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

Abstract

Formulations for collecting biological samples of saliva or nasal fluid and capturing nucleic acids in the collected samples have non-toxic chaotropic agents, ethanol, and colorants and / or flavoring agents. The formulation can be contained within the oral cavity or nasal cavity to collect a sample, and the non-toxic chaotropic agent lyses sample cells. A device has a sample port for receiving a sample-containing formulation and a solid membrane in fluid communication with the sample port. A first pump causes a sample-containing formulation to flow through the solid membrane and into the waste chamber. The ethanol binds nucleic acids in lysed cells of the sample to the solid state membrane. A second pump causes eluent to flow from the eluent chamber through the membrane, eluting the captured nucleic acid from the membrane, and flowing along with the captured nucleic acid into the eluent reservoir.

Description

[0001] Cross-references to related patent applications

[0002] This patent application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 268,742, filed on March 1, 2022, 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 a part of the present disclosure. Technical Field

[0003] The present invention relates to devices and methods for separating, concentrating, amplifying and detecting nucleic acids in biological samples such as saliva or mucus, and more specifically, to collecting oral or nasal samples, compositions containing non-toxic sample preparation materials for collecting and preparing samples to capture nucleic acids therein, and for capturing nucleic acids in biological samples and amplifying the captured nucleic acids in a reaction chamber. Background Art

[0004] Saliva is an attractive sample type for clinical diagnosis because it is non-invasive and can be used to detect many diagnostic targets that are traditionally collected by nasal swabs, sputum or blood samples. However, for use in microfluidic devices for molecular diagnosis (diagnosis based on RNA or DNA targets), saliva has several problems. First, the viscosity of saliva can vary significantly, which can affect the fluid dynamics of the device. Second, due to the inaccurate sample collection method and the different amounts of saliva that individuals can produce and spit out, it is difficult to standardize the saliva volume collected and used. In addition, saliva can foam, thereby introducing bubbles into the microfluidic channels, which can disturb the flow of fluids in the device.

[0005] To address some of these issues, sample preparation (cell lysis) of saliva is often performed outside the device or microfluidic system, where the user adds saliva to a container containing sample preparation chemicals outside the system. This can help standardize the viscosity of the sample before introducing it into the device and can help address foaming issues, but it does not address the varying volumes of saliva collected and can expose the user to potentially toxic sample preparation chemicals.

[0006] It is an object of the present invention and / or its presently preferred embodiments to overcome one or more of the above-mentioned disadvantages and / or shortcomings of the prior art. Summary of the invention

[0007] According to a first aspect, the present invention relates to a device comprising: (i) a sample port or chamber for receiving a sample-containing mixture therein, wherein the sample-containing mixture contains a biological sample; (ii) a solid membrane, which is fluidically connected to the sample port or chamber and is configured to receive the sample-containing mixture therefrom and allow the sample-containing mixture to pass through the membrane and capture nucleic acids in the biological sample on the membrane; (iii) a first pump fluidically connected to the solid membrane and a waste chamber fluidically connected to the pump, wherein actuation of the first pump causes the sample-containing mixture to flow through the solid membrane and into the waste chamber; (iv) an eluent chamber containing an eluent; (v) an eluent reservoir, which is fluidically connected to the solid membrane; and (vi) a second pump, which is fluidically connected to the solid membrane and the eluent chamber, wherein actuation of the second pump causes the eluent to flow from the eluent chamber through the solid membrane, elute the captured nucleic acids from the solid membrane, and flow into the eluent reservoir together with the captured nucleic acids.

[0008] In some embodiments of the present invention, the first pump is a syringe comprising a barrel and a plunger contained in the barrel. The barrel defines a waste chamber therein. Movement of the plunger draws or pushes, or pushes, the sample-containing mixture through the solid membrane and into the waste chamber of the barrel.

[0009] In some embodiments of the present invention, the second pump can be moved between the non-actuated position and the actuated position. The eluent chamber comprises a fragile or breakable wall, which can be broken due to the movement of the second pump between the non-actuated position and the actuated position, so as to pump the eluent from the eluent chamber through the solid film and into the eluent reservoir. In some such embodiments, the second pump is a plunger. The movement of the plunger from the non-actuated position to the actuated position causes the fragile or breakable wall of the eluent chamber to be destroyed, and pushes or pulls the eluent through the solid film and into the eluent reservoir. In other embodiments, the second pump is a syringe comprising a barrel and a plunger contained in the barrel. The movement of the plunger pushes or pulls the eluent through the solid film and into the eluent reservoir.

[0010] Some embodiments of the invention further comprise a first valve in fluid communication between the sample port or chamber and the solid state membrane and configured to allow the sample-containing mixture to flow in a direction from the sample port or chamber to the solid state membrane but prevent liquid flow in the opposite direction.

[0011] Some embodiments of the invention further comprise a second valve in fluid communication between the solid state membrane and the eluent reservoir and configured to allow fluid flow in a direction from the solid state membrane to the eluent reservoir but prevent fluid flow in the opposite direction.

[0012] Some embodiments of the present invention further comprise at least one reaction chamber and at least one conduit (e.g., a capillary conduit) in fluid communication between the eluent reservoir and the reaction chamber. The capillary conduit is configured to allow the eluent with the captured nucleic acid to flow through the capillary conduit and into the reaction chamber by capillary action.

[0013] According to another aspect, the present invention relates to an apparatus comprising: (i) a first device (mean), which is used to receive a sample-containing mixture therein, wherein the sample-containing mixture contains a biological sample; (ii) a second device, which is fluidically connected to the first device, and is used to receive the sample-containing mixture therefrom, so as to allow the sample-containing mixture to pass through the second device, and to capture nucleic acids in the biological sample on the second device; (iii) a third device, which is used to receive and hold the sample-containing mixture after passing through the second device; (iv) a fourth device, which is fluidically connected to the second device and the third device, and is used to pump the sample-containing mixture through the second device and the third device. into a third device; (v) a fifth device for containing an eluent therein and for allowing the eluent to flow through the second device after the sample-containing mixture passes through the second device, and removing the nucleic acids captured from the biological sample from the second device with the eluent; (vi) a sixth device, which is connected to the solid membrane fluid, for receiving and collecting the eluent having the nucleic acids captured from the biological sample therein; and (vii) a seventh device, which is connected to the second device and the fifth device fluid, for pumping the eluent from the fifth device through the second device, eluting the captured nucleic acids from the second device, and flowing into the sixth device together with the captured nucleic acids.

[0014] In some embodiments of the invention, the first device is a sample port or chamber, the second device is a solid state membrane, the third device is a waste chamber, the fourth device is a first pump, the fifth device is an eluent chamber, the sixth device is an eluent reservoir, and the seventh device is a second pump.

