Microfluidic devices and related methods

Abstract

To provide microfluidic devices that facilitate rapid detection of target nucleic acids present in influenza and / or RSV viruses.SOLUTION: A microfluidic device 100 includes an inlet port configured to receive a sample, a first reaction chamber fluidically coupled to the inlet port, a first pump fluidically coupled to the inlet port, a second pump fluidically coupled to a mixing chamber, a metering channel fluidically coupled to the first reaction chamber and to the mixing chamber, and one or more second reaction chambers fluidically coupled to the mixing chamber. The first pump is configured to move fluid from the inlet port to the first reaction chamber and from the first pump to the inlet port. The second pump is configured to move fluid from the second pump to the mixing chamber, from the first reaction chamber to the mixing chamber, and from the mixing chamber to the one or more second reaction chambers.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 464,576, filed February 28, 2017, entitled "Microfluidic Devices and Related Methods," which is incorporated herein by reference in its entirety.

[0002] Government Interest Statement This invention was made with government support under HHSO100201400011C awarded by the U.S. Department of Health and Human Services. The government has certain rights in this invention.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates to microfluidic devices, and more particularly to microfluidic devices used to perform diagnostic assays. [Background technology]

[0004] Microfluidic devices are designed to precisely control fluid flow within geometrically constrained networks for many applications. In some instances, microfluidic devices are used to perform specific molecular diagnostic assays, such as assays based on isothermal nucleic acid amplification methods (e.g., Recombinase Polymerase Amplification (RPA) or Nicking and Extension Amplification Reaction (NEAR)), to detect trace levels of nucleic acids. In some cases, microfluidic devices can facilitate point-of-care (POC) testing, improving the availability and speed of diagnostic assays, such as assays capable of detecting influenza (Flu) and respiratory syncytial virus (RSV). For example, microfluidic devices may facilitate the rapid detection of target nucleic acids present in influenza and / or RSV viruses. Summary of the Invention [Means for solving the problem]

[0005] The microfluidic devices disclosed herein are designed to perform diagnostic assays capable of detecting one or more target nucleic acids (e.g., assays capable of detecting influenza (Flu) and respiratory syncytial virus (RSV)). For example, the microfluidic device includes a cartridge assembly, an elastomeric layer, and a lid. The cartridge assembly and elastomeric layer together provide a series of fluidly connected ports, channels, chambers, reservoirs, valves, and associated components capable of performing assays to detect the presence of one or more target nucleic acids in a sample provided to the microfluidic device. The lid covers the cartridge assembly and provides a layer within the cartridge assembly where one or more target nucleic acids can be detected.

[0006] The cartridge assembly includes a cartridge, a cap, a seal, a plug, a magnet, a first reaction pellet, and a second reaction pellet. The cartridge defines the shape of a microfluidic network and a sample chamber that provides an inlet port to the microfluidic network. The sample chamber is sized and shaped to accommodate a sample collection device (e.g., a cotton swab) and includes a relatively wide first portion and a relatively narrow second portion so that the tip of the cotton swab within the sample chamber is fully wetted when a liquid reagent is delivered to the sample chamber.

[0007] The cartridge also defines two onboard pumps operable to push or pull fluids into or from the microfluidic network. The cartridge defines a first reaction chamber primed with a first reactant pellet, a mixing chamber, and multiple second reaction chambers primed with respective second reactant pellets, enabling multiplexing within the microfluidic device. Mixing within the second reaction chambers can occur via one or both of mixing and acoustic microstreaming. Each second reaction chamber contains an identical air spring, which allows for equal distribution of fluid within the second reaction chamber, ensuring that fill levels between the second reaction chambers automatically equilibrate as a result of the backpressure generated when the second reaction chambers are filled with fluid. Therefore, the second reaction chambers can be filled with precisely equivalent volumes of fluid and achieve equivalent pressures.

[0008] Furthermore, the volume formed between the cartridge and the lid, external to the microfluidic network, provides a waste reservoir (e.g., an air reservoir) that buffers air pressure within the microfluidic network, eliminating the need for a separate pressure balancing mechanism in the microfluidic device. Cavities within the cartridge and corresponding regions of the elastomer layer located along the cavities cooperate to form valves at selected locations along the microfluidic network to control fluid flow. Compared to conventional devices used to perform similar analyses, the microfluidic device is configured to provide a closed system such that the risk of leakage contamination to the surrounding environment is significantly reduced. Due, at least in part, to the configuration of the microfluidic device, a Flu / RSV analysis can be performed using the microfluidic device in less than approximately 15 minutes.

[0009] For example, in some embodiments, a microfluidic device is provided herein, comprising: an inlet port configured to receive a sample; a first reaction chamber fluidly coupled to the inlet port; a first pump fluidly coupled to the inlet port; a second pump fluidly coupled to a mixing chamber; a metering channel fluidly coupled to the first reaction chamber and the mixing chamber; and one or more second reaction chambers fluidly coupled to the mixing chamber, wherein the first pump is configured to move fluid from the inlet port to the first reaction chamber and from the first pump to the inlet port; and the second pump is configured to move fluid from the second pump to the mixing chamber, from the first reaction chamber to the mixing chamber, and from the mixing chamber to the one or more second reaction chambers. In some embodiments, the microfluidic device further comprises a waste reservoir configured to regulate fluid pressure within the microfluidic device. In some embodiments, the first reaction chamber contains a first set of amplification reagents (e.g., recombinase polymerase amplification (RPA) reagents). In some embodiments, the RPA reagents are lyophilized. In some embodiments, the first reaction chamber further comprises a catalytic reagent (e.g., magnesium). In some embodiments, the first amplification reagent set comprises oligomers. In some embodiments, the mixing chamber comprises a second amplification reagent set (e.g., RPA reagents). In some embodiments, the RPA reagents are lyophilized. In some embodiments, one or more second reaction chambers each comprise a second amplification reagent set (e.g., RPA reagents). In some embodiments, the RPA reagents are lyophilized. In some embodiments, the second amplification reagent set comprises oligomers. In some embodiments, the first pump comprises a first buffer. In some embodiments, the first pump comprises a first buffer and a lysing agent. In some embodiments, the second pump comprises a second buffer. In some embodiments, the second pump comprises a second buffer and a lysing agent. In some embodiments, the first pump comprises a catalytic reagent. In some embodiments, the second pump comprises a catalytic reagent. In some embodiments, the catalytic reagent comprises magnesium.In some embodiments, each of the one or more second reaction chambers is a detection chamber. In some embodiments, a portion of each detection chamber is optically transparent. In some embodiments, the first reaction chamber is configured to be coupled to a heating unit. In some embodiments, the inlet port is configured to be coupled to a heating unit. In some embodiments, the first reaction chamber comprises or is coupled to a mixing means. In some embodiments, the mixing chamber includes or is coupled to a mixing means. In some embodiments, each of the one or more second reaction chambers includes or is coupled to a mixing means. In some embodiments, the mixing means is a magnet. In some embodiments, the mixing means operates by acoustic streaming. In some embodiments, the inlet port contains a sample, and the first pump contains a first buffer solution, and the first pump is configured to pump the first buffer solution from the first pump to the inlet port to generate a diluted sample comprising the reagents and the first buffer solution. In some embodiments, the first reaction chamber contains a first set of amplification reagents, and the first pump is configured to provide a portion of the diluted sample from the inlet port to the first reaction chamber to generate a first reaction mixture comprising the diluted sample and the first set of amplification reagents. In some embodiments, the second pump is configured to provide a portion of the first reaction mixture from the first reaction chamber to the mixing chamber via the metering channel. In some embodiments, the second pump includes a second buffer, and the second pump is configured to deliver the second buffer from the second pump to the mixing chamber via the metering channel, combining the second buffer with the portion of the first reaction mixture to form a diluted first reaction mixture. In some embodiments, each of the one or more second reaction chambers includes a second set of amplification reagents, and the second pump is configured to deliver a portion of the diluted first reaction mixture from the mixing chamber to each of the one or more second reaction chambers to form a second reaction mixture comprising the diluted first reaction mixture and the second set of amplification reagents. In some embodiments, the second set of amplification reagents includes oligomers.In some embodiments, the mixing chamber contains a second set of amplification reagents, the second reagent chamber contains a second buffer, and the second pump is configured to deliver the second buffer from the second reagent chamber to the mixing chamber through the metering channel, where the second buffer combines with a portion of the first reaction mixture and the second amplification reagent set to produce a second reaction mixture. In some embodiments, the second pump is configured to deliver a portion of the second reaction mixture to each of one or more second reaction chambers. In some embodiments, each of the one or more second reaction chambers contains an oligomer. In some embodiments, the microfluidic device includes two, three, four, five, six, seven, or eight second reaction chambers. In some embodiments, the microfluidic device further includes a series of valves. In some embodiments, the microfluidic device further includes alignment holes for connecting the microfluidic device to a reader configured to process the sample and deliver the sample to the microfluidic device. In some embodiments, the connection port is configured to lockably engage with the reader. In some embodiments, the microfluidic device is a disposable cartridge. In some embodiments, the first pump and the second pump are syringe pumps. In some embodiments, the inlet port includes a cap. In some embodiments, the cap includes a gasket including gasket sealing ribs. In some embodiments, the cap includes a detent feature to secure the cap in an open position.

