual ethanol on the glassy or solid membrane surface or in the fluidic channels may be carried into the reaction chamber 32, 32 when the nucleic acid is eluted from the glassy or solid membrane surface. To solve this problem, the fluidic channels and glass surfaces may be washed with a long-chain (>4) fatty alcohol, such as 2-dodecanol, which is transparent and hydrophobic. This provides several advantages: a) the fatty alcohol displaces and solubilizes the residual ethanol in the system, b) the residual fatty alcohol does not inhibit the amplification reaction or the resulting visualization, and c) the residual fatty alcohol can be used to provide a barrier against evaporation of water from the reaction solution. Other long-chain fatty alcohols that may be used include, but are not limited to, the following (used individually or in any combination): 1) dodecanol, 2) Octanol, 3) stearyl alcohol, 4) Lauryl alcohol, 5) Cetyl alcohol, 6) Oleyl alcohol, and 7) Butyl alcohol.

[0023] There are several options for using the above formulations as mouthwash or nasal spray or rinse, including:

[0024] Option 1: Mouthwash / nasal spray or rinse is coated onto gauze or other small mass of absorbent material used to swab the mouth or nose. The swab is then pressed by syringe 18A at the inlet 14 of the microfluidic device 12, from which the material is expelled into the device. Syringe 18A may also contain 2-dodecanol 36 (about 50 to about 500 μL (or other long chain fatty alcohol, as indicated above)) to improve assay performance. The 2-dodecanol or other long chain fatty alcohol(s) 36 is present in the chamber of syringe 18A to be introduced into device 12 after saliva is mixed with the mouthwash / nasal spray.

[0025] Option 2 (saliva only): The user swirls the mouthwash in the mouth and then spits the mouthwash together with the saliva into the microfluidic device with the aid of a funnel 18B or similar device.

[0026] Option 3 (saliva only): The user swirls the mouthwash in the mouth and then spits the mouthwash along with the saliva into a second collection container or cup 18C that is preloaded with additional chaotropic agent and / or ethanol 46 that is released before, during, or after sealing of the cup. For example, the collection container 18C can include a chamber having a frangible or breakable wall that contains the additional chaotropic agent and / or ethanol 46. The container closure can include a piercing member such that upon closing the container 18C with the closure, the piercing member breaks the wall, thereby mixing the mouthwash and saliva with the additional chaotropic agent and / or ethanol 46 in the container. The user then agitates cup 18C (e.g., by shaking it) so that the lysate and chaotrope sample mixture is contained within the container, and the mixture is then introduced from the container into inlet 14 of microfluidic device 12, e.g., via another transfer device, e.g., syringe 18A, also containing 2-dodecanol (about 50 to about 500 μL (or other long chain fatty alcohol)) 36, to improve assay performance. 2-dodecanol 36 may be present in the chamber of syringe 18A so that it is introduced into device 12 after the saliva-containing mixture.

[0027] As typically shown in FIG. 3, the following are the main or key components of the device 10. Such components are located outside the microfluidic device 12: 1) a sample collection preparation or solution 16 (e.g., a mouthwash / nasal spray or wash); 2) a collection device (e.g., a collection cup or container 18C or a swab disposed at the distal end of the syringe 18A for absorbing saliva therein and releasing the saliva when compressed upon actuation of the syringe); and 3) A transfer device (e.g., syringe 18A or funnel 18B).