[0015] According to another aspect, the present invention relates to a preparation for collecting a biological sample of saliva or nasal fluid and capturing nucleic acids in the collected biological sample on a solid membrane. The preparation comprises: (i) one or more non-toxic chaotropic agents; (ii) ethanol; and (iii) a colorant and / or a flavoring agent. The preparation can be contained in the oral cavity or nasal cavity to collect a biological sample of saliva or nasal fluid therefrom. If necessary, the one or more non-toxic chaotropic agents lyse the cells of the biological sample, and the ethanol binds the nucleic acids in the lysed cells of the biological sample to the solid membrane.

[0016] Some embodiments of the present invention comprise about 0.1% to about 40% w / v non-toxic chaotropic agent and about 5% to about 30% w / v ethanol. In some such embodiments, the non-toxic chaotropic agent is selected from the group comprising the following alone or in any combination: (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.

[0017] In some embodiments of the invention, the formulation is provided in combination with a long chain fatty alcohol detergent configured to flow through the solid membrane after the formulation to substantially eliminate any remaining ethanol of the formulation on the solid membrane.

[0018] One advantage of the present invention and / or its embodiments is that it can provide a solution to the saliva collection problem encountered in the above-mentioned prior art. Another advantage is that the system allows the use of a mouthwash or nasal wash, which the user can gargle or spray into the nasal cavity before introducing the saliva or nasal fluid into the microfluidic or other nucleic acid separation and purification system. Another advantage is that the mouthwash or nasal wash contains non-toxic sample preparation chemicals, thereby allowing partial and / or complete sample preparation (e.g., cell lysis) before introduction into the microfluidic or other nucleic acid separation and purification system or into an external sample collection cup. Another advantage of the present invention and / or its embodiments is that it can standardize the volume of the collected sample, eliminate or substantially eliminate viscosity and foaming problems, and eliminate, substantially eliminate or minimize user exposure to toxic chemicals. Another advantage is that the present invention can be extended to nasal washes for easier collection of nasal samples.

[0019] Other objects and advantages of the present invention and / or its embodiments will become more apparent in view of the following detailed description of the embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A1 is a somewhat schematic diagram of one embodiment of a microfluidic system of the present invention, generally indicated by reference numeral 10, comprising a microfluidic device 12 for receiving a sample mixture 16 through an inlet port 14. The sample mixture 16 contains a biological sample (e.g., saliva or nasal fluid (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 container 18C, and through a sample introduction tube 18D. The inlet port 14 is in fluid communication with a capture membrane 20 (e.g., a glass or solid membrane), and a syringe or other pump 22 is actuated to draw or pull the sample mixture 16 through or through the solid membrane 20, wherein RNA and / or DNA in the sample is captured by the membrane and collected on the membrane, and the remainder of the sample mixture is pulled to the syringe 22 as waste. The elution bubble or other pump 24 containing an eluent 26, such as water (outlined in blue), is then pressed or actuated to break the bubble and release and pump the eluent through the solid membrane 20 to remove the RNA and / or DNA captured on the membrane and carry the captured RNA and / or DNA to the eluent reservoir 28, where the eluent with the captured RNA and / or DNA is allowed to be collected or combined. A plurality of capillaries 30, 30 are connected in a fluidic manner 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 to the reaction chambers and the negative control chambers by capillary action.

[0021] Figure 1B yes Figure 1A A simplified schematic diagram of a syringe 18A, a funnel 18B and a sample collection container 18C is shown, each of which is preferably connected or connectable to a sample introduction tube 18D in fluid communication via a lockable or locking connector 50.

[0022] Figure 2 1 is a simplified schematic diagram of another embodiment of the microfluidic system 10, in which the microfluidic device 12 includes an elution syringe or other pump 24' pre-filled with an eluent 26, such as water (strike-in blue), instead of the elution bubble 24 of FIG1. ​​The elution syringe 24' is actuated to release and pump the eluent 26 through the solid membrane 20 to remove RNA and / or DNA captured on the membrane and carry the captured RNA and / or DNA to the eluent reservoir 28, wherein the eluent 26 with the captured RNA and / or DNA is allowed to be collected or combined before being transferred to the reaction chambers 32, 32 by capillary action.

[0023] Figure 3is a simplified schematic diagram of the microfluidic system 10 of FIG. 1 , which highlights within rectangle 34 those features or portions of the system that focus on sample collection, RNA / DNA separation and purification.

[0024] Figure 4 1 is a simplified schematic diagram of the microfluidic system 10 showing the first step of using the device 12, wherein a user collects a biological sample and introduces a sample mixture 16 into the microfluidic device according to any of three options 18A, 18B or 18C.

[0025] Figure 5 1 is a simplified schematic diagram of the microfluidic system 10 illustrating a second step in using the device 10, wherein a patient or other user pulls an empty syringe 22 of the microfluidic device 10 to in turn pull a sample mixture 16 (lined in green) through the capture membrane 20 and into the barrel of the syringe 22, which acts as a waste container.

[0026] Figure 6 1 is a simplified schematic diagram of the microfluidic system 10, which shows the third step of using the device 12, in which the patient or other user continues to pull the plunger of the syringe 22 of the device 10, which pulls dodecanol (or other long chain fatty alcohol) 36 (lined in red) through the membrane 20 and / or air through the membrane 20 to remove substantially all of the remaining ethanol from the membrane.

[0027] Fig. 7A1 is a simplified schematic diagram of the microfluidic system 10, which shows the fourth step of using the device 12, in which the patient or other user presses the elution bubble 24, which pushes the elution buffer 26 through the membrane 20 to elute the captured DNA and RNA from the membrane 20 and into the capture reservoir 28 (where the eluate with the captured DNA and RNA is underlined in blue). The fluid flow is controlled by such one-way valves 38, 38 (shown as arrows underlined in red): (i) fluid communication between the inlet port 14 and the inlet side of the membrane 20; and (ii) fluid communication between the outlet side of the membrane 20 and the capture reservoir 28. The valves 38, 38 prevent liquid from flowing back into the membrane 20 from the capture reservoir 28 and / or the reaction chambers 32, 32, and prevent liquid and / or other substances from flowing back through the inlet port 14 and into the transport / collection device 18A, 18B, 18C and / or 18D. Once the captured RNA and DNA are in the capture reservoir 28, the reaction chambers 32, 32 are filled by capillary action via capillary conduits 30, 30 extending in fluid communication between the capture reservoir 28 and the respective reaction chambers 32, 32 in which the eluent is dissolved as lyophilized beads pre-stored in the reaction chambers, and the negative control chamber 40. Pressing the elution bubble 24 also activates the device heating element 42 in thermal communication with the reaction chambers 32, 32 and the negative control chamber 40 for heating the reaction chambers to the temperature required for the reaction.