[0010] Additional embodiments provide a reader configured to accept a microfluidic device described herein, the reader comprising a detector for detecting the presence of a second reaction product in one or more second reaction chambers. In some embodiments, the microfluidic device or reader includes a cap position detection component configured to detect cap closure or leakage. In some embodiments, the cap position detection component includes one or more components (e.g., an optical cap closure sensor and / or a pressure sensor). In some embodiments, the optical cap closure sensor includes a light beam that is interrupted when the cap is in a closed, sealed position. In some embodiments, the pressure sensor evaluates the cap's ability to withstand pressure. In some embodiments, the pressure is generated using a pump of the device. In some embodiments, a pressure outside a predetermined range indicates an unsealed cap. In some embodiments, the reader is configured to stop operation of the reader or microfluidic device if the cap is not identified as sealed.

[0011] Yet another embodiment is 1. A method comprising: providing a sample fluid containing a target nucleic acid, the target nucleic acid comprising at least one target polynucleotide sequence, to a microfluidic device; amplifying at least one target polynucleotide sequence under isothermal conditions; Including, Amplifying performing a first round of amplification on the target polynucleotide sequence to produce a first amplification product comprising a first amplified polynucleotide sequence; performing a second round of amplification on the first amplified polynucleotide sequence to produce a second amplification product comprising a second amplified polynucleotide sequence, the second amplified polynucleotide sequence comprising a smaller sequence entirely contained within the first amplified polynucleotide sequence produced during the first round of amplification; A method comprising: to provide. In some embodiments, the method further includes detecting a second amplification product. In some embodiments, detecting the second amplification product includes labeling the second amplification product with a first oligonucleotide linked to a fluorophore and a quencher to produce a labeled second product, cleaving the quencher from the labeled second amplification product, and optically detecting a signal from the fluorophore, wherein the detectable signal indicates the presence of the second amplification product. In some embodiments, cleavage of the quencher is performed using a nuclease. In some embodiments, the nuclease targets double-stranded DNA. In some embodiments, the nuclease is formamidopyrimine-DNA glycosylase. In some embodiments, the amplifying step includes performing a first round of amplification, wherein the amplification is RPA. In some embodiments, the amplifying step includes performing a second round of amplification, wherein the amplification is RPA. In some embodiments, the amplifying step includes performing a first round of amplification, wherein the amplification is RPA, and a second round of amplification, wherein the amplification is RPA. In some embodiments, the sample is blood, sputum, mucus, saliva, tears, or urine. In some embodiments, the method further comprises obtaining the sample from an animal. In some embodiments, the sample is obtained from an animal, and the animal is a human. In some embodiments, the target nucleic acid is a target nucleic acid of an animal pathogen. In some embodiments, the animal pathogen is a single-stranded DNA virus, a double-stranded DNA virus, or a single-stranded RNA virus. In some embodiments, the animal pathogen is a bacterium. In some embodiments, the target nucleic acid is double-stranded DNA, single-stranded DNA, or RNA. In some embodiments, the target nucleic acid is selected from, for example, genomic DNA, plasmid DNA, viral DNA, mitochondrial DNA, cDNA, synthetic double-stranded DNA, or synthetic single-stranded DNA. In some embodiments, the target nucleic acid is viral DNA or viral RNA. In some embodiments, the animal pathogen is influenza A virus, influenza B virus, or respiratory syncytial virus (RSV).In some embodiments, the target nucleic acid comprises two target polynucleotide sequences. In some embodiments, the target nucleic acid comprises three target polynucleotide sequences. In some embodiments, the method further comprises mixing the sample with an RPA reagent prior to providing the sample to the microfluidic device. In some embodiments, the second amplification product is detected less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes after providing the sample to the microfluidic device. In some embodiments, the second amplification product is detected in real time. In some embodiments, the method further comprises lysing the sample prior to amplification. In some embodiments, the lysing step comprises combining the sample with a lysing agent. In some embodiments, the lysing agent is an enzyme. In some embodiments, the lysing step comprises mechanical means. In some embodiments, the lysing step comprises heating the sample.

[0012] A further embodiment is 1. A method comprising: providing a sample containing a target nucleic acid, the target nucleic acid comprising at least one target polynucleotide sequence, to a microfluidic device; amplifying at least one target polynucleotide sequence, wherein amplifying comprises performing a first round of amplification on the target polynucleotide sequence to produce a first amplification product comprising a first amplified polynucleotide sequence; performing one or more additional successive amplifications of the first amplified polynucleotide sequence to form additional amplification products, wherein the amplification products from each successive n+1 amplifications comprise amplified polynucleotide sequences that are smaller sequences that are entirely contained within the amplified polynucleotide sequences generated during the previous n amplifications; performing a final round of amplification on the penultimate amplified polynucleotide sequence to produce a final amplification product; Detecting the final amplification product; A method comprising: to provide.

[0013] Other features and advantages will become apparent from the following detailed description, figures, and claims.

[0014] This patent or patent application publication contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the USPTO upon request and payment of the associated fee. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows an exploded view of the microfluidic device used to perform the analysis. [Figure 2] FIG. 2 shows a front view of the microfluidic device of FIG. [Figure 3] FIG. 2 shows a rear view of the microfluidic device of FIG. 1. [Figure 4] FIG. 2 shows a top view of the cartridge of the microfluidic device of FIG. 1. [Figure 5] FIG. 5 shows a bottom view of the cartridge of FIG. 4. [Figure 6] 5 shows a cross-sectional view of the pump chamber of the cartridge of FIG. 4. [Figure 7] 7 shows a cross-sectional view of the plug in the pump chamber of FIG. 6. [Figure 8] 5 shows a cross-sectional side view of the reaction chamber of the cartridge of FIG. 4. [Figure 9] 9 shows a cross-sectional perspective view of the reaction chamber of FIG. 8. [Figure 10] 9 shows a top view of the reaction chamber of FIG. 8. [Figure 11] 5 shows a top view of the valve along with the cartridge of FIG. 4. [Figure 12] 12 shows a perspective cross-sectional view of the valve of FIG. 11. [Figure 13] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 14]5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 15] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 16] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 17] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 18] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 19] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 20] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 21] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 22] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 23] 5 shows a series of schematic diagrams of the cartridge of FIG. 4 corresponding to a method of performing an analysis using the microfluidic device of FIG. 1. [Figure 24] 1 illustrates an exemplary microfluidic device. [Figure 25] 1 illustrates an exemplary cap design embodiment. [Figure 26] 1 illustrates an exemplary cap closure detection component. [Figure 27] 1 illustrates an exemplary lid design embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1-3 show exploded perspective, front (top), and rear (bottom) views, respectively, of a microfluidic device 100 for performing assays (e.g., multiplexed diagnostics for influenza (Flu) and respiratory syncytial virus (RSV) such as Flu / RSV assays). Microfluidic device 100 can be coupled to a reader that controls specific functions of microfluidic device 100 (e.g., valving, mixing, heating, pumping, and monitoring of such functions). Microfluidic device 100 includes a cartridge assembly 102, an elastomeric layer 104, and a lid 106. Together, cartridge assembly 102 and elastomeric layer 104 provide a series of fluidly connected ports, channels, chambers, reservoirs, valves, and associated components capable of performing assays to detect the presence of one or more target nucleic acids in a sample provided to microfluidic device 100. Lid 106 covers cartridge assembly 102 and provides a layer within cartridge assembly 102 where one or more target nucleic acids may be detected. The cartridge assembly 102 includes a cartridge 108 and several ancillary components (not shown in Figures 2 and 3), including a cap 110, a seal 112 (e.g., an O-ring seal), four plugs 114, a first magnet 116, an optional second magnet 118, a plurality (e.g., eight) of third magnets 120, a first reaction pellet 122, an optional intermediate pellet 123, and a plurality (e.g., eight) of second reaction pellets 124.