[0028] The following such components are disposed on or within the microfluidic device 12: 1) Tubes / channels 18D connecting the microfluidic device 12 to the transfer devices 18A, 18B and / or 18C; 2) DNA / RN capture membrane (or solid membrane) 20. In the illustrated embodiment of the solid membrane, the inlet or inlet side of the membrane is at the top and the outlet or outlet side of the membrane is at the bottom. 3) a syringe 22 that "pulls" material from the transfer device / inlet port 14, across a solid membrane 20, and into a syringe 22 (which defines a waste chamber therein); 4) One-way valves 38, 38 for controlling fluid flow into, across, and from the solid membrane 20 to a waste chamber in the syringe 22 or to a reservoir 28 for capillary transfer to the reaction chamber(s) 32, 32; 5) A blister mechanism 24, or syringe 24' or other pumping / dispensing device, preloaded with elution buffer 26 (e.g., water). Although standard pull-type syringes 22, 24' are shown, each syringe may be a counter pressure syringe or other type of syringe or similar pumping / dispensing device now known or later to become known. 6) A reservoir 28 for pooling elution 26 prior to loading into the reaction chambers 32, 32, so that fluid flows from the solid membrane 20 into the reservoir 28 and the elution is pooled in the reservoir before flowing into the reaction chamber(s) 32, 32. One advantage of the reservoir 28 is that it can be used to pool purified RNA / DNA, for example, facilitating obtaining a substantially consistent target concentration across multiple reaction chambers. 7) Capillary tubes or conduits 30, 30 that transfer the eluate from the reservoir 28 into the reaction chambers 32, 32 and the negative control chamber 40. One advantage of using capillary tubes is that they provide a capillary filling action into the reaction chamber(s), which in turn facilitates bubble management. 8) Reaction chambers 32, 32 pre-filled with lyophilized amplification reagents and a negative control chamber 40; 9) a heater 42 for the amplification reaction, and 10) Gas permeable membranes (i.e., gas only valves) 48,48 at the distal ends of the reaction chambers 32,32 and the negative control chamber 40 to allow air to escape and liquid to fill the respective chambers. As shown in the illustrated embodiment, multiple vent lines extend in fluid communication between the reaction chambers 32,32 and the negative control chamber 40 and the respective gas only valves 48,48.

[0029] In operation of the microfluidic system 10, with reference to Figs. 1A, 1B and 2, the sample-containing mixture 16 is introduced into the device 12 using options 18A, 18B or 18C and an elution buffer 26 (e.g., water) enclosed or contained within an elution blister 24 (Fig. 1A) or a pre-filled elution syringe 24' (Fig. 2). Three options are illustrated for sample collection and introduction into the device 12. Using option 18A, a collection and transfer syringe is used. If dodecanol 36 is required, it is contained within the enclosed section of the syringe 18A and is introduced into the device 12 as soon as the sample is introduced by a lockable or locking mechanism between the syringe and the introduction tube 18D (e.g., flexible tube). Using option 18B, a funnel is connected to the device 12 via a lockable or locking flexible tube 18D. As can be seen, dodecanol is not used in this particular embodiment. Funnel 18B preferably connects directly to introduction tube 18D via a locking mechanism of a type known to those of skill in the relevant art. With option 18C, if dodecanol is required, a chamber within sample collection container 18C contains dodecanol 36 and releases it when the container is closed. The mixed contents of the sample collection chamber can be "pulled" into the device via syringe 18A, if desired. The connection between collection cup 18C and inlet port 14 of device 12 is flexible tubing 18D having a lockable or locking connection 50 (FIG. 1B) of a type known to those of skill in the relevant art.

[0030] As shown in Figure 4, the first step of using the device 12 involves the patient or other user withdrawing a biological sample 16 and introducing the sample mixture 16 into the microfluidic device 12 according to any of options 18A, 18B or 18C above. In a second step, as shown by the arrow in Figure 5, the patient or other user pulls on the plunger of an empty syringe 22, which then draws the sample mixture 16 across the capture membrane 20 and into the barrel of the syringe, which acts as a waste container. In a third step, as shown in Figure 6, the patient or other user continues to pull on the plunger of the syringe 22 until the plunger hits a stop within the syringe, thus pulling the dodecanol 36 across the membrane 20 and removing any residual ethanol. In another embodiment, dodecanal is not required and in a third step, syringe 22 pulls air across membrane 20 so that any residual air (e.g., residual air in syringe 18A, funnel 18B, or collection cup 18C) displaces the ethanol.