[0028] Figure 7B yes Fig. 7A A cross-sectional view of the syringe barrel of the device showing the collected waste dodecanol 36 (strike-through in red) on top of the waste sample mixture 16 (strike-through in green) within the barrel of the syringe 22.

[0029] Figure 8 yes Figure 21 is a simplified schematic diagram of a microfluidic system 10, which shows the fourth step of using the device 12, in which the patient or other user presses or otherwise actuates the elution syringe 24', which pushes the elution buffer 26 through the membrane 20 to elute the captured DNA and RNA from the membrane and into the capture reservoir 28 (where the elution buffer with the captured DNA and RNA is underlined in blue). Fluid flow is controlled by such one-way valves 38, 38 (shown as arrows underlined in red): (i) fluid communication between the inlet port 14 and the inlet side of the membrane 20; and (ii) fluid communication between the outlet side of the membrane 20 and the capture reservoir 28. The valves 38, 38 prevent fluid from flowing back into the membrane 20 from the capture reservoir 28 and / or the reaction chambers 32, 32, and prevent any material from flowing back into the transport / collection device 18A, 18B, 18C and / or 18D through the inlet port 14. Once the RNA and DNA are in the capture reservoir 28, the reaction chambers 32, 32 and the negative control chamber 40 are filled by capillary action via the capillary conduits 30, 30, which dissolves the reaction chemicals pre-stored as lyophilized beads in the reaction chambers. Figure 8 In the embodiment, the pre-stored reaction chemicals are shown as circles in each empty reaction chamber 32, 32, and in the Fig. 9 In the embodiment of the present invention, the reaction chemicals are not shown because at this stage, the reaction chemicals are dissolved in the elution buffer along with the captured DNA and RNA. Pressing the plunger of the elution syringe 24' also activates the heating element 42 of the device 10 for heating the reaction chambers 32, 32 and the negative control chamber 40 to the temperature required for the reaction.

[0030] Fig. 9 1 is a simplified schematic diagram of the microfluidic system 10, which shows the fifth step of using the device 12, wherein 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 / translucent windows 44, 44 of the reaction chambers 32, 32. For example, Fig. 9 As shown in the figure, the positive amplification in each reaction chamber 32, 32 is marked green and can be seen through the transparent / translucent windows 44, 44 of the reaction chambers 32, 32, while the negative control in the negative control chamber 40 is marked blue and can be seen through the transparent / translucent window 44 of the negative control chamber. As can be recognized by a person of ordinary skill in the relevant art based on the teachings herein, positive amplification can be detected in any of a number of different ways that are currently known or become known later, such as by fluorescence, turbidity changes, or by digital image capture via an embedded CCD camera or other sensor or an external device (such as a smart phone).

[0031] Fig.10 is a perspective view of another apparatus embodying the present invention and generally indicated by reference numeral 112A for use in sample collection, RNA / DNA separation and purification.

[0032] Fig.11 yes Fig.10 A side view of the device with its lower manifold 152 shown in phantom to illustrate its interior.

[0033] Fig.12 1 is a top plan view of a multiplex amplification chip or device 112B that can be used with the above-mentioned device 112A to amplify RNA / DNA separated and purified in the device 112A and to detect target RNA / DNA therein.

[0034] Fig.13A yes Fig.12 Another top plan view of the multiplex amplification device 112B is shown showing a single vent membrane assembly 149 in cross-hatched form, which covers a plurality of vent ports 148, 148 in fluid communication with respective reaction chambers 132, 132 and negative control chamber 140 for venting the chambers therethrough.

[0035] Fig. 13B yes Fig.12 Another top plan view of the multiplex amplification device 112B is shown with a one-piece optically transparent material 144 in a cross-hatched form covering each reaction chamber 132, 132 and negative control chamber 140, thereby forming a transparent / translucent observation window above each reaction chamber or negative control chamber for observing the interior of each chamber therethrough.

[0036] Fig.14 yes Fig.12 A cross-sectional view of the multiplex amplification device 112B before heating and initiating the amplification reaction.

[0037] Fig.15 yes Fig.12 Another cross-sectional view of the multiplex amplification device 112B prior to heating and initiating an amplification reaction, and showing lyo-beads or other reaction chemicals pre-stored in one of the reaction chambers 132, 132, and a wax pellet 172 located in a corresponding fluid conduit 130 that is fluidly connected between the eluent reservoir 128 and the corresponding reaction chamber 132 to prevent fluid communication therethrough prior to heating for the amplification reaction.

[0038] Fig.16 yes Fig.12A cross-sectional view of a multiplex amplification device 112B is shown showing the device after amplification when the lyo-beads or other reaction chemicals are dissolved and the wax pellets are melted, and includes a heater 142 (e.g., an electric heater or a chemical heater) thermally connected to the reaction chambers 132, 132 and an excitation light emitting diode ("LED") 174 or other light source that causes the positive indicator to fluoresce such that when observed by the operator's eye 176, the fluorescence indicates positive detection of the target DNA / RNA.

[0039] Fig.17 yes Fig.12 A cross-sectional view of another version of the multiplex amplification device 112B showing the device after amplification when the lyo-beads or other reaction chemicals are dissolved and the wax pellets are melted, and including an optical sensor 174 configured to measure the emission wavelength and intensity (e.g., color and / or power) within the reaction chambers 132, 132 to sense positive detection of the target DNA / RNA and transmit a signal to, for example, a smartphone or other display (not shown) to indicate it. DETAILED DESCRIPTION

[0040] In a currently preferred embodiment, the sample collection preparation 16 is provided in the form of a mouthwash or nasal spray or rinse. The sample collection preparation 16 comprises the following components:

[0041] 1) Non-toxic chaotropic agents for cell lysis, used alone or in combination. Such agents include, but are not limited to: urea for artificial saliva; sodium lauryl sulfate for toothpaste; and / or ammonium trichloroacetate for treating lesions on the skin and mucous membranes. The amount of urea 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. The amount of sodium lauryl sulfate is preferably in the range of about 0.1% to about 3% w / v, more preferably in the range of about 0.2% to about 1.5% w / v, and even more preferably in the range of about 0.4% to about 0.7% w / v. The amount of ammonium trichloroacetate is preferably in the range of about 2% to about 40% w / v, more preferably in the range of about 4% to about 20% w / v, and even more preferably in the range of about 8% to about 10% w / v. As can be recognized by a person skilled in the art based on the teachings herein, some applications may not require cell lysis, for example in the presence of free-floating nucleic acids. However, such applications may still require non-toxic chaotropic agents to chaotropic cells and promote nucleic acid binding to solid membranes or other nucleic acid capture devices.