[0017] 4 and 5 show top and bottom views, respectively, of cartridge 108. Cartridge 108 defines the shape of a microfluidic network through which fluid flows during analysis. Cartridge 108 defines a sample chamber 126 that forms an opening 128. Sample chamber 126 provides an inlet port to the microfluidic network and is sized and shaped to accommodate various sample collection devices, such as swabs (e.g., cotton swabs) and collection tubes. For example, sample chamber 126 includes a relatively wide first portion 130 and a relatively narrow second portion 132. First portion 130 has a length of about 5 mm to about 20 mm (e.g., about 13 mm) and a relatively large inner diameter of about 8 mm to about 20 mm (e.g., about 16 mm), allowing first portion 130 to accommodate commonly used sample collection swabs or tubes. The first portion 130 narrows into a second portion 132 having a length of about 10 mm to about 30 mm (e.g., about 16 mm) and a relatively small inner diameter of about 6 mm to about 10 mm (e.g., about 8 mm) so that the tip of the swab is thoroughly wetted within the sample chamber 126 when liquid reagent is delivered to the sample chamber 126. The cap 110 and seal 112 of the cartridge assembly 102 are sized to be inserted into the opening 128 of the sample chamber 126 (e.g., so that the seal 112 surrounds the cap 110) to close and seal the sample chamber 126.

[0018] The cartridge 108 also defines a first pump chamber 134 and a second pump chamber 136. FIG. 6 shows a cross-sectional view of the first pump chamber 134 and the second pump chamber 136. A plug 114 is disposed within each end region of the first pump chamber 134 and the second pump chamber 136. FIG. 7 shows a cross-sectional view of one of the plugs 114 disposed away from the microfluidic network (i.e., the plug 114 on the left in FIG. 6). Each plug 114 defines an undercut 138 formed to engage (e.g., retain) a leader rod 140 such that the rod can exert a force (e.g., push or pull) on the plug 114 to push fluid in the pump chambers 134, 136 into the microfluidic network or draw fluid from the microfluidic network into the pump chambers 134, 136. The pump chambers 134, 136, together with the two plugs 114, respectively, form a first pump 135 and a second pump 137 (e.g., an on-board integrated syringe pump) that are actuated by the leader rod 140 to pump fluid flow within the microfluidic network.

[0019] In an initial closed state (a) of the first pump 135 and the second pump 137, the plug 114 positioned adjacent to the microfluidic network blocks fluid communication between the ports 142, 144 of the pump chambers 134, 136 and the microfluidic network. In an activated state (b) of the first pump 135 and the second pump 137, the plug 114 positioned adjacent to the microfluidic network allows fluid communication between the ports 142, 144 of the pump chambers 134, 136 and the microfluidic network. Each pump chamber 134, 136 has a length of about 50 mm to about 100 mm (e.g., about 80 mm) and an inner diameter of about 4 mm to about 8 mm (e.g., about 6 mm). Excluding the volume of the plug 114, each pump chamber 134, 136 can accommodate a fluid volume of about 1 mL to about 5 mL (e.g., about 2 mL).

[0020] 4 and 5 , the cartridge 108 defines a first reaction chamber 146 primed with a first reaction pellet 122 and housing a first magnet 116. The first reaction chamber 146 has an inner diameter of about 15 mm to about 20 mm (e.g., about 18 mm) and a volume of about 1 mL to about 2 mL (e.g., about 1.6 mL). The cartridge 108 further defines a mixing chamber 148 housing a second magnet 118 and an intermediate pellet 123. The mixing chamber 148 has an inner diameter of about 12 mm to about 18 mm (e.g., about 15 mm) and a volume of about 0.7 mL to about 1.7 mL (e.g., about 1.1 mL). The first magnet 116 can be actuated (e.g., rotated) by a reader to dissolve the first reaction pellet 122 in the liquid reagent in the first reaction chamber 146. The second magnet 118 can be actuated (e.g., rotated) by the reader to mix the fluid within the mixing chamber 148. The first reaction chamber 146 and the mixing chamber 148 can withstand magnet rotation speeds of up to approximately 60 rad / sec. During an analysis, the first magnet 116 and the second magnet 118 can be rotated by the reader at angular velocities ranging from approximately 6 rad / sec to approximately 30 rad / sec. Additionally, the first reaction chamber 146 and the mixing chamber 148 can withstand temperatures up to approximately 80°C and can be heated to temperatures of approximately 37°C to approximately 60°C by the respective adjacent heating elements of the reader during an analysis.

[0021] The cartridge 108 also defines a set of multiple (e.g., eight) second reaction chambers 150 so that multiplexing can be performed within the microfluidic device 100. Each second reaction chamber 150 is primed with a second reaction pellet 124 and contains an optional third magnet 120. Each second reaction chamber 150 has an internal width of about 2.0 mm to about 4.0 mm (e.g., about 3.0 mm), an internal length of about 5.0 mm to about 15.0 mm (e.g., about 10.0 mm), and an internal depth of about 1.0 mm to about 3 mm (e.g., about 2.2 mm). Thus, each second reaction chamber 150 has a volume of about 10 μL to about 200 μL (e.g., about 66 μL). In some embodiments, the third magnet 120 is actuated (e.g., rotated) by a reader to dissolve the second reaction pellet 124 in the liquid reagent within the second reaction chamber 150. The second reaction chamber 150 can withstand magnet rotation speeds of up to about 60 rad / s. In some embodiments, the third magnet 120 can be moved vertically within the second reaction chamber 150 at a rate of up to about 5 Hz. During analysis, the third magnet 120 can be rotated by the reader at angular velocities ranging from about 6 rad / s to about 30 rad / s, or raised and lowered within the chamber at a rate of about 1 Hz to about 5 Hz. Additionally, the second reaction chamber 150 can withstand temperatures of up to about 80°C and can be heated to temperatures of about 37°C to about 60°C by the reader's respective adjacent heating elements during analysis. In addition to, or instead of, mixing with the third magnet 120 within the second reaction chamber 150, mixing within the second reaction chamber 150 can be achieved by acoustic microstreaming.