[0031] In the fourth step, as shown in FIG. 7, the patient or other user presses down on the elution blister 24, which forces the elution buffer 26 across the membrane 20 and elutes all the DNA and RNA from the membrane and into the capture reservoir 28. Fluid flow is controlled by one-way valves 38, 38 to prevent fluid from flowing back from the reaction chambers 32, 32 into the membrane 20 and to prevent any material from flowing back into the transfer / collection device(s) 18A, 18B, 18C and / or 18D. Once the RNA and DNA enter the capture reservoir 28, the reaction chambers 32, 32 fill by capillary action, which dissolves the reaction chemicals previously stored as lyophilized beads in the reaction chambers. Pressing down on the elution blister also activates the device heating element 42 to heat the reaction chambers 32, 32 to the required temperature for the reaction. As shown in FIG. 8, in another fourth step of the device 10, the patient or other user presses down on the plunger of the elution syringe 24′, which forces the elution buffer 26 across the membrane 20 and elutes all the DNA and RNA from the membrane and into the capture reservoir 28. Fluid flow is controlled by one-way valves 38, 38 to prevent fluid from flowing back from the reaction chambers 32, 32 into the membrane 20 and to prevent any material from flowing back into the transfer / collection devices 18A, 18B, 18C and / or 18D. Once the RNA and DNA enter the capture reservoir 28, the reaction chambers 32, 32 fill by capillary action, which dissolves the reaction chemicals previously stored as lyophilized beads in the reaction chambers. Pressing down on the elution syringe 24′ also activates the device heater 42.

[0032] As shown in Figure 5, in the fifth step of the device 10, heating of the reaction chambers 32, 32 initiates an amplification reaction in which target RNA / DNA, if present, is amplified. Positive amplification is detected by user visualization (e.g., by a color change from blue to green, fluorescence, or turbidity change) or by digital image capture by an external device such as an embedded CCD camera, a photosensor, or a smartphone. In the device shown, the heater 42 is an electric heater, although chemical heating or other types of heating mechanisms now known or that later become known could equally be used.

[0033] In Figures 10 and 11, another apparatus embodying the present invention for sample collection and nucleic acid or RNA / DNA isolation and purification is generally designated by the reference number 112A. Apparatus 112A is similar to corresponding portions of apparatus 12 described above in connection with Figures 1 through 9, and thus like reference numbers preceded by the numeral "1" are used to indicate like elements. Apparatus 112A utilizes a manifold assembly and fluidic channels containing a solid purification medium, and serves as a structural chassis for a series of manually actuated valves, fluidic reservoirs, and fluidic actuated syringes that draw sample and reagents from the fluidic reservoirs, through membranes, and into waste and elution syringes. The elution fluids are then transferred to an amplification apparatus for thermal incubation, as described above in connection with Figure 9 or below in connection with Figures 12 through 17.

[0034] Apparatus 112A includes a waste syringe 122 in fluid communication with the outlet side of solid, purification membrane 120 through a waste draw valve 138 and a lower manifold 152. As shown in FIG. 11 , the lower manifold 152 defines a fluidic channel 154 therein that extends in fluid communication between the outlet of the solid membrane 120 and the waste valve 138. An elution syringe 124 is connected in fluid communication to the fluidic channel 154 through the elution valve 138. An upper manifold 156 is mounted on the lower manifold 152 on the inlet side of the solid membrane 120 and defines fluidic channel(s) (not shown) therein that are in fluid communication with the inlet side of the solid membrane.

[0035] As shown in FIG. 10, a plurality of reservoirs and associated valves are mounted on the top surface of the upper manifold 156. A sample-containing mixture reservoir 138 is mounted on the upper manifold 156 and is connected in fluid communication therewith through a respective sample valve 138. A first wash fluid reservoir 158 is mounted on the upper manifold 156 adjacent to the sample reservoir 138 and is connected in fluid communication therewith through a respective first wash valve 138. A second wash fluid reservoir 160 is mounted on the upper manifold 156 adjacent to the first wash fluid reservoir 158 and is connected in fluid communication therewith through a respective second wash valve 138. An elution fluid reservoir 162 is mounted on the upper manifold 156 adjacent to the second wash fluid reservoir 160 and is connected in fluid communication therewith through a respective elution valve 138. In the illustrated embodiment, the individual reservoirs are defined by individual syringes, with the reservoirs defined within the barrels of the syringes. Each syringe reservoir may include a luer lock or other type of connector at its distal end, for removably connecting the syringes to and disconnecting them from individual valves or manifolds. As can be recognized by those skilled in the relevant art based on the teachings herein, the reservoirs may take the form of any of a number of different devices now known or later to become known for storing, pumping, dispensing, and / or otherwise releasing the individual liquids therein, such as a depressible blister having a frangible wall, or an electrically actuated liquid dispenser. Also, in the illustrated embodiment, the valves 138, 138 are manually operable valves, with each valve including an individual knob that is manually rotated or turned to move the valve between a closed position that prevents fluid flow through the valve and an open position that allows fluid flow through the valve.As can be appreciated by one of ordinary skill in the relevant art, the valves 138, 138 can take the form of any of many different types of valves that can be operated or actuated in any of many different manners now known or that later become known, including valves that are electrically or electronically operated or actuated.