[0042] 2) Ethanol for facilitating the 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.

[0043] 3) Colorants and / or flavoring agents.

[0044] The following is a representative recipe:

[0045] 1) about 20% w / v ethanol;

[0046] 2) about 0.1% w / v sodium lauryl sulfate;

[0047] 3) about 20% w / v ammonium trichloroacetate;

[0048] 4) about 0.042% w / v menthol; and

[0049] 5) Water.

[0050] The device and method can also use long-chain fatty alcohol detergents. Ethanol is an inhibitor of many amplification reactions. In the system disclosed herein, ethanol is used to bind nucleic acids to the surface of glass or solid membrane 20. When nucleic acids are eluted from the glass or solid membrane surface, the remaining ethanol on the glass or solid membrane surface or in the fluid channel can be carried to the reaction chamber 32, 32. To solve this problem, transparent and hydrophobic long-chain (>4) fatty alcohols (such as 2-dodecanol) can be used to wash the fluid channel and glass surface. This provides several advantages: a) fatty alcohol replaces and dissolves the remaining ethanol in the system; b) the remaining fatty alcohol does not inhibit the amplification reaction or the visualization produced; and c) the remaining fatty alcohol can be used to provide a barrier to prevent water evaporation in the reaction solution. Other long-chain fatty alcohols that can be used include, but are not limited to, the following (used alone or in any combination):

[0051] 1) dodecanol;

[0052] 2) Octanol;

[0053] 3) stearyl alcohol;

[0054] 4) Lauryl alcohol;

[0055] 5) Cetyl alcohol;

[0056] 6) oleyl alcohol; and

[0057] 7) Butanol.

[0058] There are several options for using the above formulations as a mouthwash or nasal spray or wash, including the following:

[0059] Option 1: Apply a mouthwash / nasal spray or detergent to a gauze or other absorbent material pad for swabbing the mouth or nose. The swab is then compressed by syringe 18A in the inlet 14 of the microfluidic device 12 to release the material therefrom and into the device. Syringe 18A may also contain 2-dodecanol 36 (about 50 to about 500 μl (or other long chain fatty alcohols, as shown above)) for improved assay performance. 2-dodecanol or other long chain fatty alcohol 36 is located in the chamber of syringe 18A so that it is introduced into the device 12 after the saliva is mixed with the mouthwash / nasal spray.

[0060] Option 2 (Saliva Only): The user swishes the mouthwash in the mouth and then spits the mouthwash along with saliva into the microfluidic device with the help of a funnel 18B or similar device.

[0061] Option 3 (Saliva Only): The user swishes the mouthwash in the mouth and then spits the mouthwash along with the saliva into a second collection container or cup 18C preloaded with additional chaotropes and / or ethanol 46, which are released before, during, or after the cup is sealed. For example, the collection container 18C may include a chamber with a frangible or breakable wall containing additional chaotropes and / or ethanol 46. The container closure may include a piercing member so that when the container 18C is closed with the closure, the piercing member breaks the wall, thereby allowing the mouthwash and saliva to mix with the additional chaotropes and / or ethanol 46 in the container. The user then agitates cup 18C (e.g., by shaking it) so that the lysate and chaotropic sample mixture is contained within the container, and then introduces the mixture from the container into inlet 14 of microfluidic device 12 via, for example, another transfer device (e.g., syringe 18A), which may also contain 2-dodecanol (about 50 to about 500 μl (or other long-chain fatty alcohols)) 36 for improving assay performance. 2-dodecanol 36 may be located in the chamber of syringe 18A so that it is introduced into device 12 after the saliva-containing mixture.

[0062] Usually Figure 3 As shown in , the following are the main or key components of the device 10. The following components are located outside the microfluidic device 12:

[0063] 1) a sample collection preparation or solution 16 (e.g., a mouthwash / nasal spray or a detergent);

[0064] 2) a collection device (e.g., a collection cup or container 18C, or a swab at the distal end of the syringe 18A that absorbs saliva therein and releases the saliva when compressed after the syringe is actuated); and

[0065] 3) A transfer device (eg, syringe 18A or funnel 18B).

[0066] The following components are located on or within the microfluidic device 12:

[0067] 1) Tubes / channels 18D connecting the microfluidic device 12 to the transfer devices 18A, 18B, and / or 18C;

[0068] 2) DNA / RNA capture membrane (or solid-state membrane) 20. In the illustrative embodiment of the solid-state 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.

[0069] 3) a syringe 22 which "pulls" material from the transfer device / inlet port 14, through the solid membrane 20, and into the syringe 22 (defining a waste chamber therein);

[0070] 4) one-way valves 38, 38, which control the flow of fluid into and through the solid membrane 20 and from the solid membrane 20 to the waste chamber or reservoir 28 in the syringe 22 for capillary transfer to the reaction chambers 32, 32;

[0071] 5) A blister machine 24 or syringe 24' or other pump / dispensing device, pre-loaded with elution buffer 26 (e.g., water). Although standard pull-type syringes 22, 24' are shown, each syringe may be a reverse pressure syringe or other type of syringe or similar pump / dispensing device now known or later becoming known.

[0072] 6) a reservoir 28 for combining the eluate 26 before loading it into the reaction chambers 32, 32, such that the fluid flows from the solid membrane 20 into the reservoir 28 and combines within the reservoir before the eluate flows into the reaction chambers 32, 32. One advantage of the reservoir 28 is that it can be used to combine purified RNA / DNA to facilitate obtaining substantially consistent target concentrations, including, for example, across multiple reaction chambers;

[0073] 7) capillaries or conduits 30, 30, which transfer eluent from reservoir 28 to reaction chambers 32, 32 and negative control chamber 40. One advantage of using capillaries is that they can provide capillary filling to the reaction chambers, which in turn facilitates bubble management;

[0074] 8) reaction chambers 32, 32 preloaded with lyophilized amplification reagents, and negative control chamber 40;

[0075] 9) a heater 42 for amplification reaction; and

[0076] 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 illustrative embodiment, a plurality of 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.