[0022] 8, 9, and 10 show cross-sectional side, cross-sectional perspective, and top views, respectively, of the second reaction chamber 150. Acoustic microstreaming can be used to achieve mixing in the second reaction chamber 150 by a piezoelectric transducer or sonotrode in the reader acting on an air pocket formed by cavities 184 located along the bottom surface 182 of the second reaction chamber 150 or above the second reaction chamber 150 on top of the lid 106 covering the cartridge 108. A plurality of cavities 184 extend from the bottom surface 182 of the second reaction chamber 150. Microbubbles 186 are trapped in the cavities 184 of the second reaction chamber 150 by filling the chamber with fluid. The surfaces of the trapped microbubbles 186 vibrate as a result of the action of the ultrasonic transducer. The movement of the microbubble surface (e.g., the bubble "skin") causes movement of the liquid in contact with the microbubbles 186. These resulting vibrations at the applied acoustic frequency induce secondary flows within the fluid volume that resemble micro-vortices. Figure 10 shows four sequential images of mixing occurring via acoustic micro-streaming within the second reaction chamber 150. As shown, near complete mixing can be achieved within the second reaction chamber 150 within approximately 7 seconds.

[0023] In addition to housing the optional third magnet 120, each of the second reaction chambers 150 contains an identical air spring that allows for uniform distribution of fluid within the second reaction chambers 150. Because each second reaction chamber 150 contains its own air spring, the fill levels between the second reaction chambers 150 automatically balance as a result of the back pressure that is generated when the second reaction chambers 150 are filled with fluid. Thus, the second reaction chambers 150 can be filled with accurate, precise, and equivalent volumes of fluid to achieve equivalent pressures.

[0024] 4 and 5, the cartridge 108 defines multiple fluid channels of the microfluidic network. Additionally, a volume formed between the cartridge 108 and the lid 106 and external to the microfluidic network defined by the cartridge 108 provides a waste reservoir 192 (e.g., an air reservoir) that buffers air pressure within the microfluidic network, so that the microfluidic device 100 does not need to include a separate pressure balancing mechanism. a fluid channel 152 extending from the pump chambers 134, 136; a fluid channel 154 extending from the second portion 132 of the sample chamber 126 to the fluid channel 152; a fluid channel 156 extending from the fluid channel 152 to the first reaction chamber 146; a fluid channel 158 extending from the fluid channel 152 to the mixing chamber 148; a terminal fluid channel 160 extending from the fluid channel 152; a fluid channel network 162 feeding the second reaction chamber 150; a fluid channel 164 extending from the mixing chamber 148 to the fluid channel network 162; The cartridge 108 defines a fluid channel 166 extending from the first reaction chamber 146 to the fluid channel 168, a fluid channel 168 extending from the fluid channel 166, a branching fluid channel 170 extending from the pumping chambers 134, 136 to the fluid channel 168 and the waste reservoir 192, a fluid channel 172 extending from the first reaction chamber 146 to the fluid channel 168, a fluid channel 174 extending from the first reaction chamber 146 to the waste reservoir 192, a fluid channel 176 extending from the mixing chamber 148 to the fluid channel 168, and a fluid channel 178 extending from the mixing chamber 148 to the waste reservoir 192. The various channels 152-178 have internal widths ranging from about 1.0 mm to about 2.5 mm and widths ranging from about 0.5 mm to about 1.5 mm. 2 ~about 1.5mm 2 and a cross-sectional area in the range of

[0025] Cartridge 108, together with elastomer layer 104, further defines a series of valves 1-13 along the microfluidic network. Valves 1-6 are formed as channel valves, and valves 7-13 are formed as paging valves. Figures 11 and 12 show top and perspective cross-sectional views of the valves along cartridge 108. Particular cavities within cartridge 108 and corresponding regions of elastomer layer 104 located along the cavities can cooperate to form valves at selected locations along the microfluidic network. For example, region 188 of elastomer layer 104 located along cavity 190 of cartridge 108 cooperates to form valve 3. In the open state of valve 3 (as shown in Figures 11 and 12), region 188 is spaced from the surface of cavity 190, allowing fluid to pass through cavity 190 between the fluid channels connected to both ends of cavity 190. At a desired point during the analysis, the leader's piston can apply a force to region 188 to push region 188 into cavity 190, bringing it into contact with cavity 190 and preventing fluid from flowing between the fluid channels coupled to both ends of cavity 190, thereby closing valve 3. When valves 1-13 are closed, no cavities exist (i.e., the cavities have no depth). When the valves are open, fluid flow forces the valves open (i.e., the fluid flow imparts depth to the cavities). Thus, the cavities along cartridge 108 may have depths ranging from about 0 μm to about 750 μm, and the leader may apply a force ranging from about 6 N to about 8 N to close the valves to achieve adequate contact between the regions of elastomeric layer 104 and the cavities adjacent to cartridge 108.

[0026] The elastomeric layer 104 and lid 106 are attached to the cartridge 108 at one or more internal locations along an elastomeric gasket (e.g., as shown by the dark lines in Figures 2 and 3 ) and along the peripheral edges of various joining structures (e.g., laser welding, ultrasonic welding, adhesive, heat welding, and some fastening structures) so that the microfluidic device 100 is hermetically sealed when the sample chamber 126 is closed with the cap 110. The microfluidic device 100 is a disposable unit that can be discarded after a single use (e.g., after being used to perform a single analysis). The microfluidic device 100 has a compact footprint and is lightweight, weighing approximately 20 g to approximately 40 g, so that it can be easily handled, packaged, transported, and stored. The microfluidic device 100 may be provided as an assembled, ready-to-use device primed with pellets and reagents. Microfluidic device 100 may be provided in controlled atmosphere packaging (e.g., a composite plastic / metal foil pouch) containing moisture / oxygen absorbent pellets (e.g., for use with such sensitive components). Microfluidic device 100 may have a shelf life of about 12 months to about 24 months, as determined by the stability of the reagents (e.g., recombinase polymerase amplification (RPA) reagents).

[0027] The cartridge 108 is a rigid structure that may be made of one or more chemically robust materials, such as polypropylene, polystyrene, polyester, polymethyl methacrylate, and polyether ether ketone. In some embodiments, the cartridge 108 has an overall length (including the sample chamber 126) of about 80 mm to about 200 mm (e.g., about 150 mm). In some embodiments, the cartridge 108 has an overall width (including the sample chamber 126) of about 50 mm to about 100 mm (e.g., about 80 mm). In some embodiments, the cartridge 108 has an overall thickness (including the sample chamber 126) of about 8 mm to about 20 mm (e.g., about 16 mm). In some embodiments, the magnets 116, 118, 120 may be made of one or more chemically robust materials, such as neodymium, Teflon, or glass. In some examples, other inert materials may be used to encapsulate the magnets 116, 118, 120. In some embodiments, instead of magnets 116, 118, 120 in these locations, metallic materials (e.g., iron, nickel, and alloys) that are attracted to an external magnetic field (i.e., from the rig) may be used. Cartridge 108 may be transparent or translucent in one or more portions (e.g., chambers 146, 148, 150) to allow visualization and / or detection. Cartridge 108 also defines one or more alignment holes 199 (e.g., shown in Figures 1-3) sized and positioned to position microfluidic device 100 within the reader. For example, associated pins in the reader are positioned to engage alignment holes 199 in the cartridge.

[0028] In some embodiments, the cap 110 and seal 112 may be made of one or more chemically robust materials, such as polypropylene or nitrile butadiene rubber. In some embodiments, the seal 112 may be made of plastic-on-plastic or an overmolded thermoplastic elastomer. In some embodiments, the plug 114 may be made of bromobutyl or another material. Due at least in part to the chemical robustness of the cartridge 108 and plug 114, the pumps 135, 137 have been found to achieve average water vapor transmission (e.g., diffusion) rates as low as approximately 0.00054 g / (package × day) when used as a liquid reservoir.

[0029] Elastomer layer 104 may be made of one or more chemically robust materials, such as thermoplastic elastomers. Such material formulations allow elastomer layer 104 to elastically (e.g., reversibly) deform to open and close valves 1-13. In some embodiments, elastomer layer 104 has a total length of about 50 mm to about 150 mm (e.g., about 100 mm). In some embodiments, elastomer layer 104 has a total width of about 20 mm to about 80 mm (e.g., about 50 mm). In some embodiments, elastomer layer 104 has a total thickness of about 0.5 mm to about 1.5 mm (e.g., about 1.0 mm).