[0036] In operation of device 112A, a sample mixture is loaded into sample reservoir 118. Waste draw valve 138 is then opened, sample reservoir valve 138 is opened, the sample reservoir is fluidly connected to the inlet side of solid membrane 120, and waste syringe 122 is placed in fluid communication with the outlet side of solid membrane 120. The user then retracts the plunger of waste draw syringe 122 a first predetermined amount, and the sample mixture is pulled or drawn from sample reservoir 118, through upper manifold 156, and across solid membrane 120. In the illustrated embodiment, waste draw syringe 122 includes graduations or similar indicia, and the predetermined amount is determined based on movement of the plunger relative to the graduations or other indicia. However, as can be appreciated by those skilled in the relevant art based on the teachings herein, the predetermined volume can be determined or dispensed using any of many different mechanisms and according to any of many different methods, which are now known or which later become known, such as by electrical or electronic metering. In one embodiment, such volume is equal to approximately 5 mL. After drawing the sample mixture from the sample reservoir 118, the sample reservoir valve 138 is closed. The first wash fluid reservoir valve 138 is then opened, placing the first wash fluid reservoir 158 in fluid communication with the inlet side of the solid membrane 120. The user then retracts the plunger of the waste draw syringe 122 a second predetermined volume, which causes the first wash fluid to be pulled or drawn from the first wash fluid reservoir, through the upper manifold 156, and across the solid membrane 120. In the illustrated embodiment, the second predetermined volume is approximately 5 mL. The user closes the first wash fluid reservoir valve 138 and opens the second wash fluid reservoir valve 138, placing the second wash fluid reservoir 160 in fluid communication with the inlet side of the solid membrane 120. The user then retracts the plunger of the waste draw syringe 122 a third predetermined amount, causing the second wash fluid to be pulled or drawn from the second wash fluid reservoir, through the upper manifold 156, and across the solid membrane 120. In the illustrated embodiment, the third predetermined amount is approximately 10 mL.The user closes the second wash fluid reservoir valve 138, closes the waste draw valve 138, and disconnects the waste draw syringe 122 from the solid membrane. The user then opens the elution valve 138 of the elution fluid reservoir 162, placing the elution fluid reservoir in fluid communication with the inlet side of the solid membrane 120, and opens the elution draw valve 138 of the elution fluid draw syringe 124, placing the elution fluid draw syringe in fluid communication with the outlet side of the solid membrane 120. The user then pulls on the plunger of the elution fluid draw syringe 124, pulling or drawing elution fluid from the elution fluid reservoir 162 across the solid membrane, eluting the RNA and DNA therein, and trapping the eluted RNA and DNA in the barrel of the elution fluid draw syringe 124. The eluate draw syringe 124 is removable from its mount on the lower manifold 152, for example by a Luer connector, and the eluate therein is transferred to the amplification apparatus for thermal incubation and amplification in the reaction chamber(s), in the manner described above in connection with FIG. 9.

[0037] In Figures 12 to 17, another device embodying the present invention is generally designated by the reference number 112B. The device 112B can be used in conjunction with the device 112A described above. As described above, the device 112A is for sample collection, and nucleic acid or RNA / DNA isolation and purification. On the other hand, the device 112B is for, for example, amplification and detection of targeted isolated and purified nucleic acid or RNA / DNA from the device 112A. The device 112B is similar to the corresponding parts of the device 12 described above in connection with Figures 1 to 9, and therefore similar reference numbers preceded by the number "1" are used to indicate similar elements.

[0038] As shown in FIG. 12, the device 112B includes a fluid inlet 164 in fluid communication with a reservoir or premix chamber 128, which is in turn in fluid communication with a plurality of reaction / amplification chambers 132, 132 through individual capillary conduits 130, 130. Each reaction / amplification chamber 132, 132 includes an individual reaction / amplification chamber vent 148, 148 in fluid communication therewith for venting the individual chamber to the ambient atmosphere. In FIG. 13A, a single vent membrane component 148 covers a plurality of vent ports 148, 148 in fluid communication with the individual reaction chambers 132, 132 and the negative control chamber 140 for venting the chambers therethrough. The vent membrane 148 allows air or other gases to flow therethrough but prevents or substantially prevents liquids from flowing therethrough.