[0077] In operation of the microfluidic system 10, and with reference to Figure 1A , 1B and 2, using option 18A, 18B or 18C, introducing the sample-containing mixture 16 into the device 12, and encapsulating or containing an elution buffer 26 (e.g., water) in an elution bubble 24 ( Figure 1A ) or pre-filled elution syringe 24'( Figure 2 ) in. Three options for collecting and introducing samples into the device 12 are shown. With option 18A, a collection and transfer syringe is used. If dodecanol 36 is needed, the dodecanol is contained in the sealed portion of the syringe 18A, and once the sample is introduced by a lockable or locking machine between the syringe and the introduction tube 18D (e.g., a flexible tube), the dodecanol is introduced into the device 12. With option 18B, the funnel is connected to the device 12 by a lockable or locking flexible tube 18D. As can be seen, dodecanol is not used in this specific embodiment. The funnel 18B is preferably directly connected to the introduction tube 18D by a locking machine of a type known to ordinary technicians in the relevant field. With option 18C, if dodecanol is needed, the chamber within the sample collection container 18C contains and releases dodecanol 36 when the container is closed. If desired, the mixed contents in the sample collection chamber can be "pulled" into the device by the syringe 18A. The connection between the collection cup 18C and the inlet port 14 of the device 12 is a flexible tube 18D having a lockable or locking connection 50 ( Figure 1B ).

[0078] like Figure 4 As shown in , the first step of using the device 12 includes a patient or other user collecting a biological sample 16 and introducing the sample mixture 16 into the microfluidic device 12 according to any of the above options 18A, 18B or 18C. Figure 5 In a second step, as shown by the arrow in , the patient or other user pulls the plunger of the empty syringe 22, which in turn pulls the sample mixture 16 through the capture membrane 20 and into the syringe barrel which serves as a waste container. Figure 6, in the third step, the patient or other user continues to pull the plunger of syringe 22 until the plunger hits a stopper in the syringe, which in turn pulls dodecanol 36 through membrane 20 to remove all remaining ethanol. In another embodiment, dodecanal is not required, and in the third step, syringe 22 pulls air through membrane 20, such that the remaining air (e.g., remaining air in syringe 18A, funnel 18B, or collection cup 18C) excludes the ethanol.

[0079] As shown in FIG. 7 , in a fourth step, the patient or other user presses the elution bubble 24, which pushes the elution buffer 26 through the membrane 20 to elute all the DNA and RNA from the membrane and into the capture reservoir 28. The fluid flow is controlled by one-way valves 38, 38, which prevent fluid from flowing back from the reaction chambers 32, 32 into the membrane 20 and prevent any material from flowing back into the transfer / collection devices 18A, 18B, 18C and / or 18D. Once the RNA and DNA are in the capture reservoir 28, the reaction chambers 32, 32 are filled by capillary action, which dissolves the reaction chemicals pre-stored in the reaction chambers as lyophilized beads. Pressing the elution bubble also activates the device heating element 42, which is used to heat the reaction chambers 32, 32 to the temperature required for the reaction. As Figure 8 As shown in FIG. 1 , in an alternative fourth step of the device 10, the patient or other user depresses the plunger of the elution syringe 24', which pushes the elution buffer 26 through the membrane 20 to elute all the DNA and RNA from the membrane and into the capture reservoir 28. The fluid flow is controlled by one-way valves 38, 38, which prevent fluid from flowing back into the membrane 20 from the reaction chambers 32, 32 and prevent any material from flowing back into the transport / collection devices 18A, 18B, 18C and / or 18D. Once the RNA and DNA are in the capture reservoir 28, the reaction chambers 32, 32 are filled by capillary action, which dissolves the reaction chemicals pre-stored in the reaction chambers as lyophilized beads. Depressing the elution syringe 24' also activates the device heater 42.

[0080] like Figure 5 As shown in FIG. 1 , in the fifth step of the apparatus 10 , heating the reaction chambers 32 , 32 initiates an amplification reaction in which the 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 as shown), or by digital image capture via an embedded CCD camera, a light sensor, or an external device (e.g., a smartphone). Although the heater 42 is an electric heater in the illustrated apparatus, chemical heating or other types of heating machines currently known or later becoming known may be equally employed.

[0081] exist Fig.10 and 11In the present invention, another device for sample collection and nucleic acid or RNA / DNA separation and purification is generally represented by the reference numeral 112A. The device 112A is similar to the corresponding parts of the device 12 described above in conjunction with Figures 1 to 9, and therefore similar reference numerals beginning with the number "1" are used to represent similar elements. The device 112A utilizes a manifold component containing solid purification media and fluid channels, and serves as a structural chassis for a series of manually actuated valves, fluid reservoirs, and fluid-actuated syringes, which draw samples and reagents from the fluid reservoirs, through the membranes and into the waste and elution syringes. The elution fluid is then transferred to the amplification device for thermal incubation, as described above in conjunction with Fig. 9 or the following combination Figures 12 to 17 described.

[0082] The device 112A includes a waste injector 122, which is coupled in fluid communication with the outlet side of the solid state purification membrane 120 through a waste suction valve 138 and a lower manifold 152. Fig.11 As shown in the figure, the lower manifold 152 defines a fluid channel 154 therein, which extends in fluid communication between the outlet of the solid membrane 120 and the waste valve 138. The elution syringe 124 is connected in fluid communication with the fluid channel 154 through the elution valve 138. The upper manifold 156 is mounted on the lower manifold 152 on the inlet side of the solid membrane 120, and defines a fluid channel (not shown) therein that is in fluid communication with the inlet side of the solid membrane.

[0083] like Fig.10As shown in , a plurality of reservoirs and associated valves are installed on the top of the upper manifold 156. The reservoir 138 containing the sample mixture is installed on the upper manifold 156 and is connected to the upper manifold fluid by a corresponding sample valve 138. The first wash reservoir 158 is installed on the upper manifold 156 adjacent to the sample reservoir 138 and is connected to the upper manifold fluid by a corresponding first wash valve 138. The second wash reservoir 160 is installed on the upper manifold 156 adjacent to the first wash reservoir 158 and is connected to the upper manifold fluid by a corresponding second wash valve 138. The eluent reservoir 162 is installed on the upper manifold 156 adjacent to the second wash reservoir 160 and is connected to the upper manifold fluid by a corresponding elution valve 138. In an illustrative embodiment, each reservoir is defined by a corresponding syringe, wherein the reservoir is defined in the barrel of the syringe. Each syringe reservoir can include a Luer Lock or other type of connector at its distal end to releasably connect the syringe to the corresponding valve or manifold and disconnect the syringe from the corresponding valve or manifold. As can be recognized by those of ordinary skill in the relevant art based on the teachings herein, the reservoir can take the form of any of the many different devices currently known or later known for storing, pumping, distributing and / or otherwise releasing the corresponding liquid therein, such as a depressible bubble cap or an electrically actuated liquid dispenser with a frangible wall. In addition, in an illustrative embodiment, valves 138, 138 are manually operable valves, wherein each valve includes a corresponding knob, and the knob is manually turned or rotated to move the valve between the closed position of the valve that prevents liquid from flowing through and the open position of the valve that allows liquid to flow through. As recognized by those of ordinary skill in the relevant art, valves 138, 138 can take the form of any of a variety of different types of valves currently known or later known to be operated or actuated in any of a variety of different ways, including electric or electronic operation or actuation valves.