[0030] The lid 106 may be made of one or more materials, including polypropylene or polycarbonate. The lid 106 is transparent or translucent, allowing for visualization and detection of reactions occurring within the chambers 146, 148, 150 of the cartridge 108. In some embodiments, the overall length of the lid 106 is about 50 mm to about 200 mm (e.g., about 130 mm). In some embodiments, the lid 106 has an overall width of about 20 mm to about 80 mm (e.g., about 50 mm). In some embodiments, the lid 106 has a total thickness of about 0.5 mm to about 1.0 mm (e.g., about 0.7 mm).

[0031] As described above, the microfluidic device 100 is configured (e.g., has a size, shape, and material composition) for use with a reader capable of receiving the microfluidic device 100. The reader can receive the microfluidic device 100 within a test port. The reader is configured to interact with the microfluidic device 100 during the operation of an analysis within the microfluidic device 100. A series of actuators contact the microfluidic device 100 near various valve structures, effectively "opening and closing" the valves. As described above, the valves are typically in a closed state when the actuators compress the elastomeric layer 104 against the underlying molded cartridge 108. Thus, when the actuators are released, the elastomeric layer 104 relaxes, thereby allowing the valves to return to an open state. The reader also includes heating elements that apply localized heating to regions of the microfluidic device 100 as needed during the execution of an analysis. The reader also includes actuators that can push or pull the plungers (e.g., rods 140) of each pump to achieve desired fluid movement within the microfluidic network. The reader also contains fluorescence detection optics that interrogate each reaction chamber to provide a measurement indicative of the presence or absence of a target species within the sample being tested. The reader may further contain a bar code or similar system to identify the test type to verify whether the microfluidic device is within a specified expiration date and to associate the test results with an electronic patient record.

[0032] 13 through 23 show sequential schematic diagrams of the cartridge 108 corresponding to a method for performing an assay to detect a target nucleic acid using the microfluidic device 100. In some embodiments, the assay is a Flu / RSV assay performed under isothermal conditions. Referring first to FIG. 13, the sample chamber 126 of the cartridge 108 is uncapped prior to the start of the assay so that the microfluidic device 100 is open to the ambient environment. The first pump 135 is in a closed state and primed with about 1 mL to about 5 mL (e.g., about 2 mL) of a first liquid reagent 194, shown in blue. In some embodiments, the first liquid reagent 194 is a lysis buffer and includes a catalytic agent, such as magnesium. In some embodiments, the lysis buffer is an enzymatic lysis agent. In some embodiments, the lysis buffer includes hydrochloric acid (HCl) at a concentration of about 10 mM to about 10 mM and TritionX 100 at a concentration of about 0.1% to about 1%.

[0033] Continuing with reference to FIG. 13 , the second pump 137 is in a closed state and primed with about 1 mL to about 5 mL (e.g., about 2 mL) of a second liquid reagent 196, shown in green. In some embodiments, the second liquid reagent 196 is typically a reaction buffer and has a near-neutral pH. The second liquid reagent may include a catalytic agent, such as magnesium. The first reaction chamber 146 and the second reaction chamber 150 are primed with a first reaction pellet 122 and a second reaction pellet 124, respectively. In some embodiments, the first reaction pellet 122 and the second reaction pellet 124 each contain one or more lyophilized amplification reagents, such as RPA reagents, a catalytic reagent (e.g., magnesium), and oligomers. The RPA reagents may include primers specific for the target of interest and, if present, at least one probe with a detectable label for visualization of the amplified target. The probe typically contains a fluorophore and a quencher that separate upon cleavage by a nuclease when the probe hybridizes to a complementary sequence, if present in the amplified sample mixture.

[0034] Microfluidic device 100 is inserted into a reader, and the reader is manually operated via one or more controls (e.g., buttons and switches) to initiate an analysis. At the start of an analysis, valves 1-13 are open while cartridge 108 is inserted into the reader. The reader is then controlled to activate valves 1-13, pumps 135, 137, magnets 116, 118, 120, and piezoelectric transducer 180 (shown in FIG. 8), as well as heat chambers 146, 148, 150 at specific times to perform an analysis using microfluidic device 100.

[0035] 14, valves 2 and 3 are opened, and first pump 135 pumps (e.g., pushes or suctions) first liquid reagent 194 from first pump chamber 134 through fluidic channels 152 and 154 into sample chamber 126 over a period of about 5 seconds to about 10 seconds (e.g., about 9 seconds). Referring to FIG. 15, valves 2 and 3 are closed, and valves 1, 12, and 13 are open. First pump 135 draws (e.g., pulls) about 1 mL to about 5 mL (e.g., about 2 mL) of air from the ambient environment into pump chamber 134 through fluidic channels 170, 168, and 166 and sample chamber 126 over a period of about 1 second to about 10 seconds (e.g., about 5 seconds).

[0036] Referring to FIG. 16 , valves 1, 12, and 13 are closed. The sample is delivered to sample chamber 126 by the user, which is then closed and sealed with cap 110 and seal 112. In some embodiments, the sample is a biological fluid or substance collected from a human or another animal. For example, the sample may include one or more of blood, sputum, mucus, saliva, tears, or urine. The sample can be obtained from a subject using an appropriate sample collection device, such as a cotton swab or collection tube. For example, a cotton swab can be used to collect a nasal, nasopharyngeal, vaginal, buccal, or tissue passage sample and applied to the ear, eye, throat, wound, or other body surface. In some embodiments, the sample can be delivered directly to sample chamber 126 immediately after collection from the subject using the sample collection device. In some embodiments, the sample can be stored in viral transport medium (VTM) or other liquid reagent after collection from the subject, and an aliquot of the stored sample can be delivered to sample chamber 126 to begin analysis. A sample obtained from a subject or stored in another medium can be applied to sample chamber 126 in substantially the same form as it was collected from the subject (e.g., without purification of the sample to isolate target nucleic acids or removal of biological matrix or other components from the sample).

[0037] In some embodiments, the sample contains a target nucleic acid comprising a target polynucleotide sequence (e.g., or more than one target polynucleotide sequence, such as two or three target polynucleotide sequences). In some embodiments, the target nucleic acid is double-stranded DNA, single-stranded DNA, or RNA. In some embodiments, the target nucleic acid is genomic DNA, plasmid DNA, viral DNA, mitochondrial DNA, cDNA, synthetic double-stranded DNA, and synthetic single-stranded DNA. In some embodiments, the target nucleic acid is viral DNA or viral RNA. In some embodiments, the target nucleic acid is derived from an animal pathogen (e.g., a single-stranded DNA virus, a double-stranded DNA virus, a single-stranded RNA virus, or a bacterium). In some embodiments, the animal pathogen is influenza A virus, influenza B virus, or RSV. In some embodiments, the sample is mixed with RPA reagents before being sent to sample chamber 126.

[0038] The sample mixes with about 400 μL to about 1500 μL (e.g., about 500 μL) of first liquid reagent 194 contained in sample chamber 126 for about 0 seconds to about 60 seconds (e.g., about 10 seconds). If first liquid reagent 194 contains a lysing agent, the sample may be lysed during this mixing period. In some embodiments, sample chamber 126 may be heated during the mixing step. Once the sample is delivered to sample chamber 126 and the cap is placed by the user, the integrity of the seal of sample chamber 126 is tested by the reader.