[0039] As shown in FIG. 13B, a one-piece optically transparent material 144 covers each reaction chamber 132, 132 and negative control chamber 140, thereby forming a transparent / semi-transparent viewing window on each reaction chamber or negative control chamber through which the interior of each chamber can be viewed. The transparent or semi-transparent window 144 is formed of an optically transparent material of a type known to those of skill in the relevant art and covers each reaction / amplification chamber 132, 132 and negative control chamber 140 through which the interior of the chamber can be viewed. As can be recognized by those of skill in the relevant art based on the teachings herein, the device 112B can include a single vent membrane component 148 and a single viewing window component 144 for all reaction chambers 132, 132 as shown, or can include any desired number of such components.

[0040] As shown in Figures 14 and 15, the fluid inlet 164 is fluidly coupled to an inlet check valve 166, which is in turn coupled to the reservoir / premix chamber 128 through a fluid-flow path 167. The fluid inlet 164 is configured to receive a distal end of an elution syringe reservoir 162 and receive elution fluid therefrom, as described above in connection with Figure 10. The reservoir / premix chamber 128 includes a vent 129 for venting the chamber during its filling. As shown in Figure 14, a pressure relief valve 148 is fluidly coupled between the reaction / premix chamber 128 and the plurality of capillary conduits 130, which are in turn fluidly coupled to the individual reaction / amplification chambers 132. As shown in FIG. 15, each capillary conduit 130 contains within it an individual wax or other meltable pellet 172 that is configured to prevent migration of liquid from the reservoir / premix chamber 128 through the conduit at room temperature (or below the incubation temperature). The wax pellets 172 are configured not to melt at room temperature, but rather to melt at temperatures close to or approaching the incubation temperature for the amplification reaction. One advantage of this feature is that it gives the solution more time to homogenize the DNA / RNA and distribute it more consistently to each reaction chamber 132, 132. When the premix chamber 128 is heated by the heater 142 (FIGS. 16 and 17), the diffusion rate increases, thus mixing the contents. Time and temperature (i.e., at or near the incubation temperature) facilitate such mixing. It should be noted that when the wax 172 melts, capillary action draws the solution into the conduits 130, 130 and reaction chambers 132, 132, while the vent 129 allows air to return to the pre-mix chamber 128, backfilling the inner volume of the pre-mix chamber as fluid is transferred out of it.Each reaction / amplification chamber 132,132 preferably contains lyophilized master mix beads or reagent mixture 168 therein, and a respective portion of a transparent or translucent viewing window 144 is present on each such chamber and the negative control chamber 140 for visual observation therethrough of the interior of each chamber after reaction / amplification. A vent channel 170 extends between a top end or region of each reaction / amplification chamber 132,132 and a respective reaction / amplification chamber vent 148 therethrough for venting the chamber.

[0041] In operation of device 112B, the check valve 166 of the fluid inlet 164 passively closes as soon as the reservoir / premix chamber 128 is filled with elution fluid 126. As elution fluid 126 enters device 112B, the reservoir / premix chamber 128 fills completely as air escapes through reservoir / premix chamber vent 148, completing the premixing process and resulting in a substantially uniform concentration of DNA / RNA throughout the fluid volume within the reservoir / premix chamber. The fluid pressure within the reservoir / premix chamber 128 increases as fluid is added thereto. As soon as the reservoir / premix chamber 128 is filled (or reaches a predetermined fill level), the pressure inside the reservoir / premix chamber 128 exceeds the relief pressure of the premix chamber pressure relief valve 148, which then allows the substantially homogenous liquid within the reservoir / premix chamber 128 to flow into each of the five amplification / reaction chambers 132,132 and the negative control chamber 140. In the amplification / reaction chambers 132,132, the substantially homogenous liquid dissolves the reagent master mix 168 while air is exhausted through the individual reaction / amplification chamber vents 148,148.