[0084] In the operation of the device 112A, the sample mixture is loaded into the sample reservoir 118. The waste aspiration valve 138 is then opened, and the sample reservoir valve 138 is opened to connect the sample reservoir to the inlet side fluid of the solid membrane 120, and the waste syringe 122 is connected to the outlet side fluid of the solid membrane 120. Then, the user retracts the plunger of the waste aspiration syringe 122 to a first predetermined amount to pull or aspirate the sample mixture from the sample reservoir 118 through the upper manifold 156 and through the solid membrane 120. In an illustrative embodiment, the waste aspiration syringe 122 includes a scale or similar indication, and the predetermined amount is determined based on the movement of the plunger relative to the scale or other indication. However, as can be recognized by a person of ordinary skill in the relevant art based on the teachings herein, the predetermined amount can be determined or dispensed according to any of many different ways with any of many different machines currently known or later becoming known, such as by electrical or electronic metering. In one embodiment, such an amount is equal to about 5 ml. After the sample mixture is drawn from the sample reservoir 118, the sample reservoir valve 138 is closed. Next, the first wash reservoir valve 138 is opened to connect the first wash reservoir 158 to the inlet side fluid of the solid membrane 120. Then, the user retracts the plunger of the waste aspiration syringe 122 to a second predetermined amount to pull or aspirate the first detergent from the first wash reservoir through the upper manifold 156 and through the solid membrane 120. In the illustrative embodiment, the second predetermined amount is about 5 ml. The user closes the first wash reservoir valve 138 and opens the second wash reservoir valve 138 to connect the second wash reservoir 160 to the inlet side fluid of the solid membrane 120. Then, the user retracts the plunger of the waste aspiration syringe 122 to a third predetermined amount to pull or aspirate the second detergent from the second wash reservoir through the upper manifold 156 and through the solid membrane 120. In the illustrative embodiment, the third predetermined amount is about 10 ml. The user closes the second wash reservoir valve 138 and closes the waste suction valve 138 to disconnect the waste suction syringe 122 from the solid-state membrane. Then, the user opens the elution valve 138 on the eluent reservoir 162 to connect the eluent reservoir to the inlet side fluid of the solid-state membrane 120, and opens the elution suction valve 138 on the elution suction syringe 124 to connect the elution suction syringe to the outlet side fluid of the solid-state membrane 120. Then, the user pulls the plunger of the elution suction syringe 124 to pull or aspirate the eluent from the eluent reservoir 162, passing it through the solid-state membrane to elute the RNA and DNA therein, and capture the eluted RNA and DNA in the barrel of the elution suction syringe 124. The elution suction syringe 124 can be disconnected from its fixing device on the lower manifold 152, for example, by a Luer connector, to transfer the eluent therein to the amplification device for use in the reaction chamber in the above combination. Fig. 9 Thermal incubation and amplification were performed as described.

[0085] exist Figures 12 to 17 In the embodiment of the present invention, another device embodying the present invention is generally indicated by the reference numeral 112B. The device 112B can be used in combination with the above-mentioned device 112A. As described above, the device 112A is used for sample collection, and nucleic acid or RNA / DNA separation and purification. On the other hand, the device 112B is used for amplification and detection of targets from, for example, the device 112A through separation and purification of nucleic acids or RNA / DNA. The device 112B is similar to the corresponding parts of the device 12 described above in conjunction with Figures 1 to 9, and therefore similar reference numerals beginning with the number "1" are used to represent similar elements.

[0086] like Fig.12 As shown in , the device 112B includes a fluid inlet 164 in fluid communication with a reservoir or premixing chamber 128, which in turn is connected in fluid communication with a plurality of reaction / amplification chambers 132, 132 through respective capillary conduits 130, 130. Each reaction / amplification chamber 132, 132 includes a respective reaction / amplification chamber vent 148, 148 in fluid communication therewith for venting the respective chamber to the ambient atmosphere. Fig.13A In the embodiment, a single vent membrane assembly 148 covers a plurality of vent ports 148, 148 in fluid communication with the corresponding 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 blocks or substantially blocks liquids from flowing therethrough.

[0087] like Fig. 13B As shown in , a one-piece optically transparent material 144 covers each reaction chamber 132, 132 and negative control chamber 140, thereby forming a transparent / translucent observation window above each reaction chamber or negative control chamber for observing the interior of each chamber therethrough. The transparent or translucent window 144 is formed of an optically transparent material of a type known to those of ordinary skill in the relevant art, which covers each reaction / amplification chamber 132, 132 and negative control chamber 140 to allow the interior of the chamber to be observed therethrough. As can be appreciated by those of ordinary skill in the relevant art based on the teachings herein, the device 112B may include a single vent membrane assembly 148 and a single observation window assembly 144 for all reaction chambers 132, 132 as shown, or may include any desired number of such assemblies.

[0088] like Fig.14 and 15 As shown in FIG. 1 , the fluid inlet 164 is in fluid communication with an inlet check valve 166, which in turn is connected to the reservoir / premix chamber 128 via a fluid flow path 167. The fluid inlet 164 is configured to receive the distal end of the elution syringe reservoir 162 for receiving eluent therefrom, as described above in conjunction with Fig.10The reservoir / premix chamber 128 includes a vent 129 for ventilating the chamber during its filling. Fig.14 As shown in , the pressure relief valve 148 is coupled in fluid communication between the reaction / premixing chamber 128 and the plurality of capillary conduits 130, 130, which in turn are connected in fluid communication with the respective reaction / amplification chambers 132, 132. Fig.15 As shown in FIG. 1 , each capillary conduit 130 contains a respective wax or other meltable pellet 172 therein that is configured to prevent liquid from being transferred from the reservoir / premixing chamber 128 through the conduit at room temperature (or a temperature below the incubation temperature). The wax pellet 172 is configured so that it cannot melt at room temperature, but rather melts at a temperature that approaches or reaches the incubation temperature of the amplification reaction. One advantage of this feature is that it gives the solution more time to homogenize and more uniformly distribute the DNA / RNA to each reaction chamber 132, 132. When the premixing chamber 128 is heated by the heater 142 ( Fig.16 and 17 ), the diffusion rate increases and thus the contents are mixed. Time and temperature (i.e., at or near the incubation temperature) promote such mixing. It should be noted that when the wax 172 melts, capillary action draws the solution into the conduit 130, 130 and the reaction chamber 132, 132, and the vent 129 allows air to return to the premixing chamber 128 to backfill the volume inside the premixing chamber when the fluid is discharged from the premixing chamber. Each reaction / amplification chamber 132, 132 preferably contains a freeze-dried master mix bead or reagent mixture 168 therein, and the corresponding portion of the transparent or translucent observation window 144 covers each such chamber and the negative control chamber 140 for visually observing the interior of each chamber through it after reaction / amplification. The ventilation channel 170 extends between the upper end or region of each reaction / amplification chamber 132, 132 and the corresponding reaction / amplification chamber vent 148 for ventilating the chamber through it.