[0039] 17 , valves 1, 3, 4, 9, 12, and 13 are opened, and first pump 135 pumps air contained in pump chamber 134 into sample chamber 126 via fluidic channels 170, 168, and 166, thereby pumping a volume of about 200 μL to about 1000 μL (e.g., about 500 μL) of sample and first liquid reagent 194 contained in sample chamber 126 into first reaction chamber 146 via fluidic channels 154, 152, and 156 over a period of about 5 seconds to about 15 seconds (e.g., about 10 seconds). A first amplification reaction occurs in first reaction chamber 146 over a period of about 300 seconds to about 600 seconds (e.g., about 480 seconds), producing a first reaction product. In some embodiments, the first amplification reaction for amplifying a target polynucleotide sequence is an RPA reaction, and the first reaction product includes a first amplified polynucleotide sequence. During the first amplification reaction, the first reaction chamber 146 is heated and the first magnet 116 within the first reaction chamber 146 rotates to dissolve the first reaction pellet 122 in the sample.

[0040] 18, valves 3, 4, 9, 12, and 13 are closed, valve 11 is open, and first pump 135 draws about 1 mL to about 5 mL (e.g., about 2 mL) of air from waste reservoir 192 and into first pump chamber 134 through fluid channel 170 while a first amplification reaction occurs in first reaction chamber 146, as indicated by the cyan color. Referring to FIG. 19, valve 11 is closed, and valves 12, 10, 4, and 6 are open. First pump 135 pumps air contained in first pump chamber 134 through fluidic channels 170, 168, and 172 into first reaction chamber 146, thereby pumping about 100 μL to about 400 μL (e.g., about 200 μL) of first reaction product from first reaction chamber 146 into the portion of fluidic channel 152 between valves 4 and 6 that defines metering channel 198, over a period of about 5 seconds to about 10 seconds (e.g., about 7 seconds). As indicated by the cyan color, excess amount 197 of first reaction product in metering channel 198 flows into terminal fluidic channel 160.

[0041] 20 , valves 1, 12, 10, 4, and 6 are closed, and valves 5 and 7 are open. Second pump 137 pumps about 200 μL to about 800 μL (e.g., about 400 μL) of second liquid reagent 196 from second pump chamber 136 through metering channel 198 and fluidic channel 158 into mixing chamber 146, thereby displacing the first reaction product in metering channel 198 and flowing into mixing chamber 148 over a period of about 5 seconds to about 10 seconds (e.g., about 8 seconds). Within metering channel 198 and mixing chamber 148, second liquid reagent 196 dilutes the first reaction product contained in metering channel 198 (e.g., about 1:50). A volume of about 500 μL to about 2000 μL (e.g., about 1000 μL) of second liquid reagent 196 remains in second pump chamber 136. Mixing chamber 148 has a volume of about 5 μL to about 20 μL (e.g., about 8 μL) of the first reaction product, shown in yellow. Mixing chamber 148 is heated, and second magnet 118 rotates, shown in yellow, to dissolve pellet 123 into the first reaction product and second liquid reagent 196. Mixing occurs over a period of about 2 seconds to about 10 seconds (e.g., about 5 seconds). Residual air in mixing chamber 148 is vented to waste reservoir 192 via valve 7.

[0042] 21, valves 5 and 7 are closed, valve 11 is open, and first pump 135 draws about 1000 μL to about 5000 μL (e.g., about 2000 μL) of air from waste reservoir 192 through fluid channel 170 into first pump chamber 134 while mixing occurs in mixing chamber 148. Referring to FIG. 22, valve 11 is closed, and valves 8, 12, and 14 are open. First pump 135 pumps about 200 μL to about 1000 μL (e.g., about 500 μL) of air from first pump chamber 134 into the mixing chamber through fluid channels 170, 168, and 176, thereby sweeping the first reaction product in mixing chamber 146 through fluid channel network 162 and into second reaction chamber 150 over a period of about 5 seconds to about 20 seconds (e.g., about 17 seconds). The first reaction product is forced against the air spring associated with the second reaction chamber 150, delivering an equal volume of the first reaction product of about 22.5 μL to about 27.5 μL (e.g., about 25.0 μL) to the second reaction chamber 150, resulting in the second reaction chamber 150 being about 30% full.

[0043] A second amplification reaction occurs in second reaction chamber 150 for a period of about 180 seconds to about 600 seconds (e.g., about 240 seconds), during which time second reaction chamber 150 is heated and third magnet 120 rotates or moves vertically to dissolve second reaction pellet 124 into the first reaction product. In some embodiments, the second amplification reaction for amplifying the first amplified polynucleotide sequence is an RPA reaction, and the second reaction product comprises a second amplified polynucleotide sequence that includes a smaller sequence that is entirely contained within the first amplified polynucleotide sequence.

[0044] 23, valves 1, 8, and 14 are closed and continuous fluorescent detection of the second amplification reaction is performed. In some embodiments, continuous fluorescent detection is performed by: labeling the second amplification product with a first oligonucleotide conjugated to a fluorophore and a quencher to produce a labeled second product; cleaving the quencher from the labeled second amplification product; and optically detecting a signal from the fluorophore (e.g., indicative of the presence of a second amplification product). In some embodiments, the quencher is cleaved using a nuclease. In some embodiments, the nuclease targets double-stranded DNA. In some embodiments, the nuclease is formamidopyrimine-DNA glycosylase. Detection can be performed using a reader adjacent to the lid 106 and cartridge 108 along the second reaction chamber 150 every about 5 seconds to about 20 seconds (e.g., about 15 seconds) for a period of about 3 minutes to about 10 minutes (e.g., about 4 minutes).

[0045] An analysis can be performed using microfluidic device 100 in a period of less than about 30 minutes (e.g., less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes) as measured from the time the sample is delivered to sample chamber 126 to the time detection is completed. Following detection, microfluidic device 100 is ejected from the reader, and microfluidic device 100 is manually removed from the reader. Due, at least in part, to the closed system configuration of microfluidic device 100 (e.g., after capping sample chamber 126), the risk of leakage contamination to the surrounding environment is significantly reduced compared to conventional devices used to perform similar analyses.

[0046] 24 through 27 illustrate additional exemplary embodiments and features of the microfluidic devices described herein. Referring to FIG. 24, exemplary dimensions of the device are shown. In some embodiments, the device is approximately 82 mm x 106 mm x 11 mm in its widest dimension, although other dimensions are specifically contemplated (e.g., ±5%, ±10%, ±15%, ±20%, ±30%, ±40%, ±50%, etc.). Still referring to FIG. 24, in some embodiments, the device comprises an interior beveled portion 204 of the first reaction chamber 146. Still referring to FIG. 24, in some embodiments, the handle 206 of the device comprises a full circle at the grip edge for a more ergonomic design.

[0047] 27, in some embodiments, the lid 106 of the device is attached to the device via a snap 207. In some embodiments, the lid 106 does not have a V-notch 208, but the body of the device retains a V-notch 208 (shown in FIG. 24).

[0048] In some embodiments, the valve configuration is arranged to minimize the size of the device (e.g., minimize the dimensions of the device). FIG. 24 shows an exemplary valve configuration, but other configurations are specifically contemplated. In some embodiments, shared welds between fluid flow regions are avoided to facilitate leak detection. For example, referring to FIG. 24, in some embodiments, region 150 has no shared welds between device components.

[0049] In some embodiments, device robustness is enhanced by molding techniques including, for example, thinner cored-out walls, strong cored-out features, and robust core pins.

[0050] In some embodiments, the device includes a leak-proof sealing cap to minimize risk and contamination of hazardous or harmful materials (e.g., biological samples containing pathogens), including avoiding contamination of detection equipment used with the device. In some embodiments, this is achieved with one or more or each of a sealing cap, a cap closure sensor, and a cap pressure sensor. Figures 25-26 show such exemplary embodiments.

[0051] Referring to FIG. 25, an embodiment of the cap 110 is shown. Referring to the top panel of FIG. 25, a detent mechanism 201 designed to secure the cap 110 in an open position is shown. With further reference to FIG. 25, a cap gasket 200 is shown. Without being limited to a particular material, in some embodiments, the cap gasket 200 is molded in place and comprises silicone. With further reference to FIG. 25, a gasket sealing rib 202 is shown. In some embodiments, the gasket and gasket sealing rib prevent sample leakage from the sample chamber 106. Also referring to FIG. 25, a snap hook 205 component for sealing the cap in the closed position is shown. In the closed position, the snap hook 205 mates with a mating structure 209 in the upper region of the sample chamber 126.