[0042] The device 112B is then incubated with a heating unit or element 142 to induce target RNA / DNA amplification in the reaction / amplification chambers. If desired, each reaction / amplification chamber 132, 132 can contain a unique master mix 168 for amplification of a different target, if desired. As shown in FIG. 16, a heater 142 (which can be, for example, an electric or chemical heater) is thermally coupled to the reaction chambers 132, 132 and the negative control chamber 140. Following activation of the heater 142 to a predetermined incubation temperature, the lyophilized beads or other reaction chemicals 168 melt and the wax pellets 172 melt. After incubating the filled reaction chambers 132, 132 at a predetermined incubation temperature and incubation period and obtaining amplification of the RNA / DNA, the excitation LED or other light source 174 fluoresces the positive indicator, so that when viewed by the operator's eye 176, the fluorescence indicates a positive detection of the target DNA / RNA. Calcein, also known as fluorexone, or fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495 nm and 515 nm, respectively. Calcein is commonly used in biological applications as a marker for the detection of certain biological materials. In device 112B, calcein is a component in the lyophilized bead master mix 168. In lyophilized bead form, calcein cannot emit the excitation wavelength (510 nm). During amplification / incubation, if a target molecule is present in the eluate in the individual reaction chambers 132, the calcein contained in the master mix (i.e., in the lyophilized beads) undergoes a chemical change that allows the emission of excitation light, and the operator can see a fluorescent glow, indicating the presence of the target molecule in the sample, i.e., a positive detection of the target. During incubation, if the target molecule is not present in the reaction chambers 132, 132, no chemical change is induced and the reactant does not glow, indicating the target molecule is not present in the sample, i.e., the result is negative.

[0043] As shown in FIG. 17, instead of the human readable embodiment of FIG. 16, the device 112B includes an optical sensor 178 configured to output a digital binary signal that indicates either a positive or negative result on a user interface that communicates the result to the user. One advantage of this embodiment is the ease of use and suitability for use by less skilled operators that may be required for some point-of-care ("POC") applications. As in FIG. 16 above, at the stage shown in FIG. 17, the wax pellet 172 has melted. In addition, the lyophilized beads 168 have melted by the time amplification has occurred, at which time the result is read. The optical sensor 178 measures the color and intensity of the emission wavelength in the individual reaction chamber 132, or in the multiple reaction chambers 132, 132. If the 515 nm output rises above a pre-determined set threshold, the result is considered positive. On the other hand, if the 515 nm output does not rise above a pre-determined set threshold, the result is considered negative. In either case, the optical sensor 178 transmits a signal indicating either a positive or negative result to a user interface, such as a display or smartphone, that communicates the result to a user. As can be recognized by one of ordinary skill in the relevant art based on the teachings herein, the device 112B may include one optical or other sensor for all the chambers, or multiple sensors for multiple chambers, where each sensor senses an emission wavelength within the interior(s) of one or more chambers.

[0044] Based on the teachings herein, as can be recognized by those skilled in the relevant art, many changes, improvements, modifications, additions and deletions are possible to the above and other embodiments of the present invention without departing from the scope of the invention. For example, the microfluidic device or its components and operation or use method, or aspects thereof, may be the same as or similar to any of the microfluidic devices or its components and methods or aspects disclosed in the following co-pending patent applications, which are assigned to the assignee of the present invention and hereby incorporated by reference in their entirety as part of this disclosure: (i) U.S. Patent Application No. 17 / 647,828, filed on January 12, 2022, entitled "Device And Method For Detecting Nucleic Acids In Biological Samples" and (ii) U.S. Patent Application No. 17 / 941,816, filed on September 9, 2022, entitled "Device And Method For Detecting Nucleic Acids In Biological Samples". In addition, the device may include fewer or additional parts than those shown and / or described herein. For example, the device may include only one pump for pumping the sample mixture, and dodecanol and air, any wash solutions, and any eluents across one or more solid membranes. Or, the device may include multiple pumps for performing such functions. Or, the device may include multiple solid membranes or other filtration mechanisms, including membranes mounted in series. Still further, the device may include multiple capture reservoirs, or in other cases, the capture reservoir may be omitted. In other embodiments, the conduit(s) present between the capture reservoir and the reaction chamber(s) and / or negative control chamber(s) need not be capillary or operate by capillary flow action. For example, flow through the conduits can be achieved by pressure differential, such as by pumping to fill the capture reservoir.In addition, the heating element does not need to be operated on / off, but rather may be operated by thermo or thermal cycling, for example for PCR or other non-lamp methods / applications. The solid membrane can also take the form of any device now known or later to become known for capturing and releasing nucleic acids, RNA and / or DNA thereon, such as glass beads, including borosilicate glass beads. Thus, the components of the device(s) and the operation or method of use of the device(s) can take any of many different forms or configurations, can be made from or use any of many materials, now known or later to become known, and features or aspects can be added or removed without departing from the scope of the invention. Thus, this detailed description of the embodiments should be considered in an illustrative and not limiting sense.