[0089] In the operation of the device 112B, once the reservoir / premixing chamber 128 is filled with the elution fluid 126, the check valve 166 on the fluid inlet 164 is passively closed. After the elution fluid 126 enters the device 112B, as air escapes from the reservoir / premixing chamber vent 148, the reservoir / premixing chamber 128 is completely filled, completing the premixing process and resulting in a substantially uniform concentration of DNA / RNA throughout the fluid volume in the reservoir / premixing chamber. The fluid pressure in the reservoir / premixing chamber 128 increases as fluid is added thereto. Once the reservoir / premixing chamber 128 is filled (or reaches a predetermined filling level), the pressure in the reservoir / premixing chamber 128 exceeds the release pressure of the premixing chamber pressure relief valve 148, which in turn allows the substantially homogeneous liquid in the reservoir / premixing 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, a substantially homogeneous liquid dissolves the reagent master mix 168, while air is exhausted through the corresponding reaction / amplification chamber vents 148, 148.

[0090] The device 112B is then incubated by the heating unit or element 142, inducing amplification of the target RNA / DNA in the reaction / amplification chamber. If desired, each reaction / amplification chamber 132, 132 may contain a unique master mix 168 for amplification of a different target, if desired. Fig.16As shown in , a heater 142 (which may be, for example, an electric heater or a chemical heater) is thermally connected to the reaction chambers 132, 132 and the negative control chamber 140. After actuating the heater 142 to a predetermined incubation temperature, the lyo-beads or other reaction chemicals 168 are dissolved and the wax pellets 172 are melted. After incubating the filled reaction chambers 132, 132 at a predetermined incubation temperature and time period and causing RNA / DNA amplification, an LED or other light source 174 is excited to cause the positive indicator to fluoresce, so that when observed by the operator's eyes 176, the fluorescence indicates a positive detection of the target DNA / RNA. Calcein, also known as calcein (fluorexon) or fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495nm and 515nm, respectively. In biological applications, calcein is commonly used as a marker for detecting certain biological agents. In device 112B, calcein is a component in the lyo-bead master mix 168. In the lyo-bead format, calcein cannot emit an excitation wavelength (510 nm). During amplification / incubation, if the target molecule is present in the eluent in each reaction chamber 132, the calcein contained in the main mixture (i.e., in the lyo-bead) will undergo a chemical change, thereby allowing the emission of excitation light, so that the operator can observe fluorescence, indicating the presence of the target molecule in the sample, i.e., positive detection of the target. During incubation, when the target molecule is not present in the reaction chambers 132, 132, no chemical change is induced and the reaction will not emit light, indicating that the target molecule is not present in the sample, i.e., a negative result.

[0091] like Fig.17 As shown in Fig.16 In a human-readable embodiment, device 112B includes a light sensor 178 configured to output a digital binary signal indicating a positive or negative result to a user interface that communicates the result to the user. One advantage of this embodiment is that it is easy to use and suitable for use by unskilled operators, which may be necessary for some point-of-care (POC) applications. Fig.16 in Fig.17At the stage shown, the wax pellet 172 is melted. In addition, when the result is read, the lyo-bead 168 will have dissolved when the amplification occurs. The light sensor 178 measures the color and intensity of the emission wavelength in each reaction chamber 132 or multiple reaction chambers 132, 132. When the 515nm power rises above a predetermined setting threshold, the result is considered positive. On the other hand, if the 515nm power does not rise above a predetermined setting threshold, the result is considered negative. In either case, the light sensor 178 transmits a signal indicating a positive result or a negative result to a user interface (such as a display screen or a smart phone), and the user interface communicates the result to the user. As can be recognized by a person of ordinary skill in the relevant art based on the teachings herein, the device 112B may include a light sensor or other sensor for all chambers, or multiple sensors for multiple chambers, wherein each sensor senses the emission wavelength inside one or more chambers.

[0092] As can be recognized by a person of ordinary skill in the relevant art based on the teachings herein, many changes, improvements, modifications, additions and deletions may be made to the above-described and other embodiments of the present invention without departing from the scope of the present invention. For example, the microfluidic device or its components, and the operation or use method or aspects thereof may be the same or similar to any of the microfluidic devices or their components and methods or aspects disclosed in the following co-pending patent applications, which are assigned to the assignee of the present invention and are hereby incorporated by reference as part of the present disclosure in their entirety: (i) U.S. patent application serial No. 17 / 647,828, entitled “Device And Method For Detecting Nucleic Acids In Biological Samples”, filed on January 12, 2022, and (ii) U.S. patent application serial No. 17 / 941,816, entitled “Device And Method For Detecting Nucleic Acids In Biological Samples.”, filed on September 9, 2022. In addition, the device may include fewer components or additional components than shown and / or described herein. For example, the device may include only one pump for pumping the sample mixture and dodecanol and air, any washing solution and any eluent through one or more solid membranes. Alternatively, the device may include multiple pumps for performing such functions. Alternatively, the device may include multiple solid membranes or other filtering machines, including membranes installed in series. In addition, the device may include multiple capture reservoirs, or in other cases, the capture reservoir may be cancelled. In other embodiments, the conduit running between the capture reservoir and the reaction chamber and / or the negative control chamber does not need to be a capillary or operated by capillary flow. For example, flowing through the conduit can be achieved by pressure difference (e.g., by pumping to fill the capture reservoir). In addition, the heating element needs to be operated in the case of on / off, but can be operated by heat or thermal cycling, such as for PCR or other non-lamp methods / applications. The solid membrane can also be in the form of any device currently known or later known for capturing and releasing nucleic acids, RNA and / or DNA thereon, such as glass beads, including, for example, borosilicate glass beads. Therefore, the components of the device and the operation or method of using the device may take any of many different forms or configurations, and may be made of or use any of many materials currently known or later becoming known, and features or aspects may be added or removed, without departing from the scope of the invention. Therefore, the detailed description of the embodiments should be regarded as illustrative rather than restrictive.