[0052] 26, there is shown a cap position detection component 203. In some embodiments, the cap position detection component 203 is configured to detect leaks and / or improperly secured caps. In some embodiments, the cap position detection component is an optical cap closure sensor and / or a pressure detector.

[0053] In some embodiments, the cap position detection component 203 includes an optical cap closure sensor. For example, in some embodiments, the cap closure detection component generates a light beam across the cap opening, which is interrupted when the cap 110 is secured in place (e.g., via a snap hook 205 or other cap sealing component). In some embodiments, the device, or an instrument functioning with the device, is configured to cease operation or sound an alarm when the cap is not in the closed, sealed position (e.g., when the light beam is not interrupted).

[0054] In some embodiments, the cap position detection component 203 includes a pressure sensor that measures the cap's ability to withstand pressure. In some embodiments, pressure force feedback (e.g., provided by the device's pump) is utilized to detect a cap that is not properly sealed or exhibiting a leak. In some embodiments, pressure outside of an expected range (e.g., indicating that the cap is not properly sealed or that the cap is leaking) results in an alarm or a cessation of operation of the device or equipment functioning with the device.

[0055] A number of embodiments of the present invention have been described. Nevertheless, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the claims which follow this detailed description.

[0056] For example, although microfluidic device 100 has been described and illustrated as including eight second reaction chambers 150, in some embodiments, a microfluidic device substantially similar in structure and function to microfluidic device 100 may include a different number of second reaction chambers, such as one, two, three, four, five, six, seven, or more than eight second reaction chambers.

[0057] Although the elastomeric layer 104, cartridge 108, and lid 106 have been described as having particular dimensions, in some embodiments, a microfluidic device substantially similar in structure and function to the microfluidic device 100 may include an elastomeric layer, a cartridge, and a lid having dimensions that differ from those shown for the elastomeric layer 104, the cartridge 108, and the lid 106.

[0058] Although cartridge 108 is described and illustrated as including an optional intermediate pellet 123, in some embodiments, a microfluidic device similar in structure and function to microfluidic device 100 may not include an optional intermediate pellet 123.

[0059] Although the methods shown in Figures 13 through 27 have been described with respect to particular time periods, fluid volumes, pellet components, and liquid reagent components applicable to particular analyses, in some embodiments, the microfluidic device 100 can be used to perform similar or different analyses involving different time periods, fluid volumes, pellet components, and liquid reagent components.

[0060] Although the apparatus and methods herein are described as applications of recombinase polymerase amplification (RPA) technology, other isothermal techniques for amplifying and detecting target nucleic acids can also be implemented in the microfluidic device 100 described herein (e.g., cleavage and extension amplification reaction (NEAR) technology). Methods of RPA amplification and detection of RPA amplification products described herein are described in detail in U.S. Patent Nos. 7,399,590, 8,580,507, 7,270,981, 7,399,590, and 7,666,598, 7,435,561, U.S. Patent Application Publication No. 2009 / 0029421, and WO 2010 / 141940. The NEAR method is described in U.S. Patent Application Publication Nos. 2009 / 0081670 and 2009 / 0017453 and U.S. Patent Nos. 9,562,263 and 9,562,264, each of which is incorporated by reference in its entirety and is considered part of this disclosure.

Claims

1. 1. A microfluidic device comprising: an inlet port configured to receive the sample; a first reaction chamber fluidly coupled to the inlet port; a first pump fluidly coupled to the inlet port; a second pump fluidly coupled to the mixing chamber; a metering channel fluidly coupled to the first reaction chamber and the mixing chamber; one or more second reaction chambers fluidly coupled to the mixing chamber; Equipped with the first pump is configured to move fluid from the inlet port to the first reaction chamber and from the first pump to the inlet port; the second pump is configured to move fluid from the second pump to the mixing chamber, from the first reaction chamber to the mixing chamber, and from the mixing chamber to one or more second reaction chambers; Microfluidic devices.

2. The microfluidic device of claim 1 , further comprising a waste reservoir configured to regulate fluid pressure within the microfluidic device.

3. The microfluidic device of claim 1 , wherein the first reaction chamber contains a first set of amplification reagents.

4. 4. The microfluidic device of claim 3, wherein the first set of amplification reagents comprises recombinase polymerase amplification (RPA) reagents.

5. The microfluidic device of claim 4 , wherein the RPA reagents are lyophilized.

6. The microfluidic device of claim 3 , wherein the first reaction chamber further comprises a catalytic reagent.

7. The microfluidic device of claim 6 , wherein the catalytic reagent comprises magnesium.

8. The microfluidic device of claim 3 , wherein the first set of amplification reagents comprises oligomers.

9. The microfluidic device of claim 1 , wherein the mixing chamber contains a second set of amplification reagents.

10. The microfluidic device of claim 9 , wherein the second set of amplification reagents comprises RPA reagents.

11. The microfluidic device of claim 10 , wherein the RPA reagents are lyophilized.

12. The microfluidic device of claim 9 , wherein the second set of amplification reagents comprises oligomers.

13. The microfluidic device of claim 1 , wherein the one or more second reaction chambers each contain a second set of amplification reagents.

14. The microfluidic device of claim 13 , wherein the second set of amplification reagents comprises RPA reagents.

15. The microfluidic device of claim 13 , wherein the RPA reagents are lyophilized.

16. The microfluidic device of claim 13 , wherein the second set of amplification reagents comprises oligomers.

17. The microfluidic device of claim 1 , wherein the first pump comprises a first buffer solution.

18. The microfluidic device of claim 1 , wherein the first pump comprises a first buffer and a lysing agent.

19. The microfluidic device of claim 1 , wherein the second pump comprises a second buffer solution.

20. The microfluidic device of claim 1 , wherein the second pump comprises a second buffer and a lysing agent.

21. The microfluidic device of claim 1 , wherein the first pump comprises a catalytic reagent.

22. The microfluidic device of claim 1 , wherein the second pump comprises a catalytic reagent.

23. 23. The microfluidic device of claim 21 or claim 22, wherein the catalytic reagent comprises magnesium.

24. The microfluidic device of claim 1 , wherein each of the one or more second reaction chambers is a detection chamber.

25. 25. The microfluidic device of claim 24, wherein a portion of each detection chamber is optically transparent.

26. The microfluidic device of claim 1 , wherein the first reaction chamber is configured to be coupled to a heating unit.

27. The microfluidic device of claim 1 , wherein the inlet port is configured to be coupled to a heating unit.

28. The microfluidic device of claim 1 , wherein the first reaction chamber comprises or is coupled to a mixing means.

29. The microfluidic device of claim 1 , wherein the mixing chamber comprises or is connected to a mixing means.

30. The microfluidic device of claim 1 , wherein the one or more second reaction chambers each comprise or are coupled to a mixing means.

31. 31. The microfluidic device of claim 28, claim 29, or claim 30, wherein the mixing means is a magnet.

32. 31. The microfluidic device of claim 30, wherein the mixing means operates by acoustic streaming.

33. 10. The microfluidic device of claim 1, wherein the inlet port contains a sample, the first pump contains a first buffer, and the first pump is configured to pump the first buffer from the first pump to the inlet port to generate a diluted sample comprising the sample and the first buffer.

34. 34. The microfluidic device of claim 33, wherein the first reaction chamber comprises a first set of amplification reagents, and the first pump is configured to provide a portion of the diluted sample through the inlet port to the first reaction chamber to generate a first reaction mixture comprising the diluted sample and the first set of amplification reagents.

35. 35. The microfluidic device of claim 34, wherein the second pump is configured to provide a portion of the first reaction mixture from the first reaction chamber to the mixing chamber via the metering channel.