Claims

1. a sample port or sample chamber for receiving therein a sample-containing mixture including a biological sample; a solid-state membrane in fluid communication with the sample port or sample chamber and configured to receive the sample-containing mixture therefrom, the solid-state membrane allowing the sample-containing mixture to pass through the membrane and capturing nucleic acids in the biological sample on the membrane; a first pump in fluid communication with the solid membrane and a waste chamber in fluid communication with the pump, wherein actuation of the first pump causes the sample-containing mixture to flow across the solid membrane and into the waste chamber; an elution fluid chamber configured to contain or having an elution fluid therein; an eluent reservoir in fluid communication with the solid membrane; a second pump in fluid communication with the solid membrane and the elution fluid chamber, wherein actuation of the second pump causes the elution fluid to flow from the elution fluid chamber across the solid membrane, eluting captured nucleic acids from the solid membrane and flowing with the captured nucleic acids into the elution fluid reservoir; An apparatus comprising:

2. 2. The apparatus of claim 1, wherein the first pump is a syringe including a barrel and a plunger housed within the barrel, the barrel defining the waste chamber therein, and movement of the plunger draws or pulls the sample-containing mixture across the solid membrane and into the waste chamber of the barrel.

3. 3. The apparatus of claim 2, wherein the second pump is movable between an inactive position and an active position, and the eluent chamber includes a frangible or breakable wall that is breakable by movement of the second pump between the inactive position and the active position, and eluent is pumped from the eluent chamber across the solid membrane and into the eluent reservoir.

4. 4. The apparatus of claim 3, wherein the second pump is a plunger, and movement of the plunger from the unactuated position to the actuated position breaks the frangible or breakable wall of the eluent chamber and forces the eluent across the solid membrane and into the eluent reservoir.

5. 3. The apparatus of claim 2, wherein the second pump is a syringe including a barrel and a plunger housed within the barrel, movement of the plunger forcing the eluent across the solid membrane and into the eluent reservoir.

6. 10. The apparatus of claim 1, further comprising a first one-way valve fluidly connected between the sample port or sample chamber and the solid membrane and configured to allow the sample-containing mixture to flow in a direction from the sample port or sample chamber to the solid membrane.

7. 10. The apparatus of claim 1, further comprising a second valve fluidly coupled between the solid-state membrane and the eluent reservoir and configured to allow fluid flow in a direction from the solid-state membrane into the eluent reservoir.

8. The apparatus of claim 1 , further comprising at least one reaction chamber and at least one conduit fluidly connected between the eluent reservoir and the reaction chamber.

9. 9. The device of claim 8, wherein the at least one conduit is a capillary conduit configured to allow the eluent along with captured nucleic acids to flow by capillary action through the capillary conduit and into the reaction chamber.

10. 2. The device of claim 1, further comprising a plurality of reaction chambers and a conduit fluidically connected between the eluent reservoir and each reaction chamber, each conduit configured such that the eluent, together with captured nucleic acids, flows through the conduit and into the respective reaction chamber.

11. 10. The apparatus of claim 8, further comprising a valve fluidly connected between the reaction chamber and an ambient atmosphere, the valve allowing gas to flow from the reaction chamber into the ambient atmosphere but preventing the flow of liquid therethrough.

12. 9. The apparatus of claim 8, further comprising a heating element thermally coupled to the reaction chamber, the heating element defining a first condition in which the heating element heats the reaction chamber to an incubation temperature and a second condition in which the heating element does not heat the reaction chamber to the incubation temperature, the heating element configured to transition from the first condition to the second condition upon or following actuation of the second pump.