Claims

1. A device comprising: a sample port or chamber for receiving a sample-containing mixture therein, the sample-containing mixture comprising a biological sample therein; a solid-state membrane in fluid communication with the sample port or chamber and configured to receive the sample-containing mixture therefrom and allow the sample-containing mixture to pass through the membrane and capture nucleic acids in the biological sample on the membrane; a first pump in fluid communication with the solid state membrane and a waste chamber in fluid communication with the pump, wherein actuation of the first pump causes the sample-containing mixture to flow through the solid state membrane and into the waste chamber; an eluent chamber configured to receive an eluent therein or containing an eluent therein; an eluent reservoir in fluid communication with the solid state membrane; as well as A second pump is fluidly connected to the solid-state membrane and the eluent chamber, wherein actuation of the second pump causes the eluent to flow from the eluent chamber through the solid-state membrane, eluting the captured nucleic acids from the solid-state membrane and flowing into the eluent reservoir together with the captured nucleic acids.

2. The device of claim 1, wherein the first pump is a syringe comprising a barrel and a plunger housed in the barrel, the barrel defining the waste chamber therein, movement of the plunger drawing or pushing the sample-containing mixture through the solid membrane and into the waste chamber of the barrel.

3. The device of claim 2, wherein the second pump is movable between a non-actuated position and an actuated position, and the eluent chamber comprises a fragile or breakable wall which can be broken due to movement of the second pump between the non-actuated position and the actuated position to pump eluent from the eluent chamber through the solid membrane and into the eluent reservoir.

4. The device of claim 3, wherein the second pump is a plunger, and movement of the plunger from a non-actuated position to an actuated position causes the fragile or breakable wall of the eluent chamber to be destroyed and pushes the eluent through the solid membrane and into the eluent reservoir.

5. The device of claim 2, wherein the second pump is a syringe comprising a barrel and a plunger housed within the barrel, movement of the plunger pushing the eluent through the solid membrane and into the eluent reservoir.

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

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

8. The device of claim 1, further comprising at least one reaction chamber and at least one conduit in fluid communication between the eluent reservoir and the reaction chamber.

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

10. The device of claim 1, further comprising a plurality of reaction chambers and conduits, wherein the conduits are fluidically connected between the eluent reservoir and each reaction chamber, wherein each conduit is configured to allow the eluent having the captured nucleic acid to flow through the conduit and into the corresponding reaction chamber.

11. The device of claim 8, further comprising a valve in fluid communication between the reaction chamber and an ambient atmosphere, wherein the valve allows gas to flow from the reaction chamber into the ambient atmosphere but prevents liquid from flowing therethrough.

12. The device of claim 8, further comprising a heating element thermally connected to the reaction chamber, wherein the heating element defines a first condition and a second condition, wherein under the first condition the heating element heats the reaction chamber to an incubation temperature, and under the second condition the heating element does not heat the reaction chamber to the incubation temperature, and the heating element is configured to transition from the first condition to the second condition upon or after actuation of the second pump.

13. The device of claim 8, further comprising an optical sensor configured to measure at least one of the emission wavelength or intensity within the reaction chamber to detect at least one of a positive detection of the target nucleic acid or a negative detection of the target nucleic acid, and transmit a signal indicating it to a user interface.

14. An apparatus comprising: a first device for receiving therein a sample-containing mixture, the sample-containing mixture containing therein a biological sample; a second device in fluid communication with the first device for receiving the sample-containing mixture therefrom, allowing the sample-containing mixture to pass through the second device, and capturing nucleic acids in the biological sample on the second device; a third device for receiving and containing the sample-containing mixture after passing through the second device; a fourth device in fluid communication with the second device and the third device for pumping the sample-containing mixture through the second device and into the third device; a fifth device for containing an eluent therein and for allowing the eluent to flow through the second device after the sample-containing mixture passes through the second device, and removing nucleic acids captured from the biological sample from the second device with the eluent; a sixth device in fluid communication with the solid state membrane for receiving and collecting therein an eluate having nucleic acids captured from the biological sample; as well as The seventh device is fluidically connected to the second device and the fifth device, and is used to pump the eluent from the fifth device through the second device, elute the captured nucleic acid from the second device, and flow into the sixth device together with the captured nucleic acid.

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

16. A preparation for collecting biological samples of saliva or nasal fluid and capturing nucleic acids in the collected biological samples on a solid membrane, comprising: one or more non-toxic chaotropic agents; ethanol; and / or colorants and / or flavoring agents, wherein the preparation can be contained in the oral cavity or nasal cavity to collect biological samples of saliva or nasal fluid therefrom, and the one or more non-toxic chaotropic agents and ethanol perform at least one of lysing cells of the biological sample or promoting binding of nucleic acids in the biological sample to the solid membrane.

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

18. The formulation of claim 17, wherein the non-toxic chaotropic agent is selected from the group comprising the following alone or in any combination: (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. The formulation of claim 16 in combination with a long chain fatty alcohol detergent configured to flow through the solid membrane after the formulation to substantially eliminate any remaining ethanol of the formulation on the solid membrane.

20. The combination of claim 19, further comprising a transfer device, the transfer device containing the formulation in a first portion thereof and the long-chain fatty alcohol detergent in a second portion thereof, wherein the first portion thereof is receivable in an oral cavity or a nasal cavity for collecting saliva or nasal fluid, and the transfer device is configured to transfer or dispense the formulation and the collected biological sample to the solid membrane, and subsequently transfer or dispense the long-chain fatty alcohol detergent to the solid membrane.

21. The combination of claim 20, wherein the transfer device is a syringe comprising gauze or other absorbent material at its distal end, the gauze or other absorbent material being receivable in the oral or nasal cavity and configured to absorb saliva or nasal fluid therein, wherein the syringe defines a first chamber or a first portion of a chamber and a second chamber or a second portion of a chamber, the first chamber or the first portion of the chamber being fluidically connected to the gauze or other absorbent material and containing the formulation therein, the second chamber or the second portion of the chamber being located on the opposite side of the first chamber or the first portion of the chamber relative to the gauze or other absorbent material and containing the long-chain fatty alcohol detergent therein, wherein actuation of the syringe causes the formulation to flow through the gauze or other absorbent material and collect the biological sample therein, and the mixture of the formulation and the biological sample is dispensed from the syringe, and further actuation of the syringe causes the long-chain fatty alcohol detergent to be dispensed from the syringe after the mixture of the formulation and the biological sample is dispensed.

22. The combination of claim 21, wherein the syringe comprises a barrel defining the first chamber and the second chamber, and comprises a frangible or breakable wall separating the first chamber and the second chamber, and the syringe further comprises a plunger housed within the barrel, actuation of which subsequently ruptures the frangible or breakable wall and dispenses the formulation and subsequently the long-chain fatty alcohol detergent from the syringe.

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