36. 36. The microfluidic device of claim 35, wherein the second pump comprises a second buffer solution, the second pump configured to pump the second buffer solution from the second pump through the metering channel to the mixing chamber, the second buffer solution combining with a portion of the first reaction mixture to produce a diluted first reaction mixture.

37. 37. The microfluidic device of claim 36, wherein the one or more second reaction chambers each contain a second set of amplification reagents, and the second pump is configured to pump a portion of the diluted first reaction mixture from the mixing chamber to each of the one or more second reaction chambers to generate a second reaction mixture containing the diluted first reaction mixture and the second set of amplification reagents.

38. 38. The microfluidic device of claim 37, wherein the second set of amplification reagents comprises oligomers.

39. 36. The microfluidic device of claim 35, wherein the mixing chamber contains a second set of amplification reagents, the second reagent chamber contains a second buffer solution, and the second pump is configured to pump the second buffer solution from the second reagent chamber through the metering channel to the mixing chamber, wherein the second buffer solution combines with a portion of the first reaction mixture and the second set of amplification reagents to form a second reaction mixture.

40. 40. The microfluidic device of claim 39, wherein a second pump is configured to pump a portion of the second reaction mixture into each of the one or more second reaction chambers.

41. 41. The microfluidic device of claim 40, wherein each of the one or more second reaction chambers contains an oligomer.

42. The microfluidic device of claim 1 , wherein the microfluidic device comprises two, three, four, five, six, seven, or eight second reaction chambers.

43. The microfluidic device of claim 1 , further comprising a series of valves.

44. 10. The microfluidic device of claim 1, further comprising alignment holes for connecting the microfluidic device to a reader configured to process the sample and deliver the sample to the microfluidic device.

45. 45. The microfluidic device of claim 44, wherein the connection port is configured for lockable engagement with a reader.

46. 42. The microfluidic device of claim 41, wherein the microfluidic device is a disposable cartridge.

47. The microfluidic device of claim 1 , wherein the first pump and the second pump are syringe pumps.

48. The microfluidic device of claim 1 , wherein the inlet port comprises a cap.

49. 49. The microfluidic device of claim 48, wherein the cap comprises a gasket comprising gasket sealing ribs.

50. 49. The microfluidic device of claim 48, wherein the cap comprises a detent feature to secure the cap in an open position.

51. 10. A reader configured to receive the microfluidic device of claim 1, the reader comprising a detector configured to detect the presence of a second reaction product in one or more second reaction chambers.

52. 52. The reader of claim 51, wherein the microfluidic device or reader comprises a cap position detection component configured to detect cap closure or cap leakage.

53. 53. The reader of claim 52, wherein the cap position detection component comprises one or more components selected from the group consisting of an optical cap closure sensor and a pressure sensor.

54. 54. The reader of claim 53, wherein the optical cap closure sensor comprises a light beam that is interrupted when the cap is in the closed, sealed position.

55. 55. The reader of claim 54, wherein the pressure sensor assesses the cap's ability to withstand pressure.

56. 56. The reader of claim 55, wherein the pressure is generated using a pump in the device.

57. 56. The reader of claim 55, wherein a pressure outside a predetermined range indicates an unsealed cap.

58. 53. The reader of claim 52, configured to stop operation of the reader or microfluidic device if the cap is not identified as sealed.

59. 1. A method comprising: providing a sample fluid containing a target nucleic acid comprising at least one target polynucleotide sequence to a microfluidic device; amplifying at least one target polynucleotide sequence under isothermal conditions, wherein the amplifying comprises: performing a first round of amplification on the target polynucleotide sequence to produce a first amplification product comprising a first amplified polynucleotide sequence; performing a second round of amplification on the first amplified polynucleotide sequence to produce a second amplification product comprising a second amplified polynucleotide sequence, the second amplified polynucleotide sequence comprising a smaller sequence entirely contained within the first amplified polynucleotide sequence produced during the first round of amplification; A method comprising:

60. 60. The method of claim 59, further comprising detecting the second amplification product.

61. detecting the second amplification product; labeling the second amplification product with a first oligonucleotide linked to a fluorophore and a quencher to produce a labeled second product; cleaving the quencher from the labeled second amplification product; optically detecting a signal from the fluorophore, wherein the detectable signal indicates the presence of a second amplification product; and 60. The method of claim 59, comprising:

62. 62. The method of claim 61, wherein cleavage of the quencher is carried out using a nuclease.

63. 62. The method of claim 61, wherein the nuclease targets double-stranded DNA.

64. 62. The method of claim 61, wherein the nuclease is formamidopyrimine-DNA glycosylase.

65. 60. The method of claim 59, wherein the amplifying step comprises performing a first round of amplification, and the amplification is RPA.

66. 60. The method of claim 59, wherein the amplifying step comprises performing a second round of amplification, wherein the amplification is RPA.

67. 60. The method of claim 59, wherein the amplifying step comprises performing a first round of amplification, wherein the amplification is RPA, and a second round of amplification, wherein the amplification is RPA.

68. 60. The method of claim 59, wherein the sample is blood, sputum, mucus, saliva, tears, or urine.

69. 60. The method of claim 59, further comprising obtaining the sample from an animal.

70. 70. The method of claim 69, wherein the sample is obtained from an animal, and the animal is a human.

71. 60. The method of claim 59, wherein the target nucleic acid is a target nucleic acid of an animal pathogen.

72. 72. The method of claim 71, wherein the animal pathogen is a single-stranded DNA virus, a double-stranded DNA virus, or a single-stranded RNA virus.

73. 72. The method of claim 71, wherein the animal pathogen is a bacterium.

74. 60. The method of claim 59, wherein the target nucleic acid is double-stranded DNA, single-stranded DNA, or RNA.

75. 60. The method of claim 59, wherein the target nucleic acid is selected from the group consisting of genomic DNA, plasmid DNA, viral DNA, mitochondrial DNA, cDNA, synthetic double-stranded DNA, and synthetic single-stranded DNA.

76. 60. The method of claim 59, wherein the target nucleic acid is viral DNA or viral RNA.

77. 72. The method of claim 71, wherein the animal pathogen is influenza A virus, influenza B virus, or respiratory syncytial virus (RSV).

78. 60. The method of claim 59, wherein the target nucleic acid comprises two target polynucleotide sequences.

79. 60. The method of claim 59, wherein the target nucleic acid comprises three target polynucleotide sequences.

80. 60. The method of claim 59, further comprising mixing the sample with RPA reagents prior to providing the sample to the microfluidic device.

81. 60. The method of claim 59, wherein the second amplification product is detected in less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes after providing the sample to the microfluidic device.

82. 82. The method of claim 81, wherein the second amplification product is detected in real time.

83. 60. The method of claim 59, further comprising lysing the sample prior to amplification.

84. 84. The method of claim 83, wherein the lysing step comprises combining the sample with a lysing agent.

85. 85. The method of claim 84, wherein the lysis agent is an enzyme.

86. 84. The method of claim 83, wherein the dissolving step comprises mechanical means.

87. 84. The method of claim 83, wherein the lysing step comprises heating the sample.

88. 1. A method comprising: providing a sample containing a target nucleic acid, the target nucleic acid comprising at least one target polynucleotide sequence, to a microfluidic device; amplifying at least one target polynucleotide sequence; Amplifying performing a first round of amplification on the target polynucleotide sequence to produce a first amplification product comprising a first amplified polynucleotide sequence; performing one or more additional successive amplifications of the first amplified polynucleotide sequence to form additional amplification products, wherein the amplification products from each successive n+1 amplifications comprise amplified polynucleotide sequences that are smaller sequences that are entirely contained within the amplified polynucleotide sequences generated during the previous n amplifications; performing a final round of amplification on the penultimate amplified polynucleotide sequence to produce a final amplification product; Detecting the final amplification product; A method comprising:

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