13. 10. The apparatus of claim 8, further comprising an optical sensor configured to measure at least one of an emission wavelength or emission intensity in the reaction chamber, detect at least one of a positive detection of the targeted nucleic acid or a negative detection of the targeted nucleic acid, and transmit a signal indicative thereof to a user interface.

14. a first means for receiving therein a sample-containing mixture including a biological sample therein; a second means in fluid communication with said first means for receiving said sample-containing mixture therefrom, said second means for passing said sample-containing mixture through said second means and capturing nucleic acids in said biological sample on said second means; a third means for receiving and retaining the sample-containing mixture after it has passed through the second means; and a fourth means in fluid communication with said second means and said third means for pumping said sample-containing mixture across said second means and into said third means; a fifth means for containing an elution fluid therein and allowing said elution fluid to flow across said second means after said sample-containing mixture has passed through said second means, and for removing nucleic acids captured from said biological sample from said second means with said elution fluid; a sixth means in fluid communication with said solid membrane for receiving and collecting said eluent together with captured nucleic acids from said biological sample therein; a seventh means in fluid communication with said second means and said fifth means for pumping said eluent across said second means from said fifth means to elute captured nucleic acids from said second means and to flow with said captured nucleic acids into said sixth means; An apparatus comprising:

15. 15. The apparatus of claim 14, wherein the first means is a sample port or sample chamber, the second means is a solid membrane, the third means is a waste chamber, the fourth means is a first pump, the fifth means is an eluent chamber, the sixth means is an eluent reservoir, and the seventh means is a second pump.

16. A formulation for collecting a biological sample of saliva or nasal secretions and capturing nucleic acids in the collected biological sample on a solid membrane, the formulation comprising one or more non-toxic chaotropic agents, ethanol, and / or coloring and / or flavoring agents, the formulation being capable of being received into the oral or nasal cavity from which the biological sample of saliva or nasal secretions is collected, the one or more non-toxic chaotropic agents and ethanol lysing at least one cell of the biological sample or promoting binding of nucleic acids in the biological sample to the solid membrane.

17. 17. The formulation of claim 16, comprising about 0.1% to about 40% w / v of a non-toxic chaotropic agent and about 5% to about 30% w / v of ethanol.

18. 18. The formulation of claim 17, wherein the non-toxic chaotropic agent is selected, individually or in any combination thereof, from the group comprising: (i) about 5% to about 30% w / v urea, about 0.1% to about 3% w / v sodium lauryl sulfate, and about 2% to about 40% w / v ammonium trichloroacetate.

19. 17. A combination of the formulation of claim 16 and a long chain fatty alcohol wash configured to flow over the solid membrane subsequent to the formulation and to substantially displace residual ethanol of the formulation on the solid membrane.

20. 20. The combination of claim 19, further comprising a transfer device comprising the formulation in a first portion thereof and the long chain fatty alcohol rinse in a second portion thereof, the first portion being receivable within the oral or nasal cavity for collecting saliva or nasal secretions, the transfer device configured to transfer or dispense the formulation and collected biological sample to the solid membrane and then transfer or dispense the long chain fatty alcohol rinse to the solid membrane.

21. 21. The combination of claim 20, wherein the transfer device is a syringe including at its distal end a gauze or other absorbent material configured to be received within the oral or nasal cavity and to absorb saliva or nasal secretions therein, the syringe being in fluid communication with the gauze or other absorbent material and defining a first chamber or portion of a chamber having the formulation therein and a second chamber or portion of a chamber disposed opposite the first chamber or portion of a chamber relative to the gauze or other absorbent material and having the long-chain fatty alcohol cleanser therein, actuation of the syringe causes the formulation to flow through the gauze or other absorbent material to obtain a biological sample therein and dispense a mixture of the formulation and the biological sample from the syringe, and further actuation of the syringe dispenses the long-chain fatty alcohol cleanser from the syringe following dispensing of the mixture of the formulation and the biological sample.

22. 22. The combination of claim 21, wherein the syringe includes a barrel defining the first chamber and the second chamber and includes a frangible or breakable wall separating the first chamber and the second chamber, the syringe further comprising a plunger housed within the barrel, wherein upon actuation of the plunger, the frangible or breakable wall is broken and the formulation, and then the long chain fatty alcohol detergent, is dispensed from the syringe.